substrate specificity of lysyl hydroxylase isoforms and...
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U N I V E R S I TAT I S O U L U E N S I SACTAA
SCIENTIAE RERUMNATURALIUM
OULU 2008
A 511
Maija Risteli
SUBSTRATE SPECIFICITYOF LYSYL HYDROXYLASE ISOFORMS AND MULTI-FUNCTIONALITY OF LYSYL HYDROXYLASE 3
FACULTY OF SCIENCE,DEPARTMENT OF BIOCHEMISTRY,BIOCENTER OULU,UNIVERSITY OF OULU;FINNISH NATIONAL GLYCOSCIENCE GRADUATE SCHOOL
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Maija R
isteli
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A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 5 1 1
MAIJA RISTELI
SUBSTRATE SPECIFICITY OF LYSYL HYDROXYLASE ISOFORMS AND MULTIFUNCTIONALITY OF LYSYL HYDROXYLASE 3
Academic dissertation to be presented, with the assent ofthe Faculty of Science of the University of Oulu, for publicdefence in Raahensali (Auditorium L10), Linnanmaa, onAugust 8th, 2008, at 12 noon
OULUN YLIOPISTO, OULU 2008
Copyright © 2008Acta Univ. Oul. A 511, 2008
Supervised byProfessor Raili Myllylä
Reviewed byProfessor Leena Bruckner-TudermanProfessor Helena Kuivaniemi
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OULU UNIVERSITY PRESSOULU 2008
Risteli, Maija, Substrate specificity of lysyl hydroxylase isoforms andmultifunctionality of lysyl hydroxylase 3Faculty of Science, Department of Biochemistry, University of Oulu, P.O.Box 3000, FI-90014University of Oulu, Finland, Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014University of Oulu, Finland; Finnish National Glycoscience Graduate School, University ofKuopio, P.O.Box 1627, FI-70211 Kuopio, Finland Acta Univ. Oul. A 511, 2008Oulu, Finland
AbstractLysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagensand collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have beenidentified from different species. In addition, LH2 has two alternatively spliced forms, LH2a andLH2b. The hydroxylysines have an important role in the formation of the intermolecular collagencrosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are furtherglycosylated.
In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzedusing synthetic peptide substrates. The data did not indicate strict amino acid sequence specificityfor the LH isoforms. However, there seemed to be a preference for some sequences to be boundand hydroxylated by a certain isoform.
Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation ofglucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme,possessing LH and GGT activities. The DXD-like motif, characteristic of manyglycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminalpart of the LH3 molecule were critical for the GGT activity, but not for the LH activity of themolecule.
The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culturemedia of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family,which was earlier reported to have a deficiency of GGT activity. In this study, we showed that thereduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Ourdata indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition andorganization of the key extracellular matrix components, collagen types VI and I and fibronectin,and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as animportant extracellular regulator.
Keywords: collagen biosynthesis, collagen glycosyltransferase, cytoskeleton,epidermolysis bullosa simplex, extracellular matrix, lysyl hydroxylase
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Acknowledgement
This work was carried out at the Department of Biochemistry and Biocenter Oulu, University of Oulu.
I want to express my deepest gratitude to my supervisor, Professor Raili Myllylä for her guidance and the opportunity to work in her research group. Her encouragement and everlasting optimism were crucial during this work. I wish to thank Professor Kalervo Hiltunen and all the other group leaders for creating friendly atmosphere and excellent research facilities at the department. I also thank the whole staff of the department for helping me in so many ways.
I am grateful to Professor Helena Kuivaniemi and Professor Leena Bruckner-Tuderman for their valuable comments on the manuscript. Doctor Deborah Kaska is acknowledged for the careful and rapid revision of the language. I am indebted to all my collaborators and coauthors. Especially, I want to thank Olli Niemitalo M.Sc., Doctor Andre Juffer, Docent Hilkka Lankinen, Docent Raija Sormunen, Doctor Naomi Baker, Doctor Shireen Lamandé and Leena Vimpari-Kauppinen M.D. for their contribution to this work.
I am thankful to Chunguang Wang, Jari Heikkinen and Antti Salo and all the former members of the RM-team for collaboration and good companionship. In particular, I want to express my gratitude to Heli Ruotsalainen and Laura Sipilä for their friendship and the discussions concerning science and everyday life. I thank Anna-Maija Koisti, Mervi Matero and Piia Mäkelä for excellent technical assistance. I also want to thank the members of “the lunch time board” for nice company.
My warmest thanks belong to my parents for the love, support and encouragement during my whole life. I give thanks to my relatives and friends for their support and interest during this work. I want to thank my mother-in-law for helping our family in every day life.
Finally, I express my dearest gratitude to my husband Jari and our son Eero for love and understanding and for keeping me in touch with the real life.
This work was financially supported by the Academy of Finland, Sigrid Juselius Foundation, Biocenter Oulu, University of Oulu, Glycoscience Graduate School, Research and Science Foundation of Farmos, Societas Scientiarum Fennica and Maud Kuistila Memorial Foundation.
Oulu, May 2008 Maija Risteli
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Abbreviations
BM basement membrane DXD aspartate-any amino acid-aspartate C carboxy cDNA complementary DNA EBS epidermolysis bullosa simplex ECM extracellular matrix EDSVI Ehlers-Danlos syndrome type VI ER endoplasmic reticulum GGT galactosylhydroxylysyl glucosyltransferase GT hydroxylysyl galactosyltransferase Hyl hydroxylysine KDEL lysine-aspartate-glutamate-leucine Km the Michaelis constant LH lysyl hydroxylase mRNA messenger RNA N amino PCR polymerase chain reaction PLOD procollagen-lysine, 2-oxoglutarate, 5-dioxygenase, gene name for
human lysyl hydroxylase SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis siRNA small interfering RNA SLRPs small leucine rich proteoglycans UDP uridine diphosphate Vmax maximum velocity X any amino acid Y any amino acid
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List of original articles
This thesis is based on the following articles, which are referred to in the text by their Roman numerals:
I Risteli M, Niemitalo O, Lankinen H, Juffer AH & Myllylä R (2004) Characterization of collagenous peptides bound to lysyl hydroxylase isoforms. J Biol Chem 279(36): 37535–37543.
II Heikkinen J, Risteli M*, Wang C*, Latvala J, Rossi M, Valtavaara M & Myllylä R (2000) Lysyl hydroxylase is a multifunctional protein possessing collagen glucosyltransferase activity. J Biol Chem 275(46): 36158–36163.
III Wang C, Risteli M, Heikkinen J, Hussa AK, Uitto L & Myllylä R (2002) Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3). J Biol Chem 277(21): 18568–18573.
IV Risteli M, Ruotsalainen H, Salo AM, Sormunen R, Baker NL, Lamandé SR, Vimpari-Kauppinen L & Myllylä R (2008) Decreased lysyl hydroxylase 3 (LH3) activity in a family with dominant epidermolysis bullosa simplex causes abnormalities in deposition and organization of extracellular matrix. Manuscript.
*Equal contribution
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Contents
Abstract Acknowledgement 5 Abbreviations 7 List of original articles 9 Contents 11 1 Introduction 15 2 Review of the literature 17
2.1 Extracellular matrix................................................................................. 17 2.2 Collagens................................................................................................. 17
2.2.1 Classification of collagen types.................................................... 18 2.2.2 Proteins with a collagenous domain ............................................. 20 2.2.3 Collagen biosynthesis................................................................... 21
2.3 Lysine modifications ............................................................................... 22 2.3.1 Lysine modifications in collagens ................................................ 23 2.3.2 Lysine modifications in collagenous proteins............................... 28
2.4 Lysyl hydroxylase ................................................................................... 29 2.4.1 Molecular characteristics of lysyl hydroxylase ............................ 29 2.4.2 Reaction catalysed by lysyl hydroxylase...................................... 30
2.5 Lysyl hydroxylase isoforms .................................................................... 32 2.5.1 Genes of lysyl hydroxylase isoforms............................................ 32 2.5.2 Expression of lysyl hydroxylase isoforms.................................... 33 2.5.3 Molecular characteristics of lysyl hydroxylase isoforms ............. 35 2.5.4 Subcellular localization of lysyl hydroxylase isoforms................ 36 2.5.5 Diseases associated with lysyl hydroxylase isoforms .................. 37
2.6 Collagen glycosyltransferases................................................................. 40 2.6.1 Characteristics of collagen glycosyltransferases .......................... 40 2.6.2 Reactions of collagen glycosyltransferases .................................. 41
3 Aims of the present work 43 4 Materials and methods 45
4.1 Cell culture (I, II, III, IV) ........................................................................ 45 4.2 Expression of the lysyl hydroxylase isoforms as recombinant
proteins (I, II, III) .................................................................................... 45 4.3 Purification of His-tagged proteins (I, II)................................................ 46 4.4 Enzyme activity measurements (I, II, III, IV) ......................................... 46 4.5 Pepspots analysis (I)................................................................................ 47
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4.6 CD spectra of peptide substrates (I) ........................................................ 47 4.7 Biocomputing analysis (I) ....................................................................... 48 4.8 RNA isolation and Northern analysis (IV) .............................................. 48 4.9 Isolation of genomic DNA, cDNA synthesis, PCR amplification
and sequence analysis (IV)...................................................................... 49 4.10 Immunoblotting (II, III, IV) .................................................................... 49 4.11 Immunofluorescence staining (IV) and immuno-
electronmicroscopy (IV) ......................................................................... 50 5 Results 51
5.1 Substrate specificity of lysyl hydroxylase isoforms (I) ........................... 51 5.1.1 Amino acids present in the collagenous sequences of type
I-XIX collagens ............................................................................ 51 5.1.2 The collagenous peptides bound by lysyl hydroxylase
isoforms ........................................................................................ 51 5.1.3 Pepspots peptides as substrates of lysyl hydroxylase
isoforms ........................................................................................ 53 5.2 Characterization of the multifunctionality of human lysyl
hydroxylase 3 and C. elegans lysyl hydroxylase (II, III) ........................ 54 5.2.1 Galactosylhydroxylysyl glucosyltransferase activity of
human lysyl hydroxylase 3 and C. elegans lysyl hydroxylase (II, III) ...................................................................... 54
5.2.2 Amino acids important for the galactosylhydroxylysyl glucosyltransferase activity (II, III) .............................................. 55
5.3 Decreased galactosylhydroxylysyl glucosyltransferase activity in a family with epidermolysis bullosa simplex (IV) .................................. 56 5.3.1 GGT activity, LH3 protein level and analysis of LH3 gene
and cDNA in the patients (IV)...................................................... 56 5.3.2 Organization of the extracellular matrix and the
cytoskeleton in cells with decreased GGT/LH3 activity (IV) ............................................................................................... 57
6 Discussion 59 6.1 Frequency of the charged amino acids is high in the triplets
following the lysine residues................................................................... 59 6.2 Lysyl hydroxylase isoforms do not have absolute amino acid
sequence specificity for their substrates.................................................. 59 6.3 Lysyl hydroxylase 3 is a multifunctional enzyme ................................... 61
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6.4 The glycosyltransferase active site is separate from the lysyl hydroxylase active site ............................................................................ 63
6.5 Decreased GGT activity and LH3 protein level in a family with epidermolysis bullosa simplex ................................................................ 64
6.6 Decreased GGT/LH3 activity causes abnormalities in the organization of extracellular matrix and cytoskeleton ............................ 65
7 Conclusions 69 References 71 Original articles 91
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1 Introduction
Collagens are the most abundant proteins of the body. At present, the collagen superfamily consists of 28 different extracellular matrix proteins, which provide tensile strength and scaffolding for tissues and organs. The biosynthesis of collagens involves an unusually large number of co- and posttranslational modifications, including hydroxylation of lysine residues and glycosylation of certain hydroxylysine residues to galactosylhydroxylysine and glucosylgalactosylhydroxylysine. The hydroxylysines have an important role in the formation of intermolecular collagen crosslinks, which stabilize the collagen fibrils. The functions of hydroxylysine-linked carbohydrates are not fully understood.
Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in -X-Lys-Gly- sequences in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The substrate specificity of the LH isoforms is not fully known. No clear differences have been found in their tissue expression and they seem to lack collagen type specificity. In this study it was shown that the LH isoforms also do not have an absolute amino acid sequence specificity. However, the LH isoforms seem to have a preference for some sequences as is also seen in diseases linked to the LH isoforms and in LH knockout models.
Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities, and the amino acids critical for GGT activity were identified. The multifunctionality of LH3 encouraged us to reexamine a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier found to have decreased GGT activity. Our data indicated that LH3 protein levels and GGT activity were decreased in skin fibroblasts of the patients, and significantly reduced in the culture media of these cells. However, no mutations in LH3 cDNA were found, and the expression of LH3 was also normal. LH3 reduction in the EBS fibroblasts caused changes in the deposition and organization of extracellular matrix proteins, and similar changes were observed in mouse embryonic fibroblasts derived from heterozygous LH3 knockout crosses.
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2 Review of the literature
2.1 Extracellular matrix
The extracellular matrix (ECM) is a three dimensional network of heterogeneous macromolecules, which provides physical support to tissues and organs by occupying the space between cells. In addition, the ECM supports cell adhesion, communicates with cells via cellular receptors and serves as storage of growth factors. The ECM is present in all tissues, but in the connective tissue and basement membrane it is highly abundant (Aszodi et al. 2006, Aumailley & Gayraud 1998, Tanzer 2006).
The molecular composition of the ECM varies greatly according to the physical properties needed in various connective tissues and is highly dynamic and under constant remodeling. In principle, the matrix consists of collagens and elastin, the fibrous polymers, embedded within an amorphous mixture of proteoglycans and noncollagenous glycoproteins. The ECM proteins are large molecules with many domains, enabling these proteins to form oligomers and interact with other matrix proteins. Furthermore, the proteins directly interact with cellular receptors, which activate intracellular signal cascades leading to the expression of genes necessary for cell differentiation, proliferation and survival (Aszodi et al. 2006, Aumailley & Gayraud 1998, Tanzer 2006).
The basement membrane (BM) is a thin sheet of the specialized form of the extracellular matrix. It is present at the epithelial/mesenchymal interface of most tissues and surrounds muscles, peripheral nerves and fat cells. With few exceptions, the BM forms an impermeable barrier to cell migration. In addition, it influences other biological activities such as tissue development and repair. The BM consists mainly of laminins, type IV collagen, nidogen and perlecan, but the composition varies in different tissues and within the same tissue during development and repair (Aszodi et al. 2006, Erickson & Couchman 2000, Tanzer 2006, Yurchenco et al. 2004).
2.2 Collagens
Collagens are the most abundant proteins in mammals. They are a family of extracellular matrix proteins that provide tensile strength and scaffolding for tissues and organs. Furthermore, they are involved in cell adhesion, chemotaxis,
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migration and, together with cells, regulate tissue remodelling, differentiation, morphogenesis and wound healing. The collagen triple helix serves a ligand for cell receptors, proteases, other ECM proteins and glycosaminoglycans (GAGs) (Brodsky & Persikov 2005, Gelse et al. 2003, Kadler et al. 2007, Kielty & Grant 2002, Myllyharju & Kivirikko 2004).
All collagens consist of three polypeptides, called α chains, which are coiled into a unique rod-like triple helical structure, resistant to most proteases. The majority of collagens are homotrimers containing three identical α chains, but there are also heterotrimers consisting of two or even three different α chains. The amino acid sequences of collagens share two exceptional features: glycines as every third residue generating a repeating Gly-X-Y sequence and a high content of proline in the X position and hydroxyproline in the Y position in the sequence. In order to form a triple helix, each α chain adopts a polyproline II-like, left-handed helix stabilized by proline and hydroxyproline. After that, the three α chains are supercoiled around a central axis in a right-handed manner to form the triple helix, held together by interchain hydrogen bonds. Glycine, the smallest amino acid, in every third position is essential for the close packing of the three chains within the triple helix. At least 6-7 Gly-X-Y repeats are needed for triple helix formation. The residues in X and Y positions of the Gly-X-Y sequence are exposed on the surface of the triple helix and thereby have the potential to interact with other molecules. Therefore, the triple helix is not just a structural motif, but it also serves as a ligand binding site (Brodsky & Persikov 2005, Brodsky & Ramshaw 1997, Gelse et al. 2003, Kadler et al. 2007, Kielty & Grant 2002, Myllyharju & Kivirikko 2004)
2.2.1 Classification of collagen types
Currently, the vertebrate collagen superfamily consists of 28 different collagen types with 43 distinct α chains (Veit et al. 2006). Each member of the collagen family contains at least one triple-helical domain and non-triple-helical N- and C-terminal domains, which are often homologous to domains of other ECM proteins. The classification of collagens is partly contradictory, but in general, collagens are referred to as ECM proteins forming supramolecular aggregates. Based on the structure and supramolecular organization, collagens can be grouped into subfamilies (Gelse et al. 2003, Kadler et al. 2007, Kielty & Grant 2002, Myllyharju & Kivirikko 2004, Ricard-Blum & Ruggiero 2005).
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Fibrillar collagens are the principal source of tensile strength in animal tissues. This subfamily comprises type I, II, III, V and XI collagens. In addition, two novel collagens, type XXIV and XXVII, are referred to as fibrillar collagens. Type I collagen is the most abundant collagen and is present in most connective tissues. Type III collagen forms heterotypic fibrils with type I collagen in skin, tendon and vessels. Type II collagen is the predominant component of hyaline cartilage. Types V and XI are located centrally in type I/III and II collagen fibrils, respectively, and thought to regulate the diameter of the fibrils. Type XXIV collagen is expressed in tissues containing type I/V collagen fibrils and type XXVII in cartilage containing type II/XI fibrils, but the function of these novel collagens is yet to be determined (Gelse et al. 2003, Kielty & Grant 2002, Ricard-Blum & Ruggiero 2005).
Fibril-associated collagens with interrupted triple helices (FACIT) is the largest subfamily of the collagen superfamily including type IX, XII, XIV, XVI, XIX, XX, XXI and XXII collagens. The FACIT collagens are not capable of forming fibrils or other supramolecular structures. Instead, type IX collagen is associated with the surface of type II collagen fibrils and type XII and XIV collagen with type I collagen fibrils. These collagens also interact with other extracellular matrix proteins. The functions of type XVI, XIX, XX, XXI and XXII collagens are still poorly understood (Canty & Kadler 2005, Kadler et al. 2007, Kielty & Grant 2002, Ricard-Blum & Ruggiero 2005).
Type IV, VIII and X collagens form networks. Type IV collagen is only found in BM, where it is the most important component. Type VIII and X collagens form hexagonal networks and are the major components of Descemet’s membrane and hypertrophic cartilage, respectively (Gelse et al. 2003, Kadler et al. 2007, Kielty & Grant 2002).
Type VII collagen is one of the largest collagens in mammals. It is the major component of the anchoring fibrils underlying the BM of epithelia and so far the only representative of this subfamily. Type VI collagen forms microfibrillar networks in virtually all connective tissues (Gelse et al. 2003, Kadler et al. 2007, Kielty & Grant 2002). The type XXVIII collagen, a component of neural basement membranes, has a sequence similarity with type VI collagen and could be a member of the same subfamily (Veit et al. 2006).
Type XV and XVIII collagens contain multiple triple helix domains with interruptions and comprise one of the collagen subfamilies. These collagens are found in most basement membrane zones. Cleavage of NC1 domains of type XV and XVIII collagens releases endostatins, which modulate cell proliferation and
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migration, induce apoptosis, and inhibit angiogenesis and tumor growth (Kadler et al. 2007, Kielty & Grant 2002, Ricard-Blum & Ruggiero 2005).
Transmembrane collagens, type XIII, XVII, XXIII and XXV, are type II transmembrane proteins, mediating cell-matrix and cell-cell contacts. Their extracellular collagenous domain can be released by shedding, thereby functioning as a soluble molecule (Kadler et al. 2007, Kielty & Grant 2002, Ricard-Blum & Ruggiero 2005).
Type XXVI collagen, found in testis and ovary, is difficult to classify in any of the existing subfamilies, since it has more similarity with the Emu family of proteins, which contain a collagenous domain (Leimeister et al. 2002, Ricard-Blum & Ruggiero 2005).
2.2.2 Proteins with a collagenous domain
The collagen triple helix is also present in a growing set of other proteins. These proteins are not considered as collagens, since they do not have a structural role in the extracellular matrix. However, the collagenous sequence clearly contributes to the structure and function of these proteins (Brodsky & Persikov 2005, Brodsky & Shah 1995, Kielty & Grant 2002).
The C1q domain containing (C1qDC) proteins include molecules controlling inflammation, adaptive immunity and energy homeostasis. Genome analysis revealed that the human genome contains 31 C1qDC genes, and 23 of these also have a collagenous domain. The collagenous proteins encoded by these genes are: adiponectin, adiponectin like protein 1 and 2, the C1q subcomponent of complement activation, the C1qTNF proteins 1-3 and 5-8, EMILIN1 and 2, gliocolin1 and 2, otolin and CRF1 and 2. In addition, type VIII and X collagens are also members of this protein family (Tom Tang et al. 2005).
Collectins and ficolins are collagenous proteins recognizing oligosaccharide structures on the surface of microorganisms. Collectins identified from humans are the mannan-binding lectin, surfactant proteins A and D and collectins L1, P1 and K1. Furthermore, conglutinin, collectin-43 and collectin-46, detected only in bovine, are members of this protein family. The ficolin family consists of three members: H-ficolin, L-ficolin and M-ficolin (Keshi et al. 2006, Lu et al. 2002, van de Wetering et al. 2004).
The human collagenous transmembrane protein family comprises type XIII, XVII, XXIII and XXV collagens, the class A macrophage scavenger receptors, the MARCO receptor, ectodysplasin A and collomin. These type II membrane
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proteins are widely expressed and are involved in cell adhesion, epithelial-mesenchymal interactions in morphogenesis, neuromuscular signaling and host defense (Franzke et al. 2005, Snellman et al. 2007).
Besides these protein families, the asymmetric acetylcholinesterase has a collagenous tail, anchoring the enzyme in the basal lamina and localizing it into the neuromuscular junction (Deprez et al. 2000).
2.2.3 Collagen biosynthesis
The biosynthesis of collagens involves an unusually large number of co- and posttranslational modifications, many of which are unique to collagens and collagenous proteins. The biosynthesis of fibrillar collagens (types I, II, III, V and XI) has been the most thoroughly studied and can be regarded as a model, exemplifying the common features of the biosynthesis of collagens and collagenous proteins (Canty & Kadler 2005, Kielty & Grant 2002, Kivirikko 1995).
Formation of the triple helical molecule
Fibrillar collagens are synthesized as soluble precursors, procollagens, which have propeptide extensions at both the N- and C-termini. Translation of the mRNA begins on free ribosomes with the synthesis of an N-terminal signal peptide, which helps to translocate the procollagen chain into the lumen of the rough endoplasmic reticulum (ER). The proline and lysine residues of the newly synthesized polypeptide are hydroxylated by prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl hydroxylase, respectively. Some of the hydroxylysines are further glycosylated by hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase to give rise to glucosylgalactosylhydroxylysine residues. These enzymatic modifications occur cotranslationally within the cisternae of the ER and continue posttranslationally until triple helix formation. The 4-hydroxyproline content of collagens is quite constant, while proline 3-hydroxylation and lysine hydroxylation and glycosylation are dependent on the amounts of active enzymes and the time available before triple helix formation (Canty & Kadler 2005, Kielty & Grant 2002, Kivirikko & Myllylä 1979, Kivirikko et al. 1992, Kivirikko 1995).
Folding and disulfide bond formation within the C-propeptides determine the chain selections and ensure association of the monomeric procollagen chains.
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After this the triple helix folds in a zipper-like manner from the C- to the N-terminus. The procollagen folding is assisted by molecular chaperones, such as HSP47, BiP, GRP94 and protein disulfide isomerase. The correctly folded procollagen molecules are secreted through the Golgi complex to the extracellular space (Canty & Kadler 2005, Hulmes 2002, Kielty & Grant 2002, Lamandé & Bateman 1999)
Collagen fibril formation
In tissues, fibrillar collagens occur as 67-nm D-periodic fibrils, where adjacent molecules overlap each other by 234 amino acid residues. The removal of the N-propeptide and especially the C-propeptide by the ADAMTS family of proteases and procollagen C-proteinases, respectively, are the key steps in the formation of the typical collagen fibrils. There are data that indicate collagen fibrillogenesis starts already in cell surface structures, fibripositors, rather than solely in the extracellular space. Collagen molecules, without the propeptides, are able to self-assemble into fibrils. However, this is probably not sufficient for the formation of the tissue-specific suprafibrillar architectures. Most likely, interactions with fibronectin and specific integrins are also required in collagen fibrillogenesis (Canty & Kadler 2002, Canty & Kadler 2005, Hulmes 2002, Kielty & Grant 2002, Trackman 2005).
Collagen fibril growth is thought to occur by accretion and by lateral and end-to-end fusion. The telopeptide part of fibrillar collagens and the formation of heterotypic fibrils containing various collagen types, affect fibril growth. In addition, small leucine rich proteoglycans (SLRPs), located at the surface of collagen fibrils, control the fibril diameter. The FACIT collagens do not directly affect fibrillogenesis, but they appear to mediate interactions between collagen fibrils. The formation of intra- and inter-molecular crosslinks, initiated by lysyl oxidase, stabilizes collagen fibrils assembled into suprafibrillar architectures (Canty & Kadler 2002, Canty & Kadler 2005, Kielty & Grant 2002, Robins 1999).
2.3 Lysine modifications
Collagens and collagenous proteins contain hydroxylysine, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, which involves an unusual α1-2-O-glycosidic bond between glucose and galactose. The lysine modifications are found in the Y position of the repeating –X-Y-Gly- sequence,
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and thus are on the surface of the triple helix and available for interactions. Hydroxylysine is also found in the non-triple-helical, –X-Hyl-Ser- or –X-Hyl-Ala-, sequences in collagen telopeptides (Kivirikko & Myllylä 1979, Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992).
2.3.1 Lysine modifications in collagens
The hydroxylation and glycosylation of lysine residues are incomplete and different collagen types show a wide variation in the content of these modifications. The hydroxylysine content is reported only for type I-XI, XIII, XVII and XXV collagens and even less is known about the glycosylation content of these collagens (Table I). The lowest hydroxylysine content has been reported in type I and III collagens, in which 17-38% of all lysines are hydroxylated and only very few of them are further glycosylated. On the other hand, in type IV and VI collagens 70-89% of all lysines are hydroxylated and most of them are also glycosylated (Ayad et al. 1998, Kivirikko & Myllylä 1979, Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992).
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Table 1. Hydroxylation and glycosylation of lysine residues in various collagen α chains
Residues/1000 amino acids Collagen type α chain
Total Lys Hyl Glu-Gal-Hyl and
Gal-Hyl
References1
I α1 37 10 0,9
α2 32 12 1,8
II α1 38 18 9
III α1 35 6 0,9
IV α1 56 49 49 (Babel & Glanville
1984, Schwarz et al.
1986)
α2 44 39 39 (Babel & Glanville
1984, Schwarz et al.
1986)
V α1 55 35 30-34 (Mizuno et al. 2001)
α2 42 24 8-14,2 (Mizuno et al. 2001)
α3 58 43 n.r.
VI α1 59 48 48 (Chu et al. 1988)
α2 83 67 67 (Chu et al. 1988)
α3 69 48 48 (Chu et al. 1988)
VII α1 59 41 n.r.
VIII α1 and α2 45 22 n.r.
IX α1 53 33 n.r.
α2 58 46 n.r.
X α1 55 35 n.r.
XI α1 56 37 n.r.
α2 57 40 n.r.
α3 36 21 n.r.
XIII α1 40a 9,3a n.r. (Tu et al. 2002)
XVII α1 27a 11,7a 0,18a (Areida et al. 2001)
XXV α1 38a 17,3a n.r. (Söderberg et al.
2005) 1Modified from (Kivirikko & Myllylä 1979, Kivirikko et al. 1992) if no other references shown.
n.r.=not reported aresidues/molecule
There is also a marked variation in the extent of hydroxylation and glycosylation of lysines within the same collagen type from different tissues and even in the same tissue in different physiological and pathological states (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992, Uzawa et al. 2003). It seems that the
25
extent of hydroxylation and glycosylation of lysine residues is age-dependent, being highest in the embryonic state (Barnes et al. 1974, Kivirikko & Myllylä 1979, Ryhänen & Kivirikko 1974). A high degree of lysine hydroxylation has been seen in many diseases, for example in osteogenesis imperfecta (Brenner et al. 1989), where the glycosylation of hydroxylysines is also increased (Tenni et al. 1993), osteoporosis (Kowitz et al. 1997), osteoarthritis (Bank et al. 2002), chondrodysplasias (Murray et al. 1989) and osteosarcoma (Fernandes et al. 2007).
The hydroxylysine residues of collagens have two important functions: they are essential for the stability of the intra- and intermolecular collagen cross-links and their hydroxyl group serves as an attachment site for carbohydrate units (Kielty & Grant 2002, Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992). In addition, the positive charge of lysine or hydroxylysine residues is essential for the binding of SLRPs (Tenni et al. 2002, Viola et al. 2007) and in collagen fibril self-assembly (Tenni et al. 2006). However, it is not known, whether lysine hydroxylation modulates these interactions. Lysine hydroxylation and glycosylation also seem to reduce the efficiency of peptide bond hydrolysis by different peptidases (Molony et al. 1998, Wu et al. 1981).
Collagen cross-links
The covalent cross-links within and between collagen molecules provide the tensile strength and mechanical stability for the collagen fibrils. In general, fibrillar collagens (types I, II, III, V and XI) have four conserved cross-linking sites at equivalent positions in the molecule, one in both the N-telopeptide and C-telopeptide and two in the triple-helical domain at or close to residues 87 and 930 (Eyre & Wu 2005, Reiser et al. 1992, Wu & Eyre 1995). Lysine or hydroxylysine derived cross-links are also present in type IV and IX collagens (Eyre & Wu 2005, Vanacore et al. 2005).
The formation of cross-links starts with the oxidative deamination of a telopeptide lysine or hydroxylysine residue. This conversion of lysine and hydroxylysine to the respective aldehydes, allysine and hydroxyallysine, is catalyzed by lysyl oxidase. The formation of the divalent cross-links and their maturation with time to the tri- and tetravalent cross-links occur spontaneously. The cross-links can also be glycosylated, if the participating helical hydroxylysine is glycosylated. The formation of the collagen cross-links through allysine and hydroxyallysine pathways is illustrated in Figure 1 (Eyre & Wu 2005, Eyre et al.
26
1984, Kielty & Grant 2002, Knott & Bailey 1998, Robins & Brady 2002, Robins 1999, Yamauchi et al. 1987).
Fig. 1. Schematic representation of collagen cross-links. For references, see text. Abbreviations: telo telopeptide, ald aldehyde, helix helical residue.
The pathway of collagen cross-linking is more related to the type of tissue and its requirement for mechanical strength than to the type of collagen. The allysine pathway occurs primarily in adult skin, cornea and sclera, whereas the hydroxyallysine pathway predominates in bone, dentin, cartilage, ligament, tendon, embryonic skin and most internal connective tissues. Especially, the hydroxylation of telopeptide lysines affects the cross-linking pattern seen in different tissues. In skin the allysine pathway dominates, since telopeptide lysines are not hydroxylated. On the other hand, in cartilage telopeptide hydroxylation is almost complete and therefore only hydroxyallysine pathway derived pyridinolines are present. The hydroxyallysine pathway derived cross-links, pyrroles and pyridinolines, are found in bone, where telopeptides are partially hydroxylated. In addition, bone contains more lysylpyridinolines and immature divalent cross-links than the other internal connective tissues. These features are probably necessary for bone mineralization (Eyre & Wu 2005, Kielty & Grant 2002, Robins & Brady 2002, Robins 1999).
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Glycosylation of hydroxylysines
The functions of the glycosylated hydroxylysines in collagens are not fully understood. The glycosylation of certain helical hydroxylysines can direct the formation of histidinohydroxylysinonorleucine (HHL) cross-links in skin, whereas tendon lacks particular glycosylations and thereby also HHL cross-links (Eyre & Wu 2005). Foremost, it has been suggested that glycosylated hydroxylysines have a role in the organization of collagen fibrils. Studies with overmodified type I collagen (Torre-Blanco et al. 1992) and recombinant type II collagen (Notbohm et al. 1999) showed that highly hydroxylated and glycosylated collagen molecules form thinner fibrils in vitro. Similar small diameter fibrils were present in fibrotic skin, with increased hydroxylysine glycosylation (Brinckmann et al. 1999). However, contradictory results were obtained in osteopenic bone, where there was no correlation between glycosylation and fibril diameter (Bätge et al. 1997). Studies with type V collagen suggest that the bulky glycosylated hydroxylysines would decrease the electrostatic and hydrophobic interactions between collagen molecules and thereby decrease interfibrillar interactions (Mizuno et al. 2001). There is also increasing evidence that assembly and secretion of type IV and VI collagens are dependent on the glycosylated hydroxylysines (Langeveld et al. 1991, Sipilä et al. 2007).
The glycosylation of hydroxylysines might modulate the interaction of collagens with cells. Melanoma cell adhesion, spreading and signaling is promoted by the binding of the CD44 receptor in the chondroitin sulfate proteoglycan form, to the 1263-1277 region in the type IV collagen α1 chain. Interestingly, the presence of the galactosylhydroxylysine residue at the position 1265 dramatically decreased melanoma cell adhesion and spreading (Lauer-Fields et al. 2003). Various collagen types are ligands for discoidin domain receptor tyrosine kinases (DDRs). It has been shown that mouse tail collagen must be glycosylated in order to stimulate DDR2 (Vogel et al. 1997). The glycosylation of hydroxylysines seems also to have a role in arthritis. It has been shown that lysines at position 264 in type II collagen derived epitopes are completely glycosylated in normal cartilage, while arthritic cartilage contains both glycosylated and non-glycosylated epitopes (Dzhambazov et al. 2005). Probably the galactosylhydroxylysyl residue at position 264 is recognized by T cells in rheumatoid arthritis as was seen in mice with collagen induced arthritis (Corthay et al. 1998).
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2.3.2 Lysine modifications in collagenous proteins
Four lysine residues in the collagenous domain of adiponectin, an adipocyte-derived hormone, are known to be hydroxylated and glycosylated to glucosylgalactosylhydroxylysine (Wang et al. 2002). It has been suggested that the glycosylated hydroxylysines regulate the formation and secretion of the high molecular weight oligomeric complex of adiponectin and thereby contribute to the insulin sensitizing activity of adiponectin (Richards et al. 2006, Wang et al. 2006).
C1q, the key subcomponent of the classical pathway of complement activation, has been shown to contain 84 hydroxylysines (Shinkai & Yonemasu 1979), which corresponds to the complete hydroxylation of lysines in the collagenous domains of the hexameric molecule (Sellar et al. 1991). Furthermore, it was found that over 80% of the hydroxylysine residues are glycosylated, mostly to glucosylgalactosylhydroxylysine (Shinkai & Yonemasu 1979). The C1q collagenous domain binds many ligands, like the C1r proenzyme (Illy et al. 1993), C1q receptor (Malhotra et al. 1993), serum amyloid P (Ying et al. 1993) and C-reactive protein (Ying et al. 1993), leading to the activation of the classical complement pathway. The binding sites of these ligands may contain modified lysine residues, but the biological significance of the hydroxylysines and glycosylated hydroxylysines of the C1q molecule is not known.
In mannan-binding lectin (MBL) that recognizes sugar structures of microorganisms, all four lysines in the collagenous domain are hydroxylated and in most cases fully glycosylated (Jensen et al. 2007). At least two of these glucosylgalactosylhydroxylysine residues are crucial; their absence leads to reduced MBL secretion, formation of high oligomers and activation of the complement pathway, causing an increased susceptibility to infections (Heise et al. 2000). The C1q receptor-binding site of MBL also contains one hydroxylysine that is probably glycosylated (Arora et al. 2001, Malhotra et al. 1993).
Surfactant proteins A and D, the pattern-recognition molecules of pulmonary innate immunity, have been shown to contain hydroxylysine (Persson et al. 1989, Sano et al. 1987) and surfactant protein D also has glycosylated hydroxylysines (Crouch et al. 1994). The C1q receptor-binding site of surfactant protein A probably contains hydroxylysine residues (Arora et al. 2001, Malhotra et al. 1993).
The lysine residues in the collagenous domains of the bovine specific collectins, conglutinin and collectin-43, are fully hydroxylated and glycosylated
29
(Lee et al. 1991, Rothmann et al. 1997), but the functions of these residues are not known.
The collagenous tail of the asymmetric acetylcholinesterase has a high content of hydroxylysines (Rosenberry & Richardson 1977). The collagenous tail binds to heparin in the basal lamina of the neuromuscular junction. The heparin binding site in the collagenous domain involves hydroxylysine residues (Deprez et al. 2000), but their function is unknown.
Interestingly, hydroxylysine is also present in some non-collagenous proteins. The anglerfish somatostatin-28, the inhibitor of the wide range of endocrine secretions and a neurotransmitter and -modulator of the central nervous system, is present in two forms, one of them containing hydroxylysine in a –X-Hyl-Gly- sequence (Andrews et al. 1984, Spiess & Noe 1985). In addition, one surface-accessible hydroxylysine has been found in –X-Hyl-Gly- sequences of recombinant human tissue plasminogen activator, CD4 receptor and a related chimeric protein (rCD4-IgG) (Molony et al. 1995).
2.4 Lysyl hydroxylase
Lysyl hydroxylase (LH, EC 1.14.11.4) is the enzyme catalyzing the formation of hydroxylysine in collagens and collagenous proteins. It belongs to the group of 2-oxoglutarate dioxygenases, as do the two other enzymes of collagen biosynthesis: prolyl-4-hydroxylase and prolyl-3-hydroxylase (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992).
2.4.1 Molecular characteristics of lysyl hydroxylase
Lysyl hydroxylase was first purified as a homogenous protein from chick embryos (Turpeenniemi-Hujanen et al. 1980) and later from human placenta (Turpeenniemi-Hujanen et al. 1981). The active enzyme is a homodimer with a molecular weight of about 190,000, consisting of two monomers with a molecular weight of about 80,000-85,000 (Turpeenniemi-Hujanen et al. 1980, Turpeenniemi-Hujanen et al. 1981). The heterogeneity in the molecular weight of the monomers is due to differences in glycosylation (Myllylä et al. 1988).
Lysyl hydroxylase was first cloned from chick, where the polypeptide consists of 710 amino acids and a signal peptide of 20 amino acids. The polypeptide contains four potential attachment sites for asparagine-linked oligosaccharides. There is no significant homology between the primary
30
structures of lysyl hydroxylase and prolyl-4-hydroxylases, despite the marked similarities in the kinetic properties (Myllylä et al. 1991). Later, the human lysyl hydroxylase was shown to consist of 709 amino acids and a signal peptide of 18 amino acids. The overall identity between the human and chick lysyl hydroxylases is 76% at the amino acid level. The C-terminal region is especially conserved, with an identity of 94%, suggesting a functional significance of this region (Hautala et al. 1992a). Furthermore, the four potential attachment sites for asparagine-linked oligosaccharides are conserved (Hautala et al. 1992a) and some of them are essential for maximal enzyme activity (Myllylä et al. 1988, Pirskanen et al. 1996).
2.4.2 Reaction catalysed by lysyl hydroxylase
Lysyl hydroxylase, like the other 2-oxoglutarate dioxygenases, requires Fe2+, 2-oxoglutarate and O2 in its reaction. This enzyme family catalyzes the coupled reaction of hydroxylation of a C-H bond in a substrate and oxidative decarboxylation of 2-oxoglutarate, leading to the release of succinate and CO2. One oxygen atom is incorporated as a hydroxyl group to the product and the other into a carboxylate group of succinate. (Hausinger 2004, Kivirikko & Pihlajaniemi 1998).
The reaction mechanism of lysyl hydroxylase involves an ordered binding of Fe2+, 2-oxoglutarate, O2 and peptide substrate and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+ (Puistola et al. 1980a, Puistola et al. 1980b). The order of the release of the hydroxylated peptide and CO2 is uncertain and Fe2+ may not leave the enzyme after each catalytic cycle (Puistola et al. 1980b). In addition, ascorbate is a specific requirement for lysyl hydroxylase, even if it is not consumed stoichiometrically in the catalytic cycle (Puistola et al. 1980a).
Catalytic mechanism, co-substrates and inhibitors
All 2-oxoglutarate dioxygenases contain a His1-X-Asp/Glu-Xn-His2 –motif, which is the Fe2+ binding site of the enzyme (Hausinger 2004). In human lysyl hydroxylase, the Fe2+ binding site involves histidines 656 and 708 and aspartate 658 (Myllylä et al. 1992, Pirskanen et al. 1996). The 2-oxoglutarate binding site of the enzyme can be divided into two subsites. In the first site a positive side chain electrostatically interacts with the C5 carboxylate of 2-oxoglutarate, and in
31
the other site the C1 carboxylate and the C2 ketone of 2-oxoglutarate chelate Fe2+ (Hanauske-Abel & Günzler 1982, Hausinger 2004) The positive side chain stabilizing the 2-oxoglutarate binding in human lysyl hydroxylase is arginine 700 (Passoja et al. 1998a). The molecular oxygen in the reaction comes from the atmosphere and enzyme bound oxygen atoms are exchangeable with water (Kikuchi et al. 1983). It is proposed that Fe2+ and O2 react to form a superoxo radical anion, which can attack the 2-oxy group of 2-oxoglutarate. This leads to the formation of CO2 and a reactive oxy-ferryl intermediate, which can hydroxylate a lysine residue in the peptide substrate, bound in proximity to the Fe2+. The hydroxylation renews Fe2+ and concludes the catalytic cycle (Hausinger 2004, Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992).
Occasionally, lysyl hydroxylase can catalyze an uncoupled decarboxylation of 2-oxoglutarate, which does not include hydroxylation of a substrate. In this reaction, the reactive oxy-ferryl intermediate is probably converted to Fe3+ and O-, inactivating the enzyme. Ascorbate is needed to reactivate the enzyme by reducing the Fe3+. In the uncoupled reaction ascorbate is also consumed stoichiometrically (Myllylä et al. 1984). The binding site of the ascorbate might be partially identical to the 2-oxoglutarate binding site, consisting of two cis-positioned coordination sites of the enzyme bound iron (Majamaa et al. 1986).
Lysyl hydroxylase can be competitively inhibited with respect to the co-substrates by many compounds. Zn2+ is the most potent competitive inhibitor of lysyl hydroxylase with respect to Fe2+. Superoxide dismutase active copper chelates inhibit lysyl hydroxylase by dismutating the active oxygen at the catalytic site (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992). Furthermore, lysyl hydroxylase can be efficiently inhibited by pyridine-2,4,-dicarboxylate, which is a 2-oxoglutarate analogue (Majamaa et al. 1985). Many peptides, malathion and malaoxon also competitively inhibit lysyl hydroxylase with respect to the peptide substrate (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992, Samimi & Last 2001).
Peptide substrates
The amino acid sequence surrounding the lysine residue, peptide length and peptide conformation affect the interactions of the peptide substrate with lysyl hydroxylase. Lysyl hydroxylase can not use free lysine as a substrate; the substrate has to contain at least the –X-Lys-Gly- triplet (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1972, Kivirikko et al. 1992). In addition, lysyl hydroxylase
32
is able to hydroxylate lysines in the sequences –X-Lys-Ser-, –X-Lys-Ala- and –X-Lys-Thr-, which is in agreement with the presence of hydroxylysine in the –X-Hyl-Ser- and -X-Hyl-Ala- sequences in the non-helical telopeptides of some collagens (Ryhänen 1975).
The chain length of the peptide substrate seems to affect the Km-value of the reaction, whereas it has less influence on the reaction rate. The triple-helical conformation of the peptide substrate completely prevents lysine hydroxylation (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992). In addition, it has been suggested that the peptide substrate has to have a β- or γ-turn conformation in order to bind to the catalytic site of lysyl hydroxylase and a poly-proline II –type structure for effective interaction in the substrate binding site (Ananthanarayanan et al. 1992, Jiang & Ananthanarayanan 1991).
2.5 Lysyl hydroxylase isoforms
The existence of collagen type or tissue specific lysyl hydroxylase isoforms was debated for years. Data concerning the patients with Ehlers-Danlos syndrome type VI, a disease with deficiency of lysyl hydroxylase activity, initially suggested the presence of lysyl hydroxylase isoforms. The patients had a reduction in the hydroxylysine content in skin, but not in cartilage, and type I and III collagens were more affected than type II, IV and V collagens (Ihme et al. 1984, Pinnell et al. 1972, Risteli et al. 1980).
The presence of lysyl hydroxylase isoforms was demonstrated, when two novel human enzymes, lysyl hydroxylase 2 (LH2) (Valtavaara et al. 1997) and lysyl hydroxylase 3, (LH3) (Passoja et al. 1998b, Valtavaara et al. 1998) were cloned. The lysyl hydroxylase cloned earlier was designated LH1. Later the presence of LH isoforms has been shown in mouse (Ruotsalainen et al. 1999), rat (Armstrong & Last 1995, Mercer et al. 2003) and zebrafish (Schneider & Granato 2006, Schneider & Granato 2007). In addition, LH2 exists as two alternatively spliced forms, LH2a (short) and LH2b (long) (Valtavaara 1999, Yeowell & Walker 1999).
2.5.1 Genes of lysyl hydroxylase isoforms
Phylogenetic analyses of amino acid sequences suggest that lysyl hydroxylase isoforms have evolved as a result of two duplication events from an ancestral gene (Ruotsalainen et al. 1999). C. elegans has only one lysyl hydroxylase
33
enzyme (Norman & Moerman 2000) and it seems that LH3 is the oldest of the LH isoforms, since it shows a closer relation to the C. elegans LH than do the other isoforms. LH1 and LH2 are probably derived by more recent gene duplications from LH3 (Ruotsalainen et al. 1999).
The gene structure has been characterized for human LH1 (PLOD1), mouse LH2 (Plod2), human and mouse LH3 (PLOD3 and Plod3) and for all zebrafish LH isoforms. LH1 and LH3 genes contain 19 exons, while LH2 genes contain 20 exons, one being alternatively spliced (Heikkinen et al. 1994, Rautavuoma et al. 2000, Ruotsalainen et al. 2001, Schneider & Granato 2006, Schneider & Granato 2007). The size of the corresponding exons of the three LH isoforms is quite similar, but the first and last exons, coding for the 5’ and 3’ ends of the coding region and the untranslated regions of mRNA, show more variation in size. The intron size shows remarkable differences between isoforms, the LH3 gene containing shorter introns than the other isoforms, which is in agreement with the evolutionary age of LH isoforms (Ruotsalainen et al. 2001, Schneider & Granato 2007). However, the LH2 introns 13 and 13A, surrounding the alternatively spliced exon, contain short identical sequences in human and mouse, suggesting their role in the control of the alternative splicing (Ruotsalainen et al. 2001).
There seem to be no similarities in the nucleotide sequences of the putative promoters of the human and mouse LH genes, suggesting differences in the regulation of the gene expression. The putative promoters lack a typical TATAA box, revealing the housekeeping nature of these genes. The human LH1 and LH3 and mouse LH3 genes contain several repetitive Alu- and retroposon-like sequences, respectively, which are potential recombination sites in these genes (Heikkinen et al. 1994, Rautavuoma et al. 2000, Ruotsalainen et al. 2001)
The human LH genes have been mapped to different chromosomes: PLOD1 to chromosome 1p36-p36.3 (Hautala et al. 1992a), PLOD2 to 3q23-q24 (Szpirer et al. 1997) and PLOD3 to 7q33 (Valtavaara et al. 1999). Similarly, the mouse genes localize to chromosomes 4, 9, and 5 (Sipilä et al. 2000) and rat genes to chromosomes 5q36 (NCBI), 8q31 and 12q12 (Mercer et al. 2003), respectively.
2.5.2 Expression of lysyl hydroxylase isoforms
The human LH mRNAs are expressed in several adult tissues. LH1 is detected from heart, brain, placenta, lung, liver and skeletal muscle and the signal was in the same range in all these tissues (Heikkinen et al. 1994). LH2 is highly expressed is skeletal muscle, placenta, heart and pancreas (Valtavaara et al. 1997),
34
while LH3 is expressed in placenta, pancreas, heart and spinal cord (Passoja et al. 1998b, Valtavaara et al. 1998). The expression of the mouse LH isoforms shows more regulation than do the human LHs (Ruotsalainen et al. 1999), while the rat LH isoforms are more constitutively expressed than human LHs (Mercer et al. 2003). In human tissues, LH2b seems to be the major form of LH2 and the only transcript in skin, dura, aorta and lung. LH2a is present, together with LH2b, in kidney, liver, spleen and cartilage (Yeowell & Walker 1999). In mouse tissues LH2b is the main form in muscle, spleen, heart and lung, whereas LH2a is the only form in kidney and testis (Salo et al. 2006a).
All LH isoforms are widely expressed during mouse and zebrafish embryogenesis, but a variation in the levels of their transcripts suggests participation in specific developmental events (Salo et al. 2006a, Schneider & Granato 2006, Schneider & Granato 2007). The LH2 alternative splicing seems to be developmentally regulated in mouse embryos, since LH2a is predominant in early stages, whereas LH2b becomes dominant later in development (Salo et al. 2006a). However, this differs from fetal human skin, where LH2b is the only form expressed throughout development (Yeowell & Walker 1999).
These is also evidence that different cell types in some tissues are specialized to express different LH isoforms (Mercer et al. 2003, Salo et al. 2006a, Wang et al. 2000). In addition, the expression of LH1 and LH2 shows remarkable differences compared with LH3 in various cells lines. No correlation was found between the expression of the LH isoforms and the most abundant collagen types. However, the expression of LH1, LH2 and the α subunit of prolyl-4-hydroxylase, the marker of collagen synthesis, were correlated with each other, but not with the expression of LH3 (Wang et al. 2000).
Many factors have been shown to regulate LH gene expression. The antihypertensive drug minoxidil decreases gene expression of LH1 and to a much lesser extent also the levels of LH2 and LH3 in skin fibroblasts (Hautala et al. 1992b, Yeowell et al. 1992, Zuurmond et al. 2005). Hypoxia seems to increase the expression of LH1 and LH2 (Brinckmann et al. 2005, Hofbauer et al. 2003, Scheurer et al. 2004) and certain cytokines up-regulate LH2b mRNA levels (Brinckmann et al. 2005, van der Slot et al. 2005). Furthermore, the paired-bicoid homeodomain protein (PITX2) most likely induces the expression of human LH1 and mouse LH2 genes (Brinckmann et al. 2005, Hjalt et al. 2001). The alternative spicing of LH2 can be regulated by cell density and cycloheximide treatment in cell cultures (Walker et al. 2005).
35
2.5.3 Molecular characteristics of lysyl hydroxylase isoforms
The human LH1, LH2a and LH3 consist of 727, 738 and 738 amino acid residues, respectively, including a putative signal sequence (Hautala et al. 1992a, Passoja et al. 1998b, Valtavaara et al. 1997, Valtavaara et al. 1998). Furthermore, the alternatively spliced form of LH2, LH2b, contains 21 additional amino acids (Valtavaara 1999, Yeowell & Walker 1999). The length of the LH isoform polypeptides shows some variation in mouse, rat and zebrafish compared with human (Armstrong & Last 1995, Mercer et al. 2003, Ruotsalainen et al. 1999, Schneider & Granato 2006, Schneider & Granato 2007), but the length of the additional amino acid sequence of LH2b is conserved (Schneider & Granato 2007).
The similarity between the human LHs is 47% at the amino acid level and over 80% in the C-terminal end of the sequences (Valtavaara et al. 1998). The amino acid sequences of the LH isoforms are well conserved between different species, since the similarity of the mouse and rat isoforms to the corresponding human isoforms is over 90% (Armstrong & Last 1995, Mercer et al. 2003, Ruotsalainen et al. 1999). The additional amino acids of the mouse and rat LH2b are identical to human (Mercer et al. 2003, Valtavaara 1999). The zebrafish LH isoforms show less similarity to the human isoforms than those of the other species, the similarity being 65-70%. The additional amino acids of the zebrafish LH2b show 90% conservation with the human sequence (Schneider & Granato 2006, Schneider & Granato 2007).
The amino acid residues essential for the co-substrate binding of lysyl hydroxylase (Passoja et al. 1998a, Pirskanen et al. 1996) are conserved in the lysyl hydroxylase isoforms of all species characterized. In addition, all LH isoforms have nine conserved cysteine residues, probably important to enzyme activity (Yeowell et al. 2000), and several potential N-glycosylation sites, which are not conserved (Armstrong & Last 1995, Hautala et al. 1992a, Mercer et al. 2003, Passoja et al. 1998b, Ruotsalainen et al. 1999, Schneider & Granato 2006, Schneider & Granato 2007, Valtavaara et al. 1997, Valtavaara et al. 1998). The glycosylation of one asparagine in human LH1 is essential for the full catalytic activity of the enzyme (Pirskanen et al. 1996). Nevertheless, the asparagine residue is only conserved in human LH2, but not in LH3, suggesting differences in the glycosylation requirements of the lysyl hydroxylase isoforms (Passoja et al. 1998b, Valtavaara et al. 1998).
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The human recombinant LH isoforms have been shown to be homodimers. Limited proteolysis experiments suggest that the polypeptides of the human LH isoforms fold to form three structural domains, of which the two C-terminal ones contribute to lysyl hydroxylase activity (Rautavuoma et al. 2002). The Km values for Fe2+, 2-oxoglutarate, ascorbate and the peptide substrate have been determined with recombinant human LH isoforms (Passoja et al. 1998b, Valtavaara 1999). The values for LH1 and LH3 are essentially identical (Passoja et al. 1998b), but the values for LH2a and LH2b differ from these. Furthermore, the additional amino acids of LH2b seem to modify the active site, especially the binding site of ascorbate and the peptide substrate, since differences were seen in the Km-values of LH2a and LH2b (Valtavaara 1999).
2.5.4 Subcellular localization of lysyl hydroxylase isoforms
The hydroxylation of lysine residues occurs co- and posttranslationally within the cisternae of the ER until triple helix formation (Kielty & Grant 2002). Lysyl hydroxylase has been localized to the rough ER membranes (Harwood et al. 1974, Peterkofsky & Assad 1979) as a peripheral protein associated with an unidentified constituent of the membrane via electrostatic interactions (Kellokumpu et al. 1994). However, the cloning of chicken and human LH1 did not reveal neither the KDEL motif, the retention signal of soluble ER proteins, nor the double-lysine motif, the retention signal of ER transmembrane proteins (Hautala et al. 1992a, Myllylä et al. 1991).
LH1 seems to be permanently localized to the ER, and is not recycled between the ER and the Golgi (Suokas et al. 2003). The 40 amino acid long C-terminal peptide of LH1 contains the information needed for LH localization (Suokas et al. 2000). In particular, the retention motif of LH1 consists of 17 amino acids that are part of the iron binding domain of the enzyme. The most critical amino acids are exposed on the surface of the protein and thereby accessible for membrane binding (Suokas et al. 2003). Furthermore, LH2 and LH3 have been localized as membrane associated molecules in the ER (Salo et al. 2006a). The similarity of the 40 amino acid peptide in different LH isoforms is over 90% suggesting that all LH isoforms utilize this novel mechanism in their ER localization (Suokas et al. 2003).
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2.5.5 Diseases associated with lysyl hydroxylase isoforms
The deficiency of LH1 is associated with Ehlers-Danlos syndrome type VI and LH2 with Bruck syndrome. In addition, the overexpression of LH2b is linked to fibrotic conditions. Diseases associated with LH3 are not currently known. These disease models have increased our knowledge concerning the functions of the LH isoforms.
Ehlers-Danlos syndrome type VI
The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable disorders of connective tissue, affecting skin, ligaments, joints, blood vessels and internal organs. The EDS type VI or the kyphoscoliotic type of EDS is characterized by muscular hypotonia, joint laxity, progressive kyphoscoliosis, scleral fragility and rupture of the ocular globe and, in addition, by tissue fragility, easy bruising, arterial rupture, marfanoid habitus, microcornea and osteopenia (Steinmann et al. 2002). EDS VIA is caused by a deficiency of lysyl hydroxylase activity (Krane et al. 1972, Pinnell et al. 1972, Sussman et al. 1974), whereas the similar condition with normal LH activity is designated as EDS VIB (Judisch et al. 1976, Steinmann et al. 2002). Recently, lysyl hydroxylase 1 knockout mice were shown to exhibit the characteristics of EDS VIA (Takaluoma et al. 2007a).
EDS VIA is inherited as an autosomal recessive trait, due to homozygous or compound heterozygous mutations of the LH1 gene (Steinmann et al. 2002, Yeowell & Walker 2000). Over 20 different mutations have been identified in the LH1 gene, which cause the LH deficiency and the clinical characteristics of EDS VIA (Giunta et al. 2005, Walker et al. 2005, Yeowell & Walker 2000). The most common mutation, seen with 18% frequency, is a seven exon duplication caused by the Alu sequences in introns 9 and 16 in the LH1 gene (Hautala et al. 1993, Heikkinen et al. 1997, Pousi et al. 1994, Yeowell et al. 2000, Yeowell et al. 2005). A second common mutation, probably originating from a single ancestral gene, is a point mutation producing a premature termination codon in exon 14 (Pousi et al. 2000, Walker et al. 1999, Yeowell & Walker 1997, Yeowell et al. 2000). In addition, several other mutations of the LH1 gene have been characterized, including eleven other point mutations (Brinckmann et al. 1998, Giunta et al. 2005, Ha et al. 1994, Hyland et al. 1992, Walker et al. 2005, Yeowell et al. 2000, Yeowell et al. 2000), four splice site mutations (Heikkinen et al. 1999, Pajunen et al. 1998, Pousi et al. 1998, Yeowell & Walker 1997), four deletions (Giunta et al.
38
2005, Ha et al. 1994, Pousi et al. 1998, Yeowell et al. 2000, Yeowell et al. 2000), two insertions (Heikkinen et al. 1999, Yeowell et al. 2000) and an abnormal sequence of exon 17 (Heikkinen et al. 1997).
The lysyl hydroxylase activity measured from the cultured skin fibroblasts of EDS VIA patients ranges from 2% to 50% of normal (Krane et al. 1972, Steinmann et al. 2002), leading to a reduced content of hydroxylysine. The amount of hydroxylysine compared to normal varies in different tissues, e.g. 5% in skin, 20% in fascia and 50% in bone, while the amount of hydroxylysine is normal in cartilage (Pinnell et al. 1972). The reduced content of hydroxylysine alters the collagen cross-linking pattern in skin, bone and, interestingly, cartilage, these tissues containing less dehydrohydroxylysinonorleucine and hydroxylysylpyridinoline cross-links (Acil et al. 1995, Eyre & Glimcher 1972, Eyre et al. 2002, Pasquali et al. 1997).
The EDS VIA data suggest that lysyl hydroxylase 1 hydroxylates the helical lysines involved in cross-links. The helical cross-linking lysines are more markedly underhydroxylated than the other helical lysines in type I collagen of bone and in type II collagen of cartilage (Eyre et al. 2002). The type I collagen, produced by EDS VIA fibroblasts, contains much less hydroxylysine in the helical domain than is found in controls, whereas the hydroxylation level of telopeptides is normal (Uzawa et al. 2003). Furthermore, increased expression of LH1 is linked to a high hydroxylysylpyridinoline/lysylpyridinoline ratio, suggesting complete hydroxylation of the helical cross-linking lysines (Fernandes et al. 2007).
The biochemical and molecular basis of EDS VIB is currently unknown, but it seems to be heterogeneous. The lysyl hydroxylase activity is normal in these patients with clinical manifestations of EDS VI (Judisch et al. 1976, Walker et al. 2004). In some cases, the mRNA levels of LH2 or LH3 are decreased (Walker et al. 2004). In addition, there is contradictory data concerning the amount of hydroxylysine in the helical and telopeptide domains of type I collagen (Uzawa et al. 2003, Walker et al. 2004).
Bruck syndrome
Bruck syndrome (OMIM 259450) is an autosomal recessive disease resembling osteogenesis imperfecta. The disease is characterized by osteoporosis, joint contractures at birth, fragile bone and short stature. Analysis of the type I collagen in bone has revealed that patients have an underhydroxylation of the telopeptide
39
lysine residues, while hydroxylation of the helical cross-linking lysines is normal (Bank et al. 1999). Furthermore, the number of hydroxyallysine derived cross-links might be decreased (Bank et al. 1999, Ha-Vinh et al. 2004).
Three Bruck syndrome families have been shown to have mutations in exon 17 of the LH2 gene, affecting the amino acids highly conserved between different LH isoforms (Ha-Vinh et al. 2004, van der Slot et al. 2003). Recently, Bruck syndrome has also been linked to chromosome 17p12, suggesting a heterogeneous basis of the disease (Bank et al. 1999).
Bruck syndrome provides evidence that LH2 might be a telopeptide lysyl hydroxylase. Earlier in vitro studies have also suggested an association of LH2 expression with telopeptide lysine hydroxylation and with the collagen cross-linking pattern (Uzawa et al. 1999).
Fibrotic conditions
Fibrosis is a complex pathophysiological process characterized by the excessive deposition of collagen due to increased collagen synthesis and resistance to remodelling and degradation. Fibrotic changes can occur in all main tissues and organs, including skin, kidney, lung and liver (Trojanowska et al. 1998, Wynn 2004). The overhydroxylation of lysine residues and the increased formation of hydroxyallysine derived cross-links have been seen in many fibrotic conditions (Brinckmann et al. 1999, Brinckmann et al. 2001, Last et al. 1990, Ricard-Blum et al. 1992, Ricard-Blum et al. 1993). Moreover, alteration of the collagen cross-linking pattern may reduce the sensitivity of collagens to degradation (Bailey & Light 1985, Bailey et al. 1975, Last et al. 1990, Ricard-Blum et al. 1992, Ricard-Blum et al. 1993, van der Slot-Verhoeven et al. 2005).
The overexpression of LH2b has been shown to be a general fibrotic phenomenon (Brinckmann et al. 2005, Wu et al. 2006, van der Slot et al. 2003, van der Slot et al. 2004). These results confirm the function of LH2b as a telopeptide lysyl hydroxylase, since the increased expression of LH2b leads to the increased formation of the hydroxyallysine derived cross-links (Brinckmann et al. 2005, Mercer et al. 2003, Wu et al. 2006, van der Slot et al. 2003, van der Slot et al. 2004). In addition, LH2b seems to stimulate the gene expression and synthesis of collagens (Wu et al. 2006). The critical role of LH2b in the determination of the collagen cross-linking pattern was further seen in osteoblastic cells overexpressing LH2b. In these cells, matrix mineralization was significantly
40
delayed due to the increased amount of hydroxyallysine derived cross-links (Pornprasertsuk et al. 2004, Pornprasertsuk et al. 2005).
2.6 Collagen glycosyltransferases
Hydroxylysyl galactosyltransferase (GT, EC 2.4.1.50) catalyzes the attachment of galactose to the hydroxyl group of a hydroxylysyl residue via a β-glycosidic bond. Galactosylhydroxylysyl glucosyltransferase (GGT, EC 2.4.1.66) can further add glucose to the C-2 of galactose in galactosylhydroxylysine to give rise to the glucosylgalactosylhydroxylysine residue, involving an unusual α1-2-O-glycosidic bond between glucose and galactose (Kivirikko & Myllylä 1979, Spiro 1967, Spiro 1969).
2.6.1 Characteristics of collagen glycosyltransferases
GT has been purified from chick embryos at about 1000-fold purification (Risteli et al. 1976) and GGT as a homogenous protein (Myllylä et al. 1977), but genes for these enzymes have not been characterized. In gel filtration, the partially purified GT activity appears in three different peaks with molecular weights of 450,000, 200,000 and 50,000 (Risteli et al. 1976). The molecular weight of the chick GGT is about 72,000-78,000 by SDS-polyacrylamide gel electrophoresis, while a lower molecular weight was obtained in gel filtration (Myllylä et al. 1977). Both enzymes seem to be glycoproteins and require free sulfhydryl groups at their active site (Myllylä et al. 1976, Myllylä et al. 1977, Risteli et al. 1976, Risteli 1978).
The collagen glycosyltransferases have been localized to the membranes of the rough ER as is lysyl hydroxylase (Blumenkrantz et al. 1984). Furthermore, GGT has also been detected in the smooth ER, the Golgi apparatus, platelets, serum and at the cell surface (Anttinen 1977, Bauvois & Roth 1985, Bortolato et al. 1990, Bortolato et al. 1992, Menashi & Grant 1979).
The collagen glycosyltransferase activities vary in different cells and tissues and in different physiological and pathological states. Their distribution agrees with the degree of collagen glycosylation, the amount of collagens and collagen synthesis. Enzyme activities are higher in embryos and young animals than in adults (Kivirikko & Myllylä 1979). Increased enzyme activities are found after experimental hepatic injury (Risteli & Kivirikko 1974). In addition, GGT activity is elevated, for instance, in experimental diabetes and liver carcinoma, and in sera
41
of patients with pulmonary fibrosis and with various rheumatic diseases (Anttinen et al. 1985, Bolarin 1991, Kivirikko & Myllylä 1979, Myllylä et al. 1989)
2.6.2 Reactions of collagen glycosyltransferases
The collagen glycosyltransferases require a corresponding UDP-glycoside and Mn2+ in their reactions (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982). The reaction mechanism of the chick GGT involves an ordered binding of Mn2+, UDP-glucose and collagen substrate. The binding site of UDP-glucose is probably separate from the binding sites of Mn2+ and the substrate. The products are released by the enzyme in the order of glycosylated collagen, UDP and Mn2+, or Mn2+ may not leave the enzyme after each catalytic cycle (Myllylä 1976). Reaction mechanism studies have not been carried out with GT.
GT can only galactosylate hydroxylysine residues in a peptide with a minimum molecular weight of 500-600 and longer peptides are better substrates (Risteli et al. 1976). GGT can glucosylate free galactosylhydroxylysine, but the enzyme has a higher affinity for longer substrates (Kivirikko & Myllylä 1979). The amino acid sequence around the hydroxylysine residue may also affect the interaction of the substrate with both enzymes. Furthermore, the substrates of both enzymes must contain a free ε-amino group of the hydroxylysine residue and molecules with a triple-helical conformation cannot serve as substrates (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982). The preferential sugar donor for both enzymes is the corresponding UDP-glycoside, and UDP and several other nucleotides act as inhibitors. Mn2+ in both reactions can be replaced to some extent with other bivalent cations (Kivirikko & Myllylä 1979).
42
43
3 Aims of the present work
Lysyl hydroxylase is an enzyme catalyzing the formation of hydroxylysine residues in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from human, mouse, rat and zebrafish. In addition, LH2 has two alternatively spliced forms, LH2a (short) and LH2b (long). Earlier studies have revealed that LH isoforms do not have collagen type specificity or clear differences in their tissue expression. The aim of the present work was to:
1. Study the substrate requirements of the human LH isoforms, and specifically to determine whether the amino acids in close proximity to the X-Lys-Gly sequences direct the substrate binding and lysine hydroxylation by the LH isoforms.
The hydroxylysines can be further glycosylated to galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Collagen galactosylhydroxylysyl glucosyltrans-ferase activity was purified from chick embryos in the 1970’s, but gene coding for this activity was unknown. The other aims of the work were to:
2. Measure the GGT activity of the LH isoforms. 3. Identify the amino acids important for the GGT activity.
The multifunctionality of LH3 encouraged us to reexamine a Finnish epidermolysis bullosa simplex family, which was earlier found to have decreased GGT activity, accompanied by a markedly decreased urinary excretion of glucosylgalactosylhydroxylysine. Therefore, the additional aims of the work were to:
4. Characterize the LH3 cDNA and LH3 protein in this family. 5. Study the effects of the decreased GGT/LH3 activity on the extracellular
matrix proteins.
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45
4 Materials and methods
The materials and methods are described in more detail in the original articles (I–IV).
4.1 Cell culture (I, II, III, IV)
Sf9 (Spodoptera frugiperda) insect cells (I, II, III) for the production of the lysyl hydroxylase isoforms were grown in Sf-900 II serum-free medium (Gibco).
COS-7 cells (II), human skin fibroblasts and mouse embryonic fibroblasts (Sipilä et al. 2007) (IV) were grown at 37°C, 5% CO2 in DMEM with Glutamax
(Gibco) supplemented with 10% fetal calf serum (Promocell), penicillin-streptomycin and 50 µg/ml ascorbic acid.
4.2 Expression of the lysyl hydroxylase isoforms as recombinant proteins (I, II, III)
The expression of the human LH isoforms in insect cells (I, II, III) was carried out by a baculovirus transfer vector pFastBacI in the BAC-TO-BACTM Expression system (Invitrogen). The vector was modified to contain the human LH1 signal peptide, a His6-tag, and a BamHI site for insertion of the desired cDNA (Valtavaara et al. 1997, Valtavaara et al. 1998). The construct of human LH1 covered the nucleotides from 255 to 2405 (Hautala et al. 1992a). The construct of human LH2a and LH2b covered the nucleotides from 76 to 2267 (Valtavaara et al. 1997), the LH2b containing the alternatively spliced exon sequence between nucleotides 1500 and 1501 (Valtavaara 1999, Yeowell & Walker 1999). The construct of human LH3 covered the nucleotides from 289 to 2455 (Valtavaara et al. 1998). The cells were harvested 48 h after infection and homogenized as described earlier (Valtavaara et al. 1998).
Expression of the human LH3 cDNA was also carried out in a mammalian pCDNA3 expression vector (Invitrogen) and, as a GFP fusion protein, in a pEGFP-N1 vector (CLONTECH) (II). The constructs were transfected into COS-7 cells using FUGENE6 (Roche). The transfected COS-7 cells were sonicated in a buffer containing 0.1% Triton X-100, 0.2 M NaCl, and 20 mM Tris-HCl, pH 7.5, for 10 s in ice, and centrifuged.
The human LH3 and C. elegans LH cDNAs were ligated into a pQE30 vector (Qiagen) (III). The recombinant proteins contained the whole coding sequence
46
without the signal sequence, and a His6-tag at the amino terminus. The constructs
were transformed into the E. coli XL1-Blue strain. The cells were lysed in a buffer containing 0.4 M NaCl, 0.5% Nonidet P-40, and 20 mM Tris-HCl, pH 7.8, and incubated in the presence of lysozyme (50 µg/ml) and RNase A (10 µg/ml) at room temperature for 30 min. Lysis was completed by sonication. The cell debris was removed by centrifugation and the supernatant was used in the measurements.
For in vitro translation (II, III), the coding sequence of LH3 cDNA was cloned into the pCITE 4a vector (Novagen) under the T7 promoter. In vitro translation was performed with the single tube protein system 3 kit (Novagen) according to the manufacturer's protocol.
The QuickChange site-directed mutagenesis kit (Stratagene) (II, III) was used to make point mutations or deletions to the LH3 cDNA sequence.
4.3 Purification of His-tagged proteins (I, II)
His-tagged recombinant proteins were purified by Ni-NTA-agarose (Qiagen) (I, II) using the batch purification protocol as described by the manufacturer. The agarose was equilibrated with 20 mM Tris-HCl, pH 7.8, 0.3 M NaCl, 5% glycerol, and 10 mM imidazole. Ni-NTA-agarose was mixed into the cell homogenate with additions of 5% glycerol, 0.3 M NaCl, and 10 mM imidazole, and the slurry was incubated for 45 min at 4°C on a rocking platform. The matrix was then washed in 20 mM Tris-HCl, pH 7.8, 0.3 M NaCl, 5% glycerol, and 20 mM imidazole. Elution was carried out stepwise, with elution buffers that contained 100, 200 and 300 mM imidazole.
4.4 Enzyme activity measurements (I, II, III, IV)
LH activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-14C]glutarate (Kivirikko & Myllylä 1982) with the synthetic peptide IKGIKGIKG used as a substrate (II, III). In the measurements of Km- and Vmax-values (I), the specific activity of 2-oxoglutarate was 74 x 105 dpm/μmol. The peptide substrates were synthesized at the Department of Biochemistry, University of Oulu, using an ABI 433A Synthesizer and an Fmoc deprotection strategy.
The GGT activity (II, III, IV) was assayed by the method based on the transfer of a tritium-labeled sugar from UDP-glucose (139 Ci/mol) to galactosyl
47
hydroxylysyl residues in a calfskin gelatin substrate (Kivirikko & Myllylä 1982, Myllylä et al. 1975).
4.5 Pepspots analysis (I)
The amino acid sequences chosen for the Pepspots analysis were obtained from the α chains of type I-XIX collagens (Ayad et al. 1998). The nine amino acid long peptides, corresponding to the XKG sequences and the triplets on both sides of the XKG sequence, were synthesized as spots (Frank 1992) onto polyethylene glycol-derivatized cellulose membranes (AIMS Scientific Products) using the peptide scanning instrument AutoSpot Robot ASP222 (Abimed Analysen-Technik).
For peptide interaction studies, the membrane was blocked with 5% non-fat milk powder in TBS-T and subsequently incubated with the baculovirus-expressed and Nickel column purified LH isoform at a concentration of 5 μg/ml in the presence of 50 mM Tris-HCl-buffer, pH 7.8, 50 mM Fe2SO4 and 1 mM ascorbate. The bound LH isoform was electroblotted onto a PVDF membrane (Millipore) using a Semiphor transfer unit (Hoefer) as recommended by Jerini bio tools GMBH, and visualized on an x-ray film (Eastman Kodak Co) according to the protocol for ECLTM Western Blotting (Amersham biosciences) using the monoclonal anti-polyHistidine clone His-1 (Sigma) and horseradish peroxidase -conjugated Anti-Mouse IgG (Zymed) antibodies.
4.6 CD spectra of peptide substrates (I)
The secondary structures of the collagenous peptides used in the activity measurements were studied with circular dichroism (CD) spectroscopy. The CD spectra were recorded with a Jasco J-715 spectropolarimeter equipped with a microprocessor for spectral accumulation and data manipulation. All measurements were made in 1 mm path length quartz cells. The peptide concentration was 0.5 mg/ml and the measurements were performed at room temperature. The ellipticity was converted to the mean residue molar ellipticity. Secondary structure percentages of peptides were calculated from the CD spectra using the CONTIN/LL method (Provencher & Glockner 1981) with the SP37A reference set of 37 soluble proteins (Sreerama et al. 2000), as provided by the CDPro software package (Sreerama & Woody 2000).
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4.7 Biocomputing analysis (I)
A Bayesian approach (Berry 1992) was chosen to analyze the Pepspots data in order to determine the qualities of the peptides promoting or inhibiting binding of LH isoforms to the Pepspots peptides. The binding probability of each set of peptides carrying a certain quality was compared against the binding probability of peptides that were not in the set. The binding probability of a set of peptides was defined as the probability that a Pepspots binding assay gives a positive result for a peptide sampled randomly from the set.
A visualization tool called Sammon mapping (Agrafiotis 1997, Sammon 1969) was used to find more qualities related to the binding of the peptides. The Pepspots peptides were mapped onto a 2-dimensional map, where similar peptides were positioned close to each other and dissimilar peptides far apart. The sequence dissimilarity (WAC dissimilarity) was calculated using the WAC similarity matrix (Wei et al. 1997), based on physicochemical properties of the microenvironments created by amino acids. The G3PCX (Deb et al. 2002) evolutionary algorithm was used to optimize the Sammon map.
The theoretical structures of the Pepspots peptides in water were solved using the biased probability Monte Carlo global optimization procedure with distance dependent electrostatics, as provided by the ICM software package (Abagyan & Totrov 1997). Various approaches were used to find correlations between the peptide structures and the Pepspots binding data. As a structural dissimilarity measure, the root mean square deviation (RMSD) of the coordinates of the corresponding backbone and Lys5 heavy atoms between all Pepspots peptide pairs were calculated and a Sammon map was created from the RMSD data. The dependency of binding probability on peptide solvation free energy was analyzed statistically. The solvation free energy was calculated using ICM software. The secondary structure analysis (Kabsch & Sander 1983) of the Pepspots peptides was carried out on the 3-D structures, in order to find a bent structure with lysine.
4.8 RNA isolation and Northern analysis (IV)
Total RNA was isolated from skin fibroblasts with TRIzol Reagent (Gibco). For Northern analysis, the RNA was fractionated in a 0.8% agarose gel containing 0.22 M formaldehyde and transferred to a nylon membrane (Sambrook & Russel 2001). The filter was hybridized with [32P]dCTP-labeled cDNA fragments of human LH3 and LH1 (Valtavaara et al. 1997, Valtavaara et al. 1998) in
49
ExpressHyb (BD Biosciences). β-Actin cDNA was used as a control probe to normalize the quantities of mRNA. Quantification of mRNA levels was performed with ImageQuant 5.2 software (Molecular Dynamics).
4.9 Isolation of genomic DNA, cDNA synthesis, PCR amplification and sequence analysis (IV)
Genomic DNA was isolated from cultured skin fibroblasts using proteinase K and phenol extraction (Sambrook & Russel 2001). mRNA was isolated from the cells with a Oligotex Direct mRNA mini kit (Qiagen) followed by first strand cDNA synthesis using AMV reverse transcriptase (Invitrogen) with an oligo (dT)15 primer.
PCR amplification of the LH3 promoter sequence was performed with promoter and exon 1 specific oligonucleotides (Rautavuoma et al. 2000) and cDNA was amplified with LH3 specific oligonucleotides (Passoja et al. 1998b, Valtavaara et al. 1998). The PCR fragments were directly sequenced using the DYEnamic ET terminator cycle sequencing premix kit (GE Healthcare) and a ABI Prism 377 sequencer (Perkin Elmer).
4.10 Immunoblotting (II, III, IV)
The recombinant LH isoforms were detected using the monoclonal anti-polyHistidine antibody (Sigma) (II, III). The endogenous LH3 was partially purified with UDP-hexanolamine agarose (Sigma) and detected using anti-mouse LH3 antibody (Salo et al. 2006b) or PLOD3 antibody (ProteinTech, Inc). For collagen analysis, cells were homogenized into buffer containing 0.2 M NaCl, 0.1 M glycine, 0.1% Triton X-100, 50 µM DTT, 10 mM EDTA, 20 mM Tris, pH7.5 and proteins in serum-free culture medium were TCA precipitated. Collagens were detected with polyclonal rabbit anti-collagen type I (Rockland) and polyclonal rabbit anti-collagen type VI (Rockland) (IV).
The proteins were separated on 7.5% or 10% SDS-PAGE and transferred to Immobilon-P PVDF-membranes (Millipore). The membranes were blocked with 5% fat-free milk powder in TBS-T. The membranes were incubated with proprietary primary antibody and further with horseradish peroxidase -conjugated anti-rabbit IgG (P.A.R.I.S) or anti-mouse IgG (Zymed) secondary antibodies. Immunocomplexes were visualized by using ECL or ECL+ reagents (GE Healthcare) and x-ray film (Eastman Kodak Co).
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4.11 Immunofluorescence staining (IV) and immuno-electronmicroscopy (IV)
For immunofluorescence staining, cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS or with 95% ethanol/5% acetic acid. After blocking with 1% BSA, 0.1% saponin in PBS, the cells were incubated with monoclonal anti-vimentin (ABR), anti-vimentin (Sigma) or monoclonal anti-α-tubulin (Sigma) primary antibodies. In collagen and fibronectin experiments, cells were incubated with polyclonal rabbit anti-collagen type I (Rockland), polyclonal rabbit anti-collagen type VI (Rockland) and anti-human fibronectin (Sigma) in the absence of saponin, and nuclei were stained with Hoechst 33258. AlexaFluor 488 conjugated anti-rabbit IgG, anti-mouse IgG or anti-goat IgG (Molecular Probes) antibodies were used for secondary detection. For actin microfilament staining, the cells were permeabilized by 1% triton X-100 and incubated with AlexaFluor 568 phalloidin (Invitrogen). The cells were then embedded in Immu-Mount (Thermo Shandon) and examined using an Olympus epifluorescence microscope or an Olympus Fluoview 1000 confocal microscope.
For immuno-electronmicroscopy, the cells were fixed in 4% paraformaldehyde in a 0.1 M phosphate buffer pH 7.3, embedded with gelatin and immersed in 2.3 M sucrose. Thin cryo sections were incubated with PLOD3 (ProteinTech, Inc) primary antibody and then with a protein A-gold complex. Sections were examined in a Philips CM100 transmission electron microscope.
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5 Results
5.1 Substrate specificity of lysyl hydroxylase isoforms (I)
5.1.1 Amino acids present in the collagenous sequences of type I-XIX collagens
The α chains of type I-XIX collagens (Ayad et al. 1998) were analyzed for the occurrence of short sequence motifs XYG, XRG, and XKG (Fig. 3a1-3, I) as nine amino acid sequences, where G in the motif was constrained in position 6 to the maximal score. The relative frequencies of the amino acids in each position were calculated and the size of the amino acid symbols in Figure 3 (I) represents their abundance in a certain position. Our analysis revealed that proline is the most abundant amino acid in the X and Y position of the type I-XIX collagens α chains. In the triplet following the XKG motif, glutamate, aspartate, alanine, serine, and proline were the most abundant amino acids in the X position, while in case of the XRG motif, proline was again the most abundant amino acid in the X position. Furthermore, our hand-picked Pepspots peptide sequences (Fig. 3a4, I) corresponded well to the set of XKG sequences found by computer analysis.
5.1.2 The collagenous peptides bound by lysyl hydroxylase isoforms
The Pepspots membranes, containing 727 peptides representing the XYGXKGXYG sequences of collagen types I-XIX (Ayad et al. 1998), were used to study the sequence specificity of the LH isoforms. Each LH isoform was bound to approximately 200 Pepspots peptides. There was some overlap in the binding of peptides, and 46 of the peptides were bound by all isoforms. However, there were some peptides bound only by one LH isoform. The bound peptides were derived from various collagen types and there were some differences in the collagen types bound by each LH isoform. The net charge of the peptide strongly influenced the binding; a net charge of 2 or greater was usually associated with a high binding probability. Our data also indicated that some amino acids in certain positions in the XYGXKGXYG sequence affected binding of LH isoforms, for example Arg or Lys in position 8 of the peptide promoted the binding of all LH isoforms. Peptide characteristics affecting the binding probability of each LH isoform are summarized in Table 2.
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Table 2. Peptide characteristics affecting the binding probability of each LH isoform
Isoform Number of
peptides
bound
Major collagen
types of the
peptides
Peptide
net
charge
Amino acids promoting
binding (position in the
sequence)
Amino acids inhibiting
binding (position in the
sequence)
LH1 196 XI, XII, XV, XVII ≥ 2 Arg, Lys (2), Glu (4), Asp,
His (7), Arg, Lys (8)
Gly (1), Ala, Asp, Pro (2),
Gln, Ile (4), Glu (7), Ala,
Glu, Pro (8)
LH2a 195 VI, VII, XVII ≥ 3 Val (1), Arg, Lys (2), Leu,
Lys (4), Asp (7), Arg, Lys
(8)
Ala (2), Ala, Glu (4), Glu
(7), Ala, Met, Pro (8)
LH2b 226 XI, XII ≥ 2 Arg, Lys (1), Arg, Lys (2),
Ala (4), Arg, His, Ile, Lys,
Ser (7), Arg, Lys (8)
Glu (1), Asp (2), Gln, Ile
(4), Asp, Glu (7), Asp, Glu,
Gln, Pro, Val (8)
LH3 242 VI,XI, XII ≥ 2 Arg, Lys (1), Ala, Thr (4),
His, Lys, Ser, Tyr (7), Arg,
Lys (8)
Glu (1), Asp (2), Asp, Gln
(4), Asp, Glu (7), Asp, Glu,
Gln, Pro (8)
Sammon maps (Fig. 2, I), based on the WAC dissimilarities of the Pepspots peptide sequences, were used to visualize peptide binding. The Sammon maps showed no absolute sequence specificity for the LH isoforms. However, a detailed analysis of the Sammon maps (Fig. 2, areas marked by 1–5, I) revealed some differences in the binding probability of LH isoforms in certain areas (Fig. 4, I). The analysis of the peptide sequences in those areas (Fig. 3b, 1–5, I) showed differences in amino acid frequencies compared with the general sequence.
Furthermore, it was found that a low solvation free energy of the peptide, favorable for an aqueous environment, was associated with a high binding probability by all LH isoforms and a high solvation free energy was associated with a low binding probability. However, the solvation free energy was found to be strongly dependent on peptide net charge (Fig. 6, I), with both positively and negatively charged peptides preferring an aqueous environment. No increase in binding was found for peptides with a hydrogen bonded turn over Lys-5, as identified in the secondary structure analysis. The Sammon map based on structural root mean square deviation dissimilarities showed no significant aggregation of binding or non-binding peptides.
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5.1.3 Pepspots peptides as substrates of lysyl hydroxylase isoforms
Twenty-eight Pepspots peptides, representing the characteristics affecting the binding probability in Pepspots analysis, were selected for in vitro activity measurements in order to test whether they could serve as substrates of the LH isoforms. The Km and Vmax values of these peptides for the LH isoforms were measured, but no correlation was found between the Km values and binding of the peptide in the Pepspots. This could be due to the different reaction conditions in the activity assay and Pepspot analysis. 2-oxoglutarate was not present in the Pepspot analysis in order to prevent hydroxylation of peptide and subsequent release of the enzyme. Furthermore, peptides on the Pepspots were immobilized, while they were in solution in the activity assay. In addition, none of the secondary structures of the peptides, analyzed by CD spectroscopy, were found to correlate with the Km or Vmax values. However, our activity assays indicated that Pepspots peptides were bound to the active site of the LH isoforms. More peptides served as substrates of LH isoforms in the activity assay than were bound by LH isoforms in the Pepspots analysis suggesting that the activity assay was more sensitive than the Pepspots binding analysis.
The Km and Vmax values of the different peptides for the LH isoforms are listed in Table III (I). The data indicated differences in the capacity of the various isoforms to hydroxylate peptides. Interestingly, none of the negatively charged peptides were hydroxylated by LH1, whereas most of them served as substrates for the other isoforms. All peptides with a positive charge were hydroxylated by the LH isoforms and increasing the amount of NaCl in the assay markedly reduced the activity, in agreement with the Pepspots analysis, suggesting that the net charge of the peptide is a key determinant for binding. Furthermore, the alternatively spliced forms of LH2, LH2a and LH2b, varied in their ability to hydroxylate collagenous peptides.
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5.2 Characterization of the multifunctionality of human lysyl hydroxylase 3 and C. elegans lysyl hydroxylase (II, III)
5.2.1 Galactosylhydroxylysyl glucosyltransferase activity of human lysyl hydroxylase 3 and C. elegans lysyl hydroxylase (II, III)
When human LH3 cDNA constructs were expressed in Sf9 insect cells and in COS-7 mammalian cells, the GGT activity in the cells started to increase about 30 h after transfection. At the same time, GGT activity was also secreted into the cell culture medium. In Sf9 cells, about 25 to 55% of the activity was secreted, whereas in COS-7 cells the corresponding value was about 80%. Antibodies
against a synthetic peptide corresponding to the amino acids 283 to 297 of the human LH3 (Wang et al. 2000) and against the chicken glucosyltransferase (Myllylä 1981) partially inhibited the GGT activity of recombinant LH3 produced in insect cells and the GGT activity of skin fibroblasts. In addition, when the cDNA of C. elegans LH was expressed in E. coli, GGT activity was present in the soluble protein fraction. The human LH3 was also expressed as a GFP fusion protein in COS-7 cells. The fusion protein was located mainly in the endoplasmic reticulum, but in some cells, the protein also entered the Golgi complex, in agreement with the secretion of GGT. Furthermore, the other lysyl hydroxylase isoforms, LH1, LH2a and LH2b, were expressed in Sf9 cells under conditions identical to LH3. Although LH activity was present in the cells, none of these recombinant proteins had detectable GGT activity.
The His-tagged LH3, expressed in insect cells, was purified with a nickel column and one major band was present on an immunoblot with a molecular weight corresponding to LH3. The purified protein had GGT activity, but no LH activity. It was shown that imidazole, a histidine analogue used in the elution of the nickel column, was a potent inhibitor of lysyl hydroxylase. The LH3 cDNA was also translated in a cell free system and the protein corresponding to LH3 was shown to possess GGT activity.
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5.2.2 Amino acids important for the galactosylhydroxylysyl glucosyltransferase activity (II, III)
The amino acid sequence alignment of the bovine, rat, chicken, mouse and human LH1, the mouse and human LH2, the mouse and human LH3 and C. elegans and Drosophila LH revealed 29 amino acids, which were conserved between the LH3 sequences and the C. elegans sequence. The amino acids were scattered evenly throughout the molecule. In addition, 11 of these 29 amino acids were conserved in the Drosophila LH. In vitro mutagenesis combined with an in vitro translation system was used to determine which of the conserved amino acids were important for GGT activity in C. elegans LH. The conserved amino acids were mutated to amino acids present in LH1/LH2. The most inhibitory changes of C. elegans and one C-terminal change as a control were further tested with human LH3 in E. coli. Two mutations, C132I and L196I in the C. elegans and the corresponding amino acids C144I and L208I in human, inhibited GGT activity markedly in both species suggesting that these amino acids are important for the GGT catalytic activity. The alkylation study revealed that Cys144 does not form a disulfide bond and probably does not participate in the enzyme catalysis directly.
The baculovirus expression system was used to determine whether mutations inhibiting the GGT activity of LH3 have an effect on the LH activity. Mutations C144I and L208I did not have any effect on the LH activity of LH3. On the other hand, a D669A mutation decreased LH activity dramatically, whereas it had no effect on the GGT activity. To determine whether the C-terminal portion of the molecule is required for the GGT activity, the translational stop codon was generated in different parts of the LH3 cDNA, and the GGT activities of the truncated LH3 molecules were assayed. None of the truncated molecules were able to hydroxylate lysine residues, whereas a 355 amino acid long N-terminal moiety still possessed low GGT activity. The amino acids of the truncated LH3 molecules and their GGT activities are summarized in Table 3.
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Table 3. GGT activity of the truncated LH3 molecules
Amino acids of truncated LH3 molecules GGT activity % of normal
Normal LH31: 33-738 100
Mutation 1: 33-231 a frame shift2 and translation stop at 356 0,1
Mutation 5: 33-389 10
Mutation 7: 33-402 22
Mutation 2: 33-521 23
Mutation 4: 33-668 a frame shift2 and translation stop at 698 47 1full lenght LH3, without a signal sequence, covering amino acids 33-738 2one nucleotide deletion causing a frame shift in the sequence
Many glycosyltransferase families have a so-called DXD motif in their sequence (Ünligil et al. 2000). There are at least three DXD-like motifs in the LH3 sequence. A point mutation D392A indicated that the DXD-like motif at position 392-394 of the human sequence is not important for GGT activity of the LH3. The analysis of the truncated molecules showed that the most C-terminal DXD-like sequence at the position 489-491 was not essential for the activity. A short conserved motif containing many aspartate residues is located in the sequence at position 187-191 and the mutation of residues to alanine eliminated the GGT activity of the LH3 (Fig.5, III).
5.3 Decreased galactosylhydroxylysyl glucosyltransferase activity in a family with epidermolysis bullosa simplex (IV)
5.3.1 GGT activity, LH3 protein level and analysis of LH3 gene and cDNA in the patients (IV)
Skin fibroblast cultures were established from two patients of a Finnish epidermolysis bullosa simplex (EBS) family, which has been earlier reported to have a deficiency of GGT activity (Savolainen et al. 1981). This family also segregate a keratin 5 mutation (Dong et al. 1993). The mouse embryonic fibroblasts derived from heterozygous LH3 knockout crosses were used for comparison (Sipilä et al. 2007). The GGT activity of the patients’ skin fibroblasts was measured, and our results were consistent with previous results (Savolainen et al. 1981), showing decreased GGT activity for patient 10 and activity in the normal range for patient 22 (Fig. 1A, IV). When the GGT activity was measured from the culture medium, reduced values were obtained for both patient cell lines.
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For comparison, the GGT activity was measured from cells and medium of heterozygous LH3 knockout mouse embryonic fibroblasts (MEFs+/-) (Fig. 1B, IV). The GGT activity in these cells was approximately 80% and in the culture medium only about 10% of the activity of the normal mouse embryonic fibroblasts (MEFs+/+).
Sequencing of PCR fragments covering the entire coding region and most of the 5’- and 3’-untranslated regions of the LH3 cDNA, did not reveal any mutations in the EBS patients. In Northern analysis, the EBS patients had normal levels of LH3 and LH1 mRNA (Fig. 2A, IV) and the size of the LH3 mRNA was also equal to the control. In addition, only three polymorphic nucleotides, present also in healthy controls, were found in the promoter and exon 1 region of the LH3 gene in EBS patients. However, the amount of LH3 protein was reduced in both patient cells and also in the culture media when analyzed by immunoblot (Fig. 2B, IV). Similarly, fewer gold particles representing LH3 were found in EBS cells than in controls in immuno-electronmicroscopy.
5.3.2 Organization of the extracellular matrix and the cytoskeleton in cells with decreased GGT/LH3 activity (IV)
Recent analyses of LH3 knockout mouse cells have indicated that the GGT activity of LH3 is crucial for the assembly and secretion of type VI collagen (Sipilä et al. 2007). Furthermore, it is known that type VI collagen controls the organization of fibronectin (Sabatelli et al. 2001), and, together with fibronectin, it plays a role in type I collagen fibrillogenesis (Li et al. 2003, Minamitani et al. 2004). In both EBS patient cell lines, the arrangement of these proteins was abnormal in the extracellular immunofluorescence stainings. Type VI collagen formed a fine and slightly interconnected three-dimensional network in patients (Fig. 3B, IV), whereas a thick and heavily cross-linked network was observed in controls (Fig. 3A, IV). In patient cells, thin fibronectin fibrils were oriented parallel to the long axis of cells (Fig. 3F, IV) and formed fewer interconnections in the three-dimensional network than in the controls (Fig. 3E, IV). In addition, type I collagen fibrils were short and faint in patient cells (Fig. 3J, IV), differing from the type I collagen fibrils in controls (Fig 3I, IV). When immunostaining was carried out with the LH3 knockout MEFs+/- (Fig. 3D, H, L, VI), the findings were similar to those of the EBS patients.
The metabolically labeled type VI collagen of the EBS fibroblasts formed tetramers, which were secreted from the cells with no obvious abnormalities when
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compared with the controls. In immunoblot analysis, type VI and I collagen α chains of the EBS patient cells and the LH3 knockout MEFs+/- migrated normally on SDS-PAGE indicating no significant changes in lysine modifications. However, in both cell lines these collagen types were not normally deposited into extracellular matrix, since more soluble collagens were present in the medium of these cells compared to controls.
At confluence, the patient cells appeared smaller and rounder, and they were not in such close contact with each other compared with the control cells. The major cytoskeletal components of fibroblasts, actin microfilaments, tubulin microtubules and vimentin intermediate filaments, were examined by immunofluorescence in order to visualize the differences in cell shape. In EBS patient fibroblasts (Fig. 4B, IV), actin stress fibers appeared tangled and thinner and did not always extend the whole long axis of the cell when compared with controls (Fig. 4A, IV). Microtubules were also faint in EBS cells (Fig. 4F, IV), and they appeared curlier than in controls (Fig. 4E, IV). The amount of vimentin intermediate filaments appeared lower in EBS cells (Fig. 4J, IV), and the filaments were shorter than those in control cells, covering only the area surrounding the nucleus. Interestingly, similar or even more dramatic changes were seen in the cytoskeletal stainings of the LH3 knockout MEFs+/- (Fig. 4D, H and L, IV).
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6 Discussion
6.1 Frequency of the charged amino acids is high in the triplets following the lysine residues
The amino acid sequences of type I-XIX collagens (Ayad et al. 1998) were analyzed in order to determine which amino acids occupied the X and Y positions of repeating XYG sequences in close proximity to lysine residues. Our analysis revealed that proline is the most common amino acid in the X and Y position of the XYG triplets. This was expected, since a high content of proline and hydroxyproline is a unique feature of collagen triple helices (Brodsky & Persikov 2005). When the frequencies of the amino acids surrounding the XKG motif were analyzed, more charged amino acids were found in the X and Y position in the triplet following the lysine than in the general triplet (Fig. 3a, I). Interestingly, when the frequencies of the amino acids surrounding the XRG motif were analyzed, no changes compared with general triplet were found. The comparison of lysine and arginine motifs, involving basic amino acids, revealed that evolution had to some extent changed the amino acid sequence surrounding the lysine residues and this might have some role in lysine hydroxylation. Other computational analyses have also indicated that XKGE/DY sequences are present at a greater frequency than expected in fibrillar collagens (types I, II, III, V and XI). This motif was suggested to be biologically important, probably by contributing to collagen triple helix stability (Persikov et al. 2005). A high content of XKGE/DY sequences is probably also a typical feature of the novel type XX-XXIX collagens, at least this has been reported for type XXVIII collagen (Veit et al. 2006).
6.2 Lysyl hydroxylase isoforms do not have absolute amino acid sequence specificity for their substrates
The substrate specificity of the LH isoforms is not fully known. The hydroxylysine content of the various collagen types vary markedly and further variation is found in the same collagen type in different tissues and in the same tissue in different physiological and pathological states (Kivirikko & Pihlajaniemi 1998, Kivirikko et al. 1992). This finding suggests that LH isoforms could be collagen type specific or tissue specific. However, earlier studies have revealed
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that LH isoforms do not have collagen type specificity (Wang et al. 2000) or clear differences in their tissue expression (Heikkinen et al. 1994, Passoja et al. 1998b, Valtavaara et al. 1997, Valtavaara et al. 1998, Yeowell & Walker 1999).
In this study, the sequence specificity of LH isoforms was tested using the Pepspots method, where nine amino acid long collagenous peptides were synthesized on the membrane and some of the peptides were also tested in activity assays. The peptides represented the XYGXKGXYG sequences in type I-XIX collagens and altogether 727 peptides were analyzed. In general, the positive net charge of the peptide and specific amino acids in close proximity to the lysine residues were key determinants in the binding and hydroxylation of the peptide by LH isoforms. Our analysis did not reveal structural motifs that would affect the binding and hydroxylation, and thus our data disagrees with earlier data (Ananthanarayanan et al. 1992, Jiang & Ananthanarayanan 1991). However, we found that peptides with a strong preference for an aqueous environment were bound by LH isoforms with a high probability. These peptides typically had also a high net charge, which most likely is a cause of the high binding probability. The finding also suggests that the LH substrate binding site might be hydrophilic, not a deep hydrophobic pocket.
The Pepspots data suggest that LH1, LH2a, LH2b and LH3 do not have absolute sequence specificity for the binding of peptides. There was an overlap in the binding of many peptides, but there were some peptides that were preferentially bound by a certain LH isoform. Our data also confirmed the earlier findings that LH isoforms lack collagen type specificity (Wang et al. 2000), since isoforms were able to bind and hydroxylate peptides representing various collagen types. Likewise, the overall substrate specificity of LH1, LH2b and LH3 for various synthetic peptides representing type I and IV collagens have been found to be mostly similar. All isoforms were also capable of hydroxylating the same amount of helical lysines of the full-length proα1(I) chain when coexpressed in insect cells (Takaluoma et al. 2007b).
Based on our results, it is probable that LH isoforms can, to some extent, compensate for each other in tissues, but the compensation is dependent on the amount of the LH isoforms in tissues and also on the amino acids surrounding the lysine residue. The cross-linking sites in fibrillar collagens (I, II, III, V and XI) have a conserved positively charged sequence motif, usually including the sequence XKGHR (Eyre & Wu 2005). Interestingly, none of the negatively charged peptides in our activity assays were hydroxylated by LH1, suggesting that LH1 prefers positively charged sequences. Analyses of the LH1 deficient
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EDS VIA patients (Eyre et al. 2002) and LH1 knockout mice (Takaluoma et al. 2007a) have revealed that LH1 hydroxylates preferentially the helical lysines in collagen cross-linking sites and the other isoforms can not fully compensate for this hydroxylation in the absence of LH1.
Our results further show that the alternatively spliced forms of LH2, LH2a and LH2b, have different abilities to bind and hydroxylate collagenous peptides suggesting that amino acids coded by the alternatively spliced exon participate in the substrate binding site or the active site of the enzyme. Although we and others (Takaluoma et al. 2007b) have shown that LH2b can hydroxylate collagenous sequences in vitro, disease models reveal that LH2b is the main LH isoform hydroxylating the telopeptide lysines (Brinckmann et al. 2005, van der Slot et al. 2003, van der Slot et al. 2004). LH2b was unable to hydroxylate synthetic N- and C-telopeptides in vitro, but hydroxylated the N-telopeptide in the full-length proα1(I) chain when coexpressed in insect cells, confirming its function as a telopeptide lysyl hydroxylase (Takaluoma et al. 2007b). The function of LH2a is yet to be determined, but it probably has a function in embryonic development (Salo et al. 2006a).
Studies with mice lacking the LH activity of LH3 have indicated that LH3 preferentially hydroxylates type IV and VI collagen lysine residues, which are further glycosylated (Sipilä et al. 2007). The glycosylated hydroxylysines in type II, IV and VI collagens (Chu et al. 1988, Lauer-Fields et al. 2003, Van den Steen et al. 2004) and in some collagenous proteins (Jensen et al. 2007, Sellar et al. 1991, Wang et al. 2002) are often present in the XKGE/D sequence suggesting that LH3 would prefer this sequence. Interestingly, in our activity assays, LH3 was more sensitive to NaCl than the other isoforms, indicating that charged amino acids in the peptide are important for the LH3 substrate binding and hydroxylation.
6.3 Lysyl hydroxylase 3 is a multifunctional enzyme
The collagen glycosyltransferase activities were purified from chick embryos in the 1970’s (Myllylä et al. 1977, Risteli et al. 1976), but genes for these activities were unknown. In this study, human LH3 was shown to possess galactosylhydroxylysyl glucosyltransferase (GGT) activity. This was seen when LH3 cDNA was expressed in insect and mammalian cells. Even though the amino acid sequences of the LH isoforms are highly similar, LH3 was the only LH isoform possessing the GGT activity. The purification of recombinant LH3
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protein and the cell-free translation experiments further supported the finding that LH and GGT activities are present in the LH3 protein. In all systems used, LH3 had the molecular weight of about 85,000 indicating that no processing of LH3 is required to generate the GGT activity. In addition, LH of C. elegans, which has only one orthologue of the enzyme in the genome (Norman & Moerman 2000), was found to have LH and GGT activities. Later studies revealed that human LH3 and C. elegans LH also have hydroxylysyl galactosyltransferase (GT) activity when recombinant proteins were analyzed in vitro (Wang et al. 2002). Furthermore, the disruption of LH3 by siRNA reduced GT and GGT activities in cells (Wang et al. 2008). Thereby the data suggest that LH3 is able to catalyze all the reactions required for the formation of a glucosylgalactosylhydroxylysine residue. The Km values for UDP-glucose and UDP-galactose of human LH3 were identical to the values determined from enzymes isolated from chick embryos. However, the recombinant LH3 worked most effectively as GGT (Wang et al. 2002).
Recent mouse studies have confirmed that LH3 is the main molecule responsible for GGT activity in vivo. The loss of LH3 in mice leads to early embryonic lethality due to the absence of basement membrane (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). Especially, the GGT activity of LH3 is essential for the formation of the functional BM during embryogenesis (Ruotsalainen et al. 2006). Furthermore, the type I, IV and VI collagens produced by LH3 knockout cells lacked all the hydroxylysine-linked disaccharides indicating that LH3 has GT and GGT activities in vivo (Sipilä et al. 2007). Similarly, the absence of the multifunctional C. elegans LH has been shown to be embryonically lethal due to disrupted secretion of type IV collagen (Norman & Moerman 2000). In addition, the zebrafish LH3 is a multifunctional enzyme. The glycosyltransferase activities of LH3 have been shown to be essential to the motor growth cone migration into the periphery and a potential substrate of LH3 is type XVIII collagen (Schneider & Granato 2006).
LH1 has been shown to be permanently localized into the rough ER membranes via the 40 amino acid long C-terminal peptide, which is highly similar in different LH isoforms (Suokas et al. 2000, Suokas et al. 2003). Nevertheless, when LH3 was overexpressed in insect and mammalian cells, it was found that GGT activity was secreted into the medium. In microscopy, the LH3-GFP fusion protein was also found to enter the Golgi complex. However at that time, we concluded that overexpression of LH3 overloads the retention capacity of the ER, and therefore the protein starts to be secreted into the medium. Later
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studies have confirmed that in fact LH3 is secreted from the cells in vivo. In addition to the ER localization, LH3 also has an extracellular localization in some tissues, like liver and pancreas (Salo et al. 2006a, Salo et al. 2006b). Furthermore, LH3 is present on the cell surface, the GGT activity of serum originates from LH3 protein and LH3 is able to modify proteins in the extracellular space (Salo et al. 2006b). More studies are required to analyze the functions of extracellular LH3.
6.4 The glycosyltransferase active site is separate from the lysyl hydroxylase active site
The amino acid sequence similarity between the human LHs is 47% and over 80% in the C-terminal part of the molecule (Valtavaara et al. 1998). In addition, the amino acid residues that are essential for the co-substrate binding of LH1 (Passoja et al. 1998a, Pirskanen et al. 1996) are conserved in the C-terminal part of the human LH isoforms (Valtavaara et al. 1997, Valtavaara et al. 1998). The amino acid sequence alignment of the LH isoforms of different species revealed 29 amino acids that were conserved between the human and mouse LH3 sequences and the C. elegans sequence, but not with LH1 and LH2. Two of these amino acids, Cys144 and Leu208 in the human sequence, were found to be important for the GGT activity of human LH3 and C. elegans LH. Cys144 was also found to be important for the GT activity of the human LH3 (Wang et al. 2002). However, mutations of these amino acids did not have any effect on the LH activity of LH3. These data suggest that the N-terminal portion of the LH3 molecule is important for the GGT activity. This was further supported by the mutation of Asp669, which corresponds the residue involved in the Fe2+ binding site of LH1 (Pirskanen et al. 1996). As expected, this mutation decreased LH activity dramatically, whereas it had no effect on the GGT activity of LH3. This seems to be the case also in vivo, since the mouse line with an Asp669Ala mutation lacks the LH activity of LH3 (Ruotsalainen et al. 2006) and only some of the glycosylated hydroxylysines are missing suggesting that glycosyltransferase activities are normal in these mice (Sipilä et al. 2007).
The location of the LH active site in the C-terminus and the GGT active site in the N-terminus were also demonstrated by truncation of the LH3 molecule. A 338 amino acid fragment from the N-terminal part still retained some GGT activity, but LH activity was lost from all truncated molecules. Similarly, proteolytic digestion of the human LH3 revealed that the N-terminal fragment
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possesses glycosyltransferase activities (Rautavuoma et al. 2002). However, the intact LH3 molecule seems to be crucial in vivo, even if the other isoforms partly compensate for loss of the LH activity of LH3 (Schneider & Granato 2006).
Our data furthermore revealed that a DXD-like sequence, a motif characteristic of many glycosyltransferase families (Ünligil et al. 2000), is required for the GGT activity of the multifunctional LH3 molecule. The motif probably stabilizes the Mn2+ ion and thus indirectly binds the diphosphate moiety of the UDP-sugar (Gastinel et al. 2001, Ünligil et al. 2000). Mutation of the aspartates in positions 187-191 eliminated the GGT activity of the human LH3 suggesting that this region might be the Mn2+ binding site of LH3. In addition, these aspartates are important for the GT activity of LH3, suggesting that the glycosyltransferase activities of LH3 are located in the same N-terminal part of the molecule (Wang et al. 2002). The same conserved aspartate motif is also important for the glycosyltransferase activities of the mouse (our unpublished data) and zebrafish LH3 (Schneider & Granato 2006). Furthermore, comparison of the LH amino acid sequences with the structural profiles of the glycosyltransferase families have revealed that only LH3 and C. elegans LH show similarity with the DXD motif containing glycosyltransferases in the N-terminal part of the molecule. However, all LH isoforms have some similarity with glycosyltransferases in the middle part of the molecule. The ordering of the LH sequences according to the confidence of the hits with the glycosyltransferase sequences gave the order C. elegans LH>LH3>LH2>LH1 (Myllylä et al. 2007), which is the same order as seen in the phylogenetic analysis (Ruotsalainen et al. 1999).
6.5 Decreased GGT activity and LH3 protein level in a family with epidermolysis bullosa simplex
Epidermolysis bullosa simplex is a skin blistering disorder involving the basal keratinocytes in the epidermis (Uitto et al. 2007). A Finnish family with dominant EBS has been earlier reported to have decreased GGT activity in serum, skin and cultured fibroblasts, accompanied by decreased urinary excretion of glucosylgalactosylhydroxylysine (Savolainen et al. 1981). This EBS family was later shown to have a L462P substitution in keratin 5, an intermediate filament protein expressed in basal keratinocytes. This substitution completely cosegregated with the clinical phenotype and was suggested to be the underlying cause of the skin blistering seen above the basement membrane in this family
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(Dong et al. 1993, Kero et al. 1982). However, Savolainen and coworkers (1981) reported that the decrease in GGT activity correlated with the severity of the disease.
In the current study, we showed that the GGT activity and LH3 protein level are decreased in this EBS family. This was the first demonstration of a decreased amount of LH3 in humans. The amount of the extracellular LH3 was more severely reduced than the intracellular LH3 in cultures of EBS patient fibroblasts. A similar remarkable decrease of extracellular GGT/LH3 activity was observed in LH3 knockout MEFs+/- suggesting that even a slight decrease in the cellular GGT/LH3 level remarkably reduces the GGT/LH3 protein level in the extracellular space. The secretion pathway of LH3 is not yet known and therefore the EBS patient cells as well as the LH3 knockout MEFs+/- provide models for further investigation of the secretion of LH3. The reason for the reduced LH3 in EBS patient cells also remains unknown. It is possible that the LH3 is regulated by other cellular factors on the translation level. Recently, a patient with the progeroid type of Ehlers-Danlos syndrome, caused by mutations of the proteoglycan biosynthetic enzyme galactosyltransferase-1 (Faiyaz-Ul-Haque et al. 2004), was demonstrated to have decreased expression of the LH2 gene. However, no mutations were detected in either the cDNA or promoter sequence of LH2 of this patient (Walker et al. 2004) suggesting the presence of other factors regulating the amount of LH isoforms.
6.6 Decreased GGT/LH3 activity causes abnormalities in the organization of extracellular matrix and cytoskeleton
Our results demonstrated that type VI collagen, fibronectin and type I collagen were abnormally deposited and organized into the extracellular matrix in EBS patient fibroblasts, as well as in the LH3 knockout MEFs+/- used for comparison. The recent results with LH3 knockout mice indicated that a total lack of glycosylated hydroxylysines prevented the tetramerization and secretion of type VI collagen. In addition, the underglycosylation of type VI collagen, in mice lacking the LH activity of LH3, led to abnormal distribution and aggregation of type VI collagen in the skin and muscle (Sipilä et al. 2007). The present study suggests that not all lysines of type VI collagen were hydroxylated and glycosylated normally in EBS patient fibroblasts and LH3 knockout MEFs+/−, since type VI collagen deposition into filament networks was disturbed in both cell lines. These results underline the crucial role of the LH3 dependent
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glycosylation of type VI collagen in its normal assembly into the beaded microfibrillar network in the ECM.
It has been shown that a dramatic reduction or total lack of expression of type VI collagen alters the organization of fibronectin fibrils in fibroblast cultures (Sabatelli et al. 2001). Our results suggests that even small changes in the type VI collagen network, due to small alterations in the glycosylation of hydroxylysine residues catalyzed by LH3, are enough to affect the organization of fibronectin fibrils in the ECM. It is possible that changes seen in type I collagen deposition in the EBS patient fibroblasts and LH3 knockout MEFs+/- are due to altered fibronectin organization (Li et al. 2003, McDonald et al. 1982, Velling et al. 2002), but changes in the type VI collagen network could also directly affect type I collagen fibrillogenesis (Kirschner et al. 2005, Minamitani et al. 2004). Thereby, our data further establish the link between the organization of type VI collagen, fibronectin and type I collagen networks.
The vital role of LH3 in the assembly of ECM is also clearly seen in the LH3 knockout mice, where homozygous mice die at an early developmental stage due to the lack of basement membranes (Ruotsalainen et al. 2006). Heterozygous adult mice show ultrastructural changes in muscle and skin (our unpublished data), similar to those seen in the mouse line lacking the LH activity of LH3 (Sipilä et al. 2007), indicating that the decreased GGT/LH3 activity also affects the development and organization of ECM in tissues. Therefore, our results could explain the more severe phenotype seen in patients with decreased GGT activity in this EBS family (Savolainen et al. 1981). In addition it is remarkable to note, that one member of this EBS family was reported to have an exceptionally poorly developed sympathetic nervous system, where only one rudimentally developed ganglion was found in the lumbar region (Sonck 1948). The GGT activity of LH3 has recently been shown to be critical for motor growth cone migration in zebrafish (Schneider & Granato 2006), therefore it is tempting to speculate that the decreased GGT activity of this EBS family might be linked to this neuronal abnormality of the patient and would further establish the crucial role of the GGT activity of LH3.
Analysis of cytoskeleton components revealed changes in the organization of actin microfilaments, tubulin microtubules and vimentin intermediate filaments in the EBS patient fibroblasts and LH3 knockout MEFs+/-. It has been shown that ECM polymerization regulates the organization of the cytoskeleton (Hocking et al. 2000, Putnam et al. 2001). In addition, there is increasing evidence that the ECM transmits environmental signals via integrins and other cell receptors to the
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cytoskeleton and these signals affect cell proliferation, differentiation and death (Aszodi et al. 2006, Geiger et al. 2001). Our data suggest that LH3 is a key enzyme in regulation of the deposition of ECM components, and thereby also in transmission of signals to the cytoskeleton. It is probable that the glycosyltransferase activities of LH3 are an especially important extracellular factor in the regulation of extracellular matrix and cytoskeletal arrangements in the EBS patient fibroblasts and LH3 knockout MEFs+/-. This is supported by recent data (Wang et al. 2008) indicating that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, results in abnormal cell morphology, abnormalities in the organization of cytoskeletal proteins, and arrest of cell growth followed by cell death. It would be interesting to further analyze, whether decreased GGT/LH3 activity affects the behavior of the EBS patient fibroblasts and LH3 knockout MEFs+/- in vivo.
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7 Conclusions
In this thesis study the effect of the amino acid sequence of the peptide substrate on the binding and hydroxylation of the peptide by different lysyl hydroxylase isoforms was analyzed. Although the data do not indicate strict sequence specificity for the LH isoforms, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Overall, the key factors promoting peptide binding of all isoforms were a positive charge of the peptide and specific amino acid residues in close proximity to the lysine. Based on our results, it is probable that the LH isoforms can compensate for each other to some extent in tissues.
Lysyl hydroxylase 3 was also found to possess galactosylhydroxylysyl glucosyltransferase activity, in addition to lysyl hydroxylase activity. This is the first identification of a cDNA of GGT, an enzyme catalyzing the glucosylation of galactosylhydroxylysine residues in collagens and collagenous proteins. In addition, the lysyl hydroxylase of C. elegans was shown to be a multifunctional enzyme. Site-directed mutagenesis revealed that the GGT active site is separate from the C-terminal LH active site. The DXD-like motif, characteristic of many glycosyltransferase families, and two conserved amino acids in the N-terminal part of the LH3 and C. elegans LH were critical for the GGT activity and did not have an effect on the LH activity of the molecule.
The LH3 protein level and GGT activity were found to be decreased in skin fibroblasts and in the culture media of cells, collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which has been earlier reported to have a deficiency of GGT activity. However, the reduction of enzyme activity was not due to a mutation or lower expression of the LH3 gene. The amount of extracellular LH3 was more severely decreased than the intracellular LH3 in the EBS patient fibroblasts as well as in the LH3 knockout MEFs+/- used for comparison in this study. The data indicate that the decreased GGT/LH3 activity in cells, probably followed by a change in the glycosylation of type VI collagen, has an effect on the deposition and organization of the key ECM components, types VI and I collagen and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator, and could explain the more severe phenotype seen in patients with decreased GGT activity in this EBS family.
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References Abagyan RA & Totrov MM (1997) Contact area difference (CAD): a robust measure to
evaluate accuracy of protein models. J.Mol.Biol. 268(3): 678-685. Acil Y, Vetter U, Brenner R, Müller PK & Brinckmann J (1995) Ehlers-Danlos syndrome
type VI: cross-link pattern in tissue and urine sample as a diagnostic marker. J.Am.Acad.Dermatol. 33(3): 522-524.
Agrafiotis DK (1997) A new method for analyzing protein sequence relationships based on Sammon maps. Protein Sci. 6(2): 287-293.
Ananthanarayanan VS, Saint-Jean A & Jiang P (1992) Conformation of a synthetic hexapeptide substrate of collagen lysyl hydroxylase. Arch.Biochem.Biophys. 298(1): 21-28.
Andrews PC, Hawke D, Shively JE & Dixon JE (1984) Anglerfish preprosomatostatin II is processed to somatostatin-28 and contains hydroxylysine at residue 23. J.Biol.Chem. 259(24): 15021-15024.
Anttinen H (1977) Collagen glucosyltransferases activity in human serum. Clin.Chim.Acta 77(3): 323-330.
Anttinen H, Terho EO, Järvensivu PM & Savolainen ER (1985) Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases. Clin.Chim.Acta 148(1): 3-8.
Areida SK, Reinhardt DP, Müller PK, Fietzek PP, Kowitz J, Marinkovich MP & Notbohm H (2001) Properties of the Collagen Type XVII Ectodomain. Evidence for N- to C-terminal triple helix folding. J. Biol. Chem. 276(2): 1594-1601.
Armstrong LC & Last JA (1995) Rat lysyl hydroxylase: molecular cloning, mRNA distribution and expression in a baculovirus system. Biochim.Biophys.Acta 1264(1): 93-102.
Arora M, Munoz E & Tenner AJ (2001) Identification of a site on mannan-binding lectin critical for enhancement of phagocytosis. J.Biol.Chem. 276(46): 43087-43094.
Aszodi A, Legate KR, Nakchbandi I & Fässler R (2006) What mouse mutants teach us about extracellular matrix function. Annu.Rev.Cell Dev.Biol. 22: 591-621.
Aumailley M & Gayraud B (1998) Structure and biological activity of the extracellular matrix. J.Mol.Med. 76(3-4): 253-265.
Ayad S, Boot-Handford RP, Humphries MJ, Kadler KE & Shuttleworth A (1998) The Extracellular Matrix. FactsBook. 2nd ed. San Diego: Academic Press.
Babel W & Glanville RW (1984) Structure of human-basement-membrane (type IV) collagen. Complete amino-acid sequence of a 914-residue-long pepsin fragment from the alpha1(IV) chain. European Journal of Biochemistry 143(3): 545-556.
Bailey AJ, Bazin S, Sims TJ, Le Lous M, Nicoletis C & Delaunay A (1975) Characterization of the collagen of human hypertrophic and normal scars. Biochim.Biophys.Acta 405(2): 412-421.
Bailey AJ & Light ND (1985) Intermolecular cross-linking in fibrotic collagen. Ciba Found.Symp. 114: 80-96.
72
Bank RA, Robins SP, Wijmenga C, Breslau-Siderius LJ, Bardoel AF, van der Sluijs HA, Pruijs HE & TeKoppele JM (1999) Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17. Proc.Natl.Acad.Sci.U.S.A. 96(3): 1054-1058.
Bank RA, Verzijl N, Lafeber FP & Tekoppele JM (2002) Putative role of lysyl hydroxylation and pyridinoline cross-linking during adolescence in the occurrence of osteoarthritis at old age. Osteoarthritis Cartilage 10(2): 127-134.
Barnes MJ, Constable BJ, Morton LF & Royce PM (1974) Age-related variations in hydroxylation of lysine and proline in collagen. Biochem.J. 139(2): 461-468.
Bätge B, Winter C, Notbohm H, Acil Y, Brinckmann J & Müller PK (1997) Glycosylation of human bone collagen I in relation to lysylhydroxylation and fibril diameter. J.Biochem.(Tokyo) 122(1): 109-115.
Bauvois B & Roth S (1985) A collagen:glucosyltransferase at the surface of malignant fibroblasts. J.Cell.Physiol. 124(2): 213-218.
Berry DA (ed) (1992) Statistics: A Bayesian Perspective. Duxbury Press, London: Wadworth Publishing Co.
Blumenkrantz N, Assad R & Peterkofsky B (1984) Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes. J.Biol.Chem. 259(2): 854-859.
Bolarin DM (1991) Liver collagen glucosyltransferase in experimental primary liver carcinoma. Acta Physiol.Hung. 77(2): 113-120.
Bortolato M, Azzar G, Farjanel J & Got R (1990) Evidence of partial localization in Golgi apparatus of UDP-glucose collagen glucosyltransferase from chick embryo liver. Int.J.Biochem. 22(9): 1039-1046.
Bortolato M, Radisson J, Azzar G & Got R (1992) Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver. Int.J.Biochem. 24(2): 243-248.
Brenner RE, Vetter U, Nerlich A, Worsdorfer O, Teller WM & Müller PK (1989) Osteogenesis imperfecta: insufficient collagen synthesis in early childhood as evidenced by analysis of compact bone and fibroblast cultures. Eur.J.Clin.Invest. 19(2): 159-166.
Brinckmann J, Acil Y, Feshchenko S, Katzer E, Brenner R, Kulozik A & Kugler S (1998) Ehlers-Danlos syndrome type VI: lysyl hydroxylase deficiency due to a novel point mutation (W612C). Arch.Dermatol.Res. 290(4): 181-186.
Brinckmann J, Kim S, Wu J, Reinhardt DP, Batmunkh C, Metzen E, Notbohm H, Bank RA, Krieg T & Hunzelmann N (2005) Interleukin 4 and prolonged hypoxia induce a higher gene expression of lysyl hydroxylase 2 and an altered cross-link pattern: important pathogenetic steps in early and late stage of systemic scleroderma? Matrix Biol. 24(7): 459-468.
73
Brinckmann J, Neess CM, Gaber Y, Sobhi H, Notbohm H, Hunzelmann N, Fietzek PP, Müller PK, Risteli J, Gebker R & Scharffetter-Kochanek K (2001) Different pattern of collagen cross-links in two sclerotic skin diseases: lipodermatosclerosis and circumscribed scleroderma. J.Invest.Dermatol. 117(2): 269-273.
Brinckmann J, Notbohm H, Tronnier M, Acil Y, Fietzek PP, Schmeller W, Müller PK & Bätge B (1999) Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin. J.Invest.Dermatol. 113(4): 617-621.
Brodsky B & Persikov AV (2005) Molecular structure of the collagen triple helix. Adv.Protein Chem. 70: 301-339.
Brodsky B & Ramshaw JA (1997) The collagen triple-helix structure. Matrix Biol. 15(8-9): 545-554.
Brodsky B & Shah NK (1995) Protein motifs. 8. The triple-helix motif in proteins. FASEB J. 9(15): 1537-1546.
Canty EG & Kadler KE (2005) Procollagen trafficking, processing and fibrillogenesis. J.Cell.Sci. 118(Pt 7): 1341-1353.
Canty EG & Kadler KE (2002) Collagen fibril biosynthesis in tendon: a review and recent insights. Comp.Biochem.Physiol.A.Mol.Integr.Physiol. 133(4): 979-985.
Chu ML, Conway D, Pan TC, Baldwin C, Mann K, Deutzmann R & Timpl R (1988) Amino acid sequence of the triple-helical domain of human collagen type VI. J.Biol.Chem. 263(35): 18601-18606.
Corthay A, Backlund J, Broddefalk J, Michaelsson E, Goldschmidt TJ, Kihlberg J & Holmdahl R (1998) Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis. Eur.J.Immunol. 28(8): 2580-2590.
Crouch E, Chang D, Rust K, Persson A & Heuser J (1994) Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly. J.Biol.Chem. 269(22): 15808-15813.
Deb K, Anand A & Joshi D (2002) A computationally efficient evolutionary algorithm for real-parameter optimization. Evol.Comput. 10(4): 371-395.
Deprez P, Doss-Pepe E, Brodsky B & Inestrosa NC (2000) Interaction of the collagen-like tail of asymmetric acetylcholinesterase with heparin depends on triple-helical conformation, sequence and stability. Biochem.J. 350(Pt 1): 283-290.
Dong W, Ryynänen M & Uitto J (1993) Identification of a leucine-to-proline mutation in the keratin 5 gene in a family with the generalized Kobner type of epidermolysis bullosa simplex. Hum.Mutat. 2(2): 94-102.
Dzhambazov B, Holmdahl M, Yamada H, Lu S, Vestberg M, Holm B, Johnell O, Kihlberg J & Holmdahl R (2005) The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans. Eur.J.Immunol. 35(2): 357-366.
Erickson AC & Couchman JR (2000) Still more complexity in mammalian basement membranes. J.Histochem.Cytochem. 48(10): 1291-1306.
74
Eyre D, Shao P, Weis MA & Steinmann B (2002) The kyphoscoliotic type of Ehlers-Danlos syndrome (type VI): differential effects on the hydroxylation of lysine in collagens I and II revealed by analysis of cross-linked telopeptides from urine. Mol.Genet.Metab. 76(3): 211-216.
Eyre DR & Glimcher MJ (1972) Reducible crosslinks in hydroxylysine-deficient collagens of a heritable disorder of connective tissue. Proc.Natl.Acad.Sci.U.S.A. 69(9): 2594-2598.
Eyre DR, Paz MA & Gallop PM (1984) Cross-linking in collagen and elastin. Annu.Rev.Biochem. 53: 717-748.
Eyre DR & Wu JJ (2005) Collagen Cross-links. In: Brinckmann J, Notbohm H & Müller PK (eds) Topics in Current Chemistry, Collagen: Primer in Structure, Processing and Assembly. Berlin-Heidelberg: Springer-Verlag, 207-229.
Faiyaz-Ul-Haque M, Zaidi SH, Al-Ali M, Al-Mureikhi MS, Kennedy S, Al-Thani G, Tsui LC & Teebi AS (2004) A novel missense mutation in the galactosyltransferase-I (B4GALT7) gene in a family exhibiting facioskeletal anomalies and Ehlers-Danlos syndrome resembling the progeroid type. Am.J.Med.Genet.A. 128(1): 39-45.
Fernandes RJ, Harkey MA, Weis M, Askew JW & Eyre DR (2007) The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells. Bone 40(5): 1343-1351.
Frank R (1992) Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron, 48(42): 9217-9232.
Franzke CW, Bruckner P & Bruckner-Tuderman L (2005) Collagenous transmembrane proteins: recent insights into biology and pathology. J.Biol.Chem. 280(6): 4005-4008.
Gastinel LN, Bignon C, Misra AK, Hindsgaul O, Shaper JH & Joziasse DH (2001) Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. EMBO J. 20(4): 638-649.
Geiger B, Bershadsky A, Pankov R & Yamada KM (2001) Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk. Nat.Rev.Mol.Cell Biol. 2(11): 793-805.
Gelse K, Pöschl E & Aigner T (2003) Collagens--structure, function, and biosynthesis. Adv.Drug Deliv.Rev. 55(12): 1531-1546.
Giunta C, Randolph A & Steinmann B (2005) Mutation analysis of the PLOD1 gene: an efficient multistep approach to the molecular diagnosis of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA). Mol.Genet.Metab. 86(1-2): 269-276.
Ha VT, Marshall MK, Elsas LJ, Pinnell SR & Yeowell HN (1994) A patient with Ehlers-Danlos syndrome type VI is a compound heterozygote for mutations in the lysyl hydroxylase gene. J.Clin.Invest. 93(4): 1716-1721.
Hanauske-Abel HM & Günzler V (1982) A stereochemical concept for the catalytic mechanism of prolylhydroxylase: applicability to classification and design of inhibitors. J.Theor.Biol. 94(2): 421-455.
75
Harwood R, Grant ME & Jackson DS (1974) Collagen biosynthesis. Characterization of subcellular fractions from embyonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases. Biochem.J. 144(1): 123-130.
Hausinger RP (2004) FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit.Rev.Biochem.Mol.Biol. 39(1): 21-68.
Hautala T, Byers MG, Eddy RL, Shows TB, Kivirikko KI & Myllylä R (1992a) Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2. Genomics 13(1): 62-69.
Hautala T, Heikkinen J, Kivirikko KI & Myllylä R (1993) A large duplication in the gene for lysyl hydroxylase accounts for the type VI variant of Ehlers-Danlos syndrome in two siblings. Genomics 15(2): 399-404.
Hautala T, Heikkinen J, Kivirikko KI & Myllylä R (1992b) Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts. Biochem.J. 283(Pt 1): 51-54.
Ha-Vinh R, Alanay Y, Bank RA, Campos-Xavier AB, Zankl A, Superti-Furga A & Bonafe L (2004) Phenotypic and molecular characterization of Bruck syndrome (osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2. Am.J.Med.Genet.A. 131(2): 115-120.
Heikkinen J, Hautala T, Kivirikko KI & Myllylä R (1994) Structure and expression of the human lysyl hydroxylase gene (PLOD): introns 9 and 16 contain Alu sequences at the sites of recombination in Ehlers-Danlos syndrome type VI patients. Genomics 24(3): 464-471.
Heikkinen J, Pousi B, Pope M & Myllylä R (1999) A null-mutated lysyl hydroxylase gene in a compound heterozygote British patient with Ehlers-Danlos syndrome type VI. Hum.Mutat. 14(4): 351.
Heikkinen J, Toppinen T, Yeowell H, Krieg T, Steinmann B, Kivirikko KI & Myllylä R (1997) Duplication of seven exons in the lysyl hydroxylase gene is associated with longer forms of a repetitive sequence within the gene and is a common cause for the type VI variant of Ehlers-Danlos syndrome. Am.J.Hum.Genet. 60(1): 48-56.
Heise CT, Nicholls JR, Leamy CE & Wallis R (2000) Impaired secretion of rat mannose-binding protein resulting from mutations in the collagen-like domain. J.Immunol. 165(3): 1403-1409.
Hjalt TA, Amendt BA & Murray JC (2001) PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome. J.Cell Biol. 152(3): 545-552.
Hocking DC, Sottile J & Langenbach KJ (2000) Stimulation of integrin-mediated cell contractility by fibronectin polymerization. J.Biol.Chem. 275(14): 10673-10682.
Hofbauer KH, Gess B, Lohaus C, Meyer HE, Katschinski D & Kurtz A (2003) Oxygen tension regulates the expression of a group of procollagen hydroxylases. Eur.J.Biochem. 270(22): 4515-4522.
76
Hulmes DJ (2002) Building collagen molecules, fibrils, and suprafibrillar structures. J.Struct.Biol. 137(1-2): 2-10.
Hyland J, Ala-Kokko L, Royce P, Steinmann B, Kivirikko KI & Myllylä R (1992) A homozygous stop codon in the lysyl hydroxylase gene in two siblings with Ehlers-Danlos syndrome type VI. Nat.Genet. 2(3): 228-231.
Ihme A, Krieg T, Nerlich A, Feldmann U, Rauterberg J, Glanville RW, Edel G & Müller PK (1984) Ehlers-Danlos syndrome type VI: collagen type specificity of defective lysyl hydroxylation in various tissues. J.Invest.Dermatol. 83(3): 161-165.
Illy C, Thielens NM & Arlaud GJ (1993) Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement. J.Protein Chem. 12(6): 771-781.
Jensen PH, Laursen I, Matthiesen F & Hojrup P (2007) Posttranslational modifications in human plasma MBL and human recombinant MBL. Biochim.Biophys.Acta 1774(3): 335-344.
Jiang P & Ananthanarayanan VS (1991) Conformational requirement for lysine hydroxylation in collagen. Structural studies on synthetic peptide substrates of lysyl hydroxylase. J.Biol.Chem. 266(34): 22960-22967.
Judisch GF, Waziri M & Krachmer JH (1976) Ocular Ehlers-Danlos syndrome with normal lysyl hydroxylase activity. Arch.Ophthalmol. 94(9): 1489-1491.
Kabsch W & Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22(12): 2577-2637.
Kadler KE, Baldock C, Bella J & Boot-Handford RP (2007) Collagens at a glance. J.Cell.Sci. 120(Pt 12): 1955-1958.
Kellokumpu S, Sormunen R, Heikkinen J & Myllylä R (1994) Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum. J.Biol.Chem. 269(48): 30524-30529.
Kero M, Peltonen L, Foidart JM & Savolainen ER (1982) Immunohistological localization of three basement membrane components in various forms of epidermolysis bullosa. J.Cutan.Pathol. 9(5): 316-328.
Keshi H, Sakamoto T, Kawai T, Ohtani K, Katoh T, Jang SJ, Motomura W, Yoshizaki T, Fukuda M, Koyama S, Fukuzawa J, Fukuoh A, Yoshida I, Suzuki Y & Wakamiya N (2006) Identification and characterization of a novel human collectin CL-K1. Microbiol.Immunol. 50(12): 1001-1013.
Kielty CM & Grant GA (2002) The Collagen Family: Structure, Assembly, and Organization in the Extracelllular Matrix. In: Royce PM & Steinmann B (eds) Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects. 2nd ed. New York: Wiley-Liss Inc, 159-221.
Kikuchi Y, Suzuki Y & Tamiya N (1983) The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase. Biochem.J. 213(2): 507-512.
77
Kirschner J, Hausser I, Zou Y, Schreiber G, Christen HJ, Brown SC, Anton-Lamprecht I, Muntoni F, Hanefeld F & Bonnemann CG (2005) Ullrich congenital muscular dystrophy: connective tissue abnormalities in the skin support overlap with Ehlers-Danlos syndromes. Am.J.Med.Genet.A. 132(3): 296-301.
Kivirikko KI (1995) Posttraslational Processing of Collagens. In: Bittar EE, Bittar N & Greenwich CT (eds) Principles pf Medical Biology.Cellular Organelles and the Extracellular Matrix. London: JAI Press Inc, 233-254.
Kivirikko KI & Myllylä R (1982) Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes. Methods Enzymol. 82(Pt A): 245-304.
Kivirikko KI & Myllylä R (1979) Collagen glycosyltransferases. Int.Rev.Connect.Tissue Res. 8: 23-72.
Kivirikko KI, Myllylä R & Pihlajaniemi T (1992) Hydroxylation of proline and lysine residues in collagens and other animal and plant proteins. In: Harding JJ & Grabbe MJC (eds) Post-Translational Modifications of Proteins. Boca Raton, Florida: CRC Press, 1-51.
Kivirikko KI & Pihlajaniemi T (1998) Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv.Enzymol.Relat.Areas Mol.Biol. 72: 325-398.
Kivirikko KI, Shudo K, Sakakibara S & Prockop DJ (1972) Studies on protocollagen lysine hydroxylase. Hydroxylation of synthetic peptides and the stoichiometric decarboxylation of -ketoglutarate. Biochemistry 11(1): 122-129.
Knott L & Bailey AJ (1998) Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone 22(3): 181-187.
Kowitz J, Knippel M, Schuhr T & Mach J (1997) Alteration in the extent of collagen I hydroxylation, isolated from femoral heads of women with a femoral neck fracture caused by osteoporosis. Calcif.Tissue Int. 60(6): 501-505.
Krane SM, Pinnell SR & Erbe RW (1972) Lysyl-protocollagen hydroxylase deficiency in fibroblasts from siblings with hydroxylysine-deficient collagen. Proc.Natl.Acad.Sci.U.S.A. 69(10): 2899-2903.
Lamandé SR & Bateman JF (1999) Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones. Semin.Cell Dev.Biol. 10(5): 455-464.
Langeveld JP, Noelken ME, Hard K, Todd P, Vliegenthart JF, Rouse J & Hudson BG (1991) Bovine glomerular basement membrane. Location and structure of the asparagine-linked oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer. J. Biol. Chem. 266(4): 2622-2631.
Last JA, King TE,Jr, Nerlich AG & Reiser KM (1990) Collagen cross-linking in adult patients with acute and chronic fibrotic lung disease. Molecular markers for fibrotic collagen. Am.Rev.Respir.Dis. 141(2): 307-313.
Lauer-Fields JL, Malkar NB, Richet G, Drauz K & Fields GB (2003) Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation. J.Biol.Chem. 278(16): 14321-14330.
78
Lee YM, Leiby KR, Allar J, Paris K, Lerch B & Okarma TB (1991) Primary structure of bovine conglutinin, a member of the C-type animal lectin family. J.Biol.Chem. 266(5): 2715-2723.
Leimeister C, Steidl C, Schumacher N, Erhard S & Gessler M (2002) Developmental expression and biochemical characterization of Emu family members. Dev.Biol. 249(2): 204-218.
Li S, Van Den Diepstraten C, D'Souza SJ, Chan BM & Pickering JG (2003) Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin, RhoA, and fibronectin polymerization. Am.J.Pathol. 163(3): 1045-1056.
Lu J, Teh C, Kishore U & Reid KB (2002) Collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system. Biochim.Biophys.Acta 1572(2-3): 387-400.
Majamaa K, Günzler V, Hanauske-Abel HM, Myllylä R & Kivirikko KI (1986) Partial identity of the 2-oxoglutarate and ascorbate binding sites of prolyl 4-hydroxylase. J.Biol.Chem. 261(17): 7819-7823.
Majamaa K, Turpeenniemi-Hujanen TM, Latipää P, Günzler V, Hanauske-Abel HM, Hassinen IE & Kivirikko KI (1985) Differences between collagen hydroxylases and 2-oxoglutarate dehydrogenase in their inhibition by structural analogues of 2-oxoglutarate. Biochem.J. 229(1): 127-133.
Malhotra R, Laursen SB, Willis AC & Sim RB (1993) Localization of the receptor-binding site in the collectin family of proteins. Biochem.J. 293(Pt 1): 15-19.
McDonald JA, Kelley DG & Broekelmann TJ (1982) Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix. J.Cell Biol. 92(2): 485-492.
Menashi S & Grant ME (1979) Studies on the collagen glucosyltransferase activity present in platelets and plasma. Biochem.J. 178(3): 777-784.
Mercer DK, Nicol PF, Kimbembe C & Robins SP (2003) Identification, expression, and tissue distribution of the three rat lysyl hydroxylase isoforms. Biochem.Biophys.Res.Commun. 307(4): 803-809.
Minamitani T, Ikuta T, Saito Y, Takebe G, Sato M, Sawa H, Nishimura T, Nakamura F, Takahashi K, Ariga H & Matsumoto K (2004) Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen. Exp.Cell Res. 298(1): 305-315.
Mizuno K, Adachi E, Imamura Y, Katsumata O & Hayashi T (2001) The fibril structure of type V collagen triple-helical domain. Micron 32(3): 317-323.
Molony MS, Quan C, Mulkerrin MG & Harris RJ (1998) Hydroxylation of Lys residues reduces their susceptibility to digestion by trypsin and lysyl endopeptidase. Anal.Biochem. 258(1): 136-137.
Molony MS, Wu S-, Keyt LK & Harris RJ (1995) The Unexpected Presence of Hydroxylysine in Non-Collagenous Proteins. In: Crabb JW (ed) Techniques in Protein Chemistry. Orlando: Academic Press, 91-98.
Murray LW, Bautista J, James PL & Rimoin DL (1989) Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias. Am.J.Hum.Genet. 45(1): 5-15.
79
Myllyharju J & Kivirikko KI (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 20(1): 33-43.
Myllylä R (1981) Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis. Biochim.Biophys.Acta 658(2): 299-307.
Myllylä R (1976) Studies on the mechanism of collagen glucosyltransferase reaction. Eur.J.Biochem. 70(1): 225-231.
Myllylä R, Anttinen H, Risteli L & Kivirikko KI (1977) Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos. Biochim.Biophys.Acta 480(1): 113-121.
Myllylä R, Becvar R, Adam M & Kivirikko KI (1989) Markers of collagen metabolism in sera of patients with various rheumatic diseases. Clin.Chim.Acta 183(3): 243-252.
Myllylä R, Günzler V, Kivirikko KI & Kaska DD (1992) Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases. Biochem.J. 286(Pt 3): 923-927.
Myllylä R, Majamaa K, Günzler V, Hanauske-Abel HM & Kivirikko KI (1984) Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. J.Biol.Chem. 259(9): 5403-5405.
Myllylä R, Pajunen L & Kivirikko KI (1988) Polyclonal and monoclonal antibodies to human lysyl hydroxylase and studies on the molecular heterogeneity of the enzyme. Biochem.J. 253(2): 489-496.
Myllylä R, Pihlajaniemi T, Pajunen L, Turpeenniemi-Hujanen T & Kivirikko KI (1991) Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. J.Biol.Chem. 266(5): 2805-2810.
Myllylä R, Risteli L & Kivirikko KI (1976) Collagen glucosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage. Eur.J.Biochem. 61(1): 59-67.
Myllylä R, Risteli L & Kivirikko KI (1975) Assay of collagen-galactosyltransferase and collagen-glucosyltransferase activities and preliminary characterization of enzymic reactions with transferases from chick-embryo cartilage. Eur.J.Biochem. 52(3): 401-410.
Myllylä R, Wang C, Heikkinen J, Juffer A, Lampela O, Risteli M, Ruotsalainen H, Salo A & Sipilä L (2007) Expanding the lysyl hydroxylase toolbox: new insights into the localization and activities of lysyl hydroxylase 3 (LH3). J.Cell.Physiol. 212(2): 323-329.
NCBI databases at http://www.ncbi.nlm.nih.gov/ Norman KR & Moerman DG (2000) The let-268 locus of Caenorhabditis elegans encodes
a procollagen lysyl hydroxylase that is essential for type IV collagen secretion. Dev.Biol. 227(2): 690-705.
80
Notbohm H, Nokelainen M, Myllyharju J, Fietzek PP, Müller PK & Kivirikko KI (1999) Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro. J.Biol.Chem. 274(13): 8988-8992.
OMIM databases at http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM Pajunen L, Suokas M, Hautala T, Kellokumpu S, Tebbe B, Kivirikko KI & Myllylä R
(1998) A splice-site mutation that induces exon skipping and reduction in lysyl hydroxylase mRNA levels but does not create a nonsense codon in Ehlers-Danlos syndrome type VI. DNA Cell Biol. 17(2): 117-123.
Pasquali M, Still MJ, Vales T, Rosen RI, Evinger JD, Dembure PP, Longo N & Elsas LJ (1997) Abnormal formation of collagen cross-links in skin fibroblasts cultured from patients with Ehlers-Danlos syndrome type VI. Proc.Assoc.Am.Physicians 109(1): 33-41.
Passoja K, Myllyharju J, Pirskanen A & Kivirikko KI (1998a) Identification of arginine-700 as the residue that binds the C-5 carboxyl group of 2-oxoglutarate in human lysyl hydroxylase 1. FEBS Lett. 434(1-2): 145-148.
Passoja K, Rautavuoma K, Ala-Kokko L, Kosonen T & Kivirikko KI (1998b) Cloning and characterization of a third human lysyl hydroxylase isoform. Proc.Natl.Acad.Sci.U.S.A. 95(18): 10482-10486.
Persikov AV, Ramshaw JAM, Kirkpatrick A & Brodsky B (2005) Electrostatic Interactions Involving Lysine Make Major Contributions to Collagen Triple-Helix Stability. Biochemistry 44(5): 1414-1422.
Persson A, Chang D, Rust K, Moxley M, Longmore W & Crouch E (1989) Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein. Biochemistry 28(15): 6361-6367.
Peterkofsky B & Assad R (1979) Localization of chick embryo limb bone microsomal lysyl hydroxylase at intracisternal and intramembrane sites. J.Biol.Chem. 254(11): 4714-4720.
Pinnell SR, Krane SM, Kenzora JE & Glimcher MJ (1972) A heritable disorder of connective tissue. Hydroxylysine-deficient collagen disease. N.Engl.J.Med. 286(19): 1013-1020.
Pirskanen A, Kaimio AM, Myllylä R & Kivirikko KI (1996) Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity. J.Biol.Chem. 271(16): 9398-9402.
Pornprasertsuk S, Duarte WR, Mochida Y & Yamauchi M (2005) Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization. J.Bone Miner.Res. 20(1): 81-87.
Pornprasertsuk S, Duarte WR, Mochida Y & Yamauchi M (2004) Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells. J.Bone Miner.Res. 19(8): 1349-1355.
81
Pousi B, Hautala T, Heikkinen J, Pajunen L, Kivirikko KI & Myllylä R (1994) Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome. Am.J.Hum.Genet. 55(5): 899-906.
Pousi B, Hautala T, Hyland JC, Schroter J, Eckes B, Kivirikko KI & Myllylä R (1998) A compound heterozygote patient with Ehlers-Danlos syndrome type VI has a deletion in one allele and a splicing defect in the other allele of the lysyl hydroxylase gene. Hum.Mutat. 11(1): 55-61.
Pousi B, Heikkinen J, Schroter J, Pope M & Myllylä R (2000) A nonsense codon of exon 14 reduces lysyl hydroxylase mRNA and leads to aberrant RNA splicing in a patient with Ehlers-Danlos syndrome type VI. Mutat.Res. 432(1-2): 33-37.
Provencher SW & Glockner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20(1): 33-37.
Puistola U, Turpeenniemi-Hujanen TM, Myllylä R & Kivirikko KI (1980a) Studies on the lysyl hydroxylase reaction. I. Initial velocity kinetics and related aspects. Biochim.Biophys.Acta 611(1): 40-50.
Puistola U, Turpeenniemi-Hujanen TM, Myllylä R & Kivirikko KI (1980b) Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism. Biochim.Biophys.Acta 611(1): 51-60.
Putnam AJ, Schultz K & Mooney DJ (2001) Control of microtubule assembly by extracellular matrix and externally applied strain. Am.J.Physiol.Cell.Physiol. 280(3): C556-64.
Rautavuoma K, Passoja K, Helaakoski T & Kivirikko KI (2000) Complete exon-intron organization of the gene for human lysyl hydroxylase 3 (LH3). Matrix Biol. 19(1): 73-79.
Rautavuoma K, Takaluoma K, Passoja K, Pirskanen A, Kvist AP, Kivirikko KI & Myllyharju J (2002) Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity. J.Biol.Chem. 277(25): 23084-23091.
Rautavuoma K, Takaluoma K, Sormunen R, Myllyharju J, Kivirikko KI & Soininen R (2004) Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice. Proc.Natl.Acad.Sci.U.S.A. 101(39): 14120-14125.
Reiser K, McCormick RJ & Rucker RB (1992) Enzymatic and nonenzymatic cross-linking of collagen and elastin. FASEB J. 6(7): 2439-2449.
Ricard-Blum S, Bresson-Hadni S, Vuitton DA, Ville G & Grimaud JA (1992) Hydroxypyridinium collagen cross-links in human liver fibrosis: study of alveolar echinococcosis. Hepatology 15(4): 599-602.
Ricard-Blum S, Esterre P & Grimaud JA (1993) Collagen cross-linking by pyridinoline occurs in non-reversible skin fibrosis. Cell.Mol.Biol.(Noisy-le-grand) 39(7): 723-727.
Ricard-Blum S & Ruggiero F (2005) The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol.Biol.(Paris) 53(7): 430-442.
82
Richards AA, Stephens T, Charlton HK, Jones A, Macdonald GA, Prins JB & Whitehead JP (2006) Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications. Mol.Endocrinol. 20(7): 1673-1687.
Risteli J & Kivirikko KI (1974) Activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in the liver of rats with hepatic injury. Biochem.J. 144(1): 115-122.
Risteli L (1978) Further characterization of collagen galactosyltransferase from chick embryos. Biochem.J. 169(1): 189-196.
Risteli L, Myllylä R & Kivirikko KI (1976) Partial purification and characterization of collagen galactosyltransferase from chick embryos. Biochem.J. 155(1): 145-153.
Risteli L, Risteli J, Ihme A, Krieg T & Müller PK (1980) Preferential hydroxylation of type IV collagen by lysyl hydroxylase from Ehlers-Danlos syndrome type VI fibroblasts. Biochem.Biophys.Res.Commun. 96(4): 1778-1784.
Robins SP (1999) Fibrillogenesis and Maturation of Collagens. In: Seibel M, Robins SP & Bilezikian J (eds) Dynamics of Bone and Cartilage Metabolism. London: Academic Press, 31-42.
Robins SP & Brady JD (2002) Collagen Cross-linking and Metabolism. In: Bilezikian J, Raisz L & Rodan G (eds) Principles of Bone Biology. London: Academic Press, 211-223.
Rosenberry TL & Richardson JM (1977) Structure of 18S and 14S acetylcholinesterase. Identification of collagen-like subunits that are linked by disulfide bonds to catalytic subunits. Biochemistry 16(16): 3550-3558.
Rothmann AB, Mortensen HD, Holmskov U & Hojrup P (1997) Structural characterization of bovine collectin-43. Eur.J.Biochem. 243(3): 630-635.
Ruotsalainen H, Sipilä L, Kerkelä E, Pospiech H & Myllylä R (1999) Characterization of cDNAs for mouse lysyl hydroxylase 1, 2 and 3, their phylogenetic analysis and tissue-specific expression in the mouse. Matrix Biol. 18(3): 325-329.
Ruotsalainen H, Sipilä L, Vapola M, Sormunen R, Salo AM, Uitto L, Mercer DK, Robins SP, Risteli M, Aszodi A, Fässler R & Myllylä R (2006) Glycosylation catalyzed by lysyl hydroxylase 3 is essential for basement membranes. J.Cell.Sci. 119(Pt 4): 625-635.
Ruotsalainen H, Vanhatupa S, Tampio M, Sipilä L, Valtavaara M & Myllylä R (2001) Complete genomic structure of mouse lysyl hydroxylase 2 and lysyl hydroxylase 3/collagen glucosyltransferase. Matrix Biol. 20(2): 137-146.
Ryhänen L (1975) Hydroxylation of lysyl residues in lysine-rich and arginine-rich histones by lysyl hydroxylase in vitro. Biochim.Biophys.Acta 397(1): 50-57.
Ryhänen L & Kivirikko KI (1974) Developmental changes in protocollagen lysyl hydroxylase activity in the chick embryo. Biochim.Biophys.Acta 343(1): 121-128.
Sabatelli P, Bonaldo P, Lattanzi G, Braghetta P, Bergamin N, Capanni C, Mattioli E, Columbaro M, Ognibene A, Pepe G, Bertini E, Merlini L, Maraldi NM & Squarzoni S (2001) Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts. Matrix Biol. 20(7): 475-486.
83
Salo AM, Sipilä L, Sormunen R, Ruotsalainen H, Vainio S & Myllylä R (2006a) The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult. Matrix Biol. 25(8): 475-483.
Salo AM, Wang C, Sipilä L, Sormunen R, Vapola M, Kervinen P, Ruotsalainen H, Heikkinen J & Myllylä R (2006b) Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling. J.Cell.Physiol. 207(3): 644-653.
Sambrook J & Russel DW (2001) Molecular Cloning, A laboratory manual. 3rd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Samimi A & Last JA (2001) Mechanism of inhibition of lysyl hydroxylase activity by the organophosphates malathion and malaoxon. Toxicol.Appl.Pharmacol. 176(3): 181-186.
Sammon JW, Jr. (1969) A Nonlinear Mapping for Data Structure Analysis. Computers, IEEE Transactions on. Computers, IEEE Transactions on C-18(5): 401-409.
Sano K, Fisher J, Mason RJ, Kuroki Y, Schilling J, Benson B & Voelker D (1987) Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A). Biochem.Biophys.Res.Commun. 144(1): 367-374.
Savolainen ER, Kero M, Pihlajaniemi T & Kivirikko KI (1981) Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex. N.Engl.J.Med. 304(4): 197-204.
Scheurer SB, Rybak JN, Rosli C, Neri D & Elia G (2004) Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: A transcriptomic and proteomic study. Proteomics 4(6): 1737-1760.
Schneider VA & Granato M (2007) Genomic structure and embryonic expression of zebrafish lysyl hydroxylase 1 and lysyl hydroxylase 2. Matrix Biol. 26(1): 12-19.
Schneider VA & Granato M (2006) The myotomal diwanka (lh3) glycosyltransferase and type XVIII collagen are critical for motor growth cone migration. Neuron 50(5): 683-695.
Schwarz U, Schuppan D, Oberbaumer I, Glanville RW, Deutzmann R, Timpl R & Kühn K (1986) Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha2(IV) chain and its comparison with the alpha1(IV) chain. European Journal of Biochemistry 157(1): 49-56.
Sellar GC, Blake DJ & Reid KB (1991) Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q. Biochem.J. 274( Pt 2): 481-490.
Shinkai H & Yonemasu K (1979) Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens. Biochem.J. 177(3): 847-852.
Sipilä L, Ruotsalainen H, Sormunen R, Baker NL, Lamandé SR, Vapola M, Wang C, Sado Y, Aszodi A & Myllylä R (2007) Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines. J.Biol.Chem. 282(46):33381-33388
Sipilä L, Szatanik M, Vainionpää H, Ruotsalainen H, Myllylä R & Guenet JL (2000) The genes encoding mouse lysyl hydroxylase isoforms map to chromosomes 4, 5, and 9. Mamm.Genome 11(12): 1132-1134.
84
Snellman A, Tuomisto A, Koski A, Latvanlehto A & Pihlajaniemi T (2007) The role of disulfide bonds and alpha-helical coiled-coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins. J.Biol.Chem. 282(20): 14898-14905.
Söderberg L, Kakuyama H, Moller A, Ito A, Winblad B, Tjernberg LO & Naslund J (2005) Characterization of the Alzheimer's disease-associated CLAC protein and identification of an amyloid beta-peptide-binding site. J.Biol.Chem. 280(2): 1007-1015.
Sonck CE (1948) On the aetiology of epidermolysis bullosa hereditaria. Acta Derm.Venereol. 28(5): 498-506.
Spiess J & Noe BD (1985) Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28. Proc.Natl.Acad.Sci.U.S.A. 82(2): 277-281.
Spiro RG (1969) Characterization and quantitative determination of the hydroxylysine-linked carbohydrate units of several collagens. J.Biol.Chem. 244(4): 602-612.
Spiro RG (1967) The structure of the disaccharide unit of the renal glomerular basement membrane. J.Biol.Chem. 242(20): 4813-4823.
Sreerama N, Venyaminov SY & Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal.Biochem. 287(2): 243-251.
Sreerama N & Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal.Biochem. 287(2): 252-260.
Steinmann B, Eyre DR & Superti-Furga A (2002) The Ehlers-Danlos syndrome. In: Royce PM & Steinmann B (eds) Connective tissue and its heritable disorders. 2nd ed. New York: Wiley-Liss Inc, 431-523.
Suokas M, Lampela O, Juffer AH, Myllylä R & Kellokumpu S (2003) Retrieval-independent localization of lysyl hydroxylase in the endoplasmic reticulum via a peptide fold in its iron-binding domain. Biochem.J. 370(Pt 3): 913-920.
Suokas M, Myllylä R & Kellokumpu S (2000) A single C-terminal peptide segment mediates both membrane association and localization of lysyl hydroxylase in the endoplasmic reticulum. J.Biol.Chem. 275(23): 17863-17868.
Sussman M, Lichtenstein JR, Nigra TP, Martin GR & McKusick VA (1974) Hydroxylysine-deficient skin collagen in a patient with a form of the Ehlers-Danlos syndrome. J.Bone Joint Surg.Am. 56(6): 1228-1234.
Szpirer C, Szpirer J, Riviere M, Vanvooren P, Valtavaara M & Myllylä R (1997) Localization of the gene encoding a novel isoform of lysyl hydroxylase. Mamm.Genome 8(9): 707-708.
Takaluoma K, Hyry M, Lantto J, Sormunen R, Bank RA, Kivirikko KI, Myllyharju J & Soininen R (2007a) Tissue-specific changes in the hydroxylysine content and cross-links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock-out mice. J.Biol.Chem. 282(9): 6588-6596.
Takaluoma K, Lantto J & Myllyharju J (2007b) Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences. Matrix Biol. 26(5): 396-403
85
Tanzer ML (2006) Current concepts of extracellular matrix. J.Orthop.Sci. 11(3): 326-331. Tenni R, Sonaggere M, Viola M, Bartolini B, Tira ME, Rossi A, Orsini E, Ruggeri A &
Ottani V (2006) Self-aggregation of fibrillar collagens I and II involves lysine side chains. Micron 37(7): 640-647.
Tenni R, Valli M, Rossi A & Cetta G (1993) Possible role of overglycosylation in the type I collagen triple helical domain in the molecular pathogenesis of osteogenesis imperfecta. Am.J.Med.Genet. 45(2): 252-256.
Tenni R, Viola M, Welser F, Sini P, Giudici C, Rossi A & Tira ME (2002) Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen. Eur.J.Biochem. 269(5): 1428-1437.
Tom Tang Y, Hu T, Arterburn M, Boyle B, Bright JM, Palencia S, Emtage PC & Funk WD (2005) The complete complement of C1q-domain-containing proteins in Homo sapiens. Genomics 86(1): 100-111.
Torre-Blanco A, Adachi E, Hojima Y, Wootton JA, Minor RR & Prockop DJ (1992) Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils. J.Biol.Chem. 267(4): 2650-2655.
Trackman PC (2005) Diverse biological functions of extracellular collagen processing enzymes. J.Cell.Biochem. 96(5): 927-937.
Trojanowska M, LeRoy EC, Eckes B & Krieg T (1998) Pathogenesis of fibrosis: type 1 collagen and the skin. J.Mol.Med. 76(3-4): 266-274.
Tu H, Sasaki T, Snellman A, Gohring W, Pirilä P, Timpl R & Pihlajaniemi T (2002) The Type XIII Collagen Ectodomain Is a 150-nm Rod and Capable of Binding to Fibronectin, Nidogen-2, Perlecan, and Heparin. J. Biol. Chem. 277(25): 23092-23099.
Turpeenniemi-Hujanen TM, Puistola U & Kivirikko KI (1981) Human lysyl hydroxylase: purification to homogeneity, partial characterization and comparison of catalytic properties with those of a mutant enzyme from Ehlers-Danlos syndrome type VI fibroblasts. Coll.Relat.Res. 1(4): 355-366.
Turpeenniemi-Hujanen TM, Puistola U & Kivirikko KI (1980) Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein. Biochem.J. 189(2): 247-253.
Uitto J, Richard G & McGrath JA (2007) Diseases of epidermal keratins and their linker proteins. Exp.Cell Res. 313(10): 1995-2009.
Ünligil UM, Zhou S, Yuwaraj S, Sarkar M, Schachter H & Rini JM (2000) X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J. 19(20): 5269-5280.
Uzawa K, Grzesik WJ, Nishiura T, Kuznetsov SA, Robey PG, Brenner DA & Yamauchi M (1999) Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro. J.Bone Miner.Res. 14(8): 1272-1280.
86
Uzawa K, Yeowell HN, Yamamoto K, Mochida Y, Tanzawa H & Yamauchi M (2003) Lysine hydroxylation of collagen in a fibroblast cell culture system. Biochem.Biophys.Res.Commun. 305(3): 484-487.
Valtavaara M (1999) Novel lysyl hydroxylase isoforms. Acta Univ Oul (A334): 1-63. Valtavaara M, Papponen H, Pirttilä AM, Hiltunen K, Helander H & Myllylä R (1997)
Cloning and characterization of a novel human lysyl hydroxylase isoform highly expressed in pancreas and muscle. J.Biol.Chem. 272(11): 6831-6834.
Valtavaara M, Szpirer C, Szpirer J & Myllylä R (1999) Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3). J.Biol.Chem. 275: 20956.
Valtavaara M, Szpirer C, Szpirer J & Myllylä R (1998) Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3). J.Biol.Chem. 273(21): 12881-12886.
van de Wetering JK, van Golde LM & Batenburg JJ (2004) Collectins: players of the innate immune system. Eur.J.Biochem. 271(7): 1229-1249.
Van den Steen PE, Proost P, Brand DD, Kang AH, Van Damme J & Opdenakker G (2004) Generation of glycosylated remnant epitopes from human collagen type II by gelatinase B. Biochemistry 43(33): 10809-10816.
van der Slot AJ, van Dura EA, de Wit EC, De Groot J, Huizinga TW, Bank RA & Zuurmond AM (2005) Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels. Biochim.Biophys.Acta 1741(1-2): 95-102.
van der Slot AJ, Zuurmond AM, Bardoel AF, Wijmenga C, Pruijs HE, Sillence DO, Brinckmann J, Abraham DJ, Black CM, Verzijl N, DeGroot J, Hanemaaijer R, TeKoppele JM, Huizinga TW & Bank RA (2003) Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis. J.Biol.Chem. 278(42): 40967-40972.
van der Slot AJ, Zuurmond AM, van den Bogaerdt AJ, Ulrich MM, Middelkoop E, Boers W, Karel Ronday H, DeGroot J, Huizinga TW & Bank RA (2004) Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon. Matrix Biol. 23(4): 251-257.
van der Slot-Verhoeven AJ, van Dura EA, Attema J, Blauw B, Degroot J, Huizinga TW, Zuurmond AM & Bank RA (2005) The type of collagen cross-link determines the reversibility of experimental skin fibrosis. Biochim.Biophys.Acta 1740(1): 60-67.
Vanacore RM, Friedman DB, Ham AJ, Sundaramoorthy M & Hudson BG (2005) Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly. J.Biol.Chem. 280(32): 29300-29310.
Veit G, Kobbe B, Keene DR, Paulsson M, Koch M & Wagener R (2006) Collagen XXVIII, a novel von Willebrand factor A domain-containing protein with many imperfections in the collagenous domain. J.Biol.Chem. 281(6): 3494-3504.
87
Velling T, Risteli J, Wennerberg K, Mosher DF & Johansson S (2002) Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1. J.Biol.Chem. 277(40): 37377-37381.
Viola M, Bartolini B, Sonaggere M, Giudici C, Tenni R & Tira ME (2007) Fibromodulin interactions with type I and II collagens. Connect.Tissue Res. 48(3): 141-148.
Vogel W, Gish GD, Alves F & Pawson T (1997) The discoidin domain receptor tyrosine kinases are activated by collagen. Mol.Cell 1(1): 13-23.
Walker LC, Marini JC, Grange DK, Filie J & Yeowell HN (1999) A patient with Ehlers-Danlos syndrome type VI is homozygous for a premature termination codon in exon 14 of the lysyl hydroxylase 1 gene. Mol.Genet.Metab. 67(1): 74-82.
Walker LC, Overstreet MA, Siddiqui A, De Paepe A, Ceylaner G, Malfait F, Symoens S, Atsawasuwan P, Yamauchi M, Ceylaner S, Bank RA & Yeowell HN (2005) A novel mutation in the lysyl hydroxylase 1 gene causes decreased lysyl hydroxylase activity in an Ehlers-Danlos VIA patient. J.Invest.Dermatol. 124(5): 914-918.
Walker LC, Overstreet MA, Willing MC, Marini JC, Cabral WA, Pals G, Bristow J, Atsawasuwan P, Yamauchi M & Yeowell HN (2004) Heterogeneous basis of the type VIB form of Ehlers-Danlos syndrome (EDS VIB) that is unrelated to decreased collagen lysyl hydroxylation. Am.J.Med.Genet.A. 131(2): 155-162.
Walker LC, Overstreet MA & Yeowell HN (2005) Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts. Matrix Biol. 23(8): 515-523.
Walker LC, Teebi AS, Marini JC, De Paepe A, Malfait F, Atsawasuwan P, Yamauchi M & Yeowell HN (2004) Decreased expression of lysyl hydroxylase 2 (LH2) in skin fibroblasts from three Ehlers-Danlos patients does not result from mutations in either the coding or proximal promoter region of the LH2 gene. Mol.Genet.Metab. 83(4): 312-321.
Wang C, Kovanen V, Raudasoja P, Eskelinen S, Pospiech H & Myllylä R (2008) The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability. J.Cell.Mol.Med. 12(2): 1-18.
Wang C, Luosujärvi H, Heikkinen J, Risteli M, Uitto L & Myllylä R (2002) The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro. Matrix Biol. 21(7): 559-566.
Wang C, Valtavaara M & Myllylä R (2000) Lack of collagen type specificity for lysyl hydroxylase isoforms. DNA Cell Biol. 19(2): 71-77.
Wang Y, Lam KS, Chan L, Chan KW, Lam JB, Lam MC, Hoo RC, Mak WW, Cooper GJ & Xu A (2006) Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J.Biol.Chem. 281(24): 16391-16400.
Wang Y, Xu A, Knight C, Xu LY & Cooper GJ (2002) Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J.Biol.Chem. 277(22): 19521-19529.
88
Wei L, Altman R & Chang J (1997) Using the radial distributions of physical features to compare amino acid enviroments and align amino acid sequences. In: Dunker AK, Altman RB, Hunter L & Klein TE (eds) Pacific Symposium on Biocombuting. Hawaii: Word Scientific Publishing Co., 465-476.
Wu GY, Pereyra B & Seifter S (1981) Specificity of trypsin and carboxypeptidase B for hydroxylysine residues in denatured collagens. Biochemistry 20(15): 4321-4324.
Wu J, Reinhardt DP, Batmunkh C, Lindenmaier W, Far RK, Notbohm H, Hunzelmann N & Brinckmann J (2006) Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts. Exp.Cell Res. 312(18): 3485-3494.
Wu JJ & Eyre DR (1995) Structural analysis of cross-linking domains in cartilage type XI collagen. Insights on polymeric assembly. J.Biol.Chem. 270(32): 18865-18870.
Wynn TA (2004) Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat.Rev.Immunol. 4(8): 583-594.
Yamauchi M, London RE, Guenat C, Hashimoto F & Mechanic GL (1987) Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen. J.Biol.Chem. 262(24): 11428-11434.
Yeowell HN, Allen JD, Walker LC, Overstreet MA, Murad S & Thai SF (2000) Deletion of cysteine 369 in lysyl hydroxylase 1 eliminates enzyme activity and causes Ehlers-Danlos syndrome type VI. Matrix Biol. 19(1): 37-46.
Yeowell HN, Ha V, Walker LC, Murad S & Pinnell SR (1992) Characterization of a partial cDNA for lysyl hydroxylase from human skin fibroblasts; lysyl hydroxylase mRNAs are regulated differently by minoxidil derivatives and hydralazine. J.Invest.Dermatol. 99(6): 864-869.
Yeowell HN & Walker LC (2000) Mutations in the lysyl hydroxylase 1 gene that result in enzyme deficiency and the clinical phenotype of Ehlers-Danlos syndrome type VI. Mol.Genet.Metab. 71(1-2): 212-224.
Yeowell HN & Walker LC (1999) Tissue specificity of a new splice form of the human lysyl hydroxylase 2 gene. Matrix Biol. 18(2): 179-187.
Yeowell HN & Walker LC (1997) Ehlers-Danlos syndrome type VI results from a nonsense mutation and a splice site-mediated exon-skipping mutation in the lysyl hydroxylase gene. Proc.Assoc.Am.Physicians 109(4): 383-396.
Yeowell HN, Walker LC, Farmer B, Heikkinen J & Myllylä R (2000) Mutational analysis of the lysyl hydroxylase 1 gene (PLOD) in six unrelated patients with Ehlers-Danlos syndrome type VI: prenatal exclusion of this disorder in one family. Hum.Mutat. 16(1): 90.
Yeowell HN, Walker LC & Neumann LM (2005) An Ehlers-Danlos syndrome type VIA patient with cystic malformations of the meninges. Eur.J.Dermatol. 15(5): 353-358.
Ying SC, Gewurz AT, Jiang H & Gewurz H (1993) Human serum amyloid P component oligomers bind and activate the classical complement pathway via residues 14-26 and 76-92 of the A chain collagen-like region of C1q. J.Immunol. 150(1): 169-176.
Yurchenco PD, Amenta PS & Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol. 22(7): 521-538.
89
Zuurmond AM, van der Slot-Verhoeven AJ, van Dura EA, De Groot J & Bank RA (2005) Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis. Matrix Biol. 24(4): 261-270.
90
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Original articles
I Risteli M, Niemitalo O, Lankinen H, Juffer AH & Myllylä R (2004) Characterization of collagenous peptides bound to lysyl hydroxylase isoforms. J Biol Chem 279(36): 37535–37543.
II Heikkinen J, Risteli M, Wang C, Latvala J, Rossi M, Valtavaara M & Myllylä R (2000) Lysyl hydroxylase is a multifunctional protein possessing collagen glucosyltransferase activity. J Biol Chem 275(46): 36158–36163.
III Wang C, Risteli M, Heikkinen J, Hussa AK, Uitto L & Myllylä R (2002) Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3). J Biol Chem 277(21): 18568–18573.
IV Risteli M, Ruotsalainen H, Salo AM, Sormunen R, Baker NL, Lamandé SR, Vimpari-Kauppinen L & Myllylä R (2008) Decreased lysyl hydroxylase 3 (LH3) activity in a family with dominant epidermolysis bullosa simplex causes abnormalities in deposition and organization of extracellular matrix. Manuscript.
Reprinted with permission from The American Society for Biochemistry and Molecular Biology, Inc. (I-III).
Original publications are not included in the electronic version of the dissertation.
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493. Donnini, Serena (2007) Computing free energies of protein-ligand association
494. Syrjänen, Anna-Liisa (2007) Lay participatory design: A way to developinformation technology and activity together
495. Partanen, Sari (2007) Recent spatiotemporal changes and main determinants ofaquatic macrophyte vegetation in large lakes in Finland
496. Vuoti, Sauli (2007) Syntheses and catalytic properties of palladium (II) complexesof various new aryl and aryl alkyl phosphane ligands
497. Alaviuhkola, Terhi (2007) Aromatic borate anions and thiophene derivatives forsensor applications
498. Törn, Anne (2007) Sustainability of nature-based tourism
499. Autio, Kaija (2007) Characterization of 3-hydroxyacyl-ACP dehydratase ofmitochondrial fatty acid synthesis in yeast, humans and trypanosomes
500. Raunio, Janne (2008) The use of Chironomid Pupal Exuvial Technique (CPET) infreshwater biomonitoring: applications for boreal rivers and lakes
501. Paasivaara, Antti (2008) Space use, habitat selection and reproductive output ofbreeding common goldeneye (Bucephala clangula)
502. Asikkala, Janne (2008) Application of ionic liquids and microwave activation inselected organic reactions
503. Rinta-aho, Marko (2008) On monomial exponential sums in certain index 2 casesand their connections to coding theory
504. Sallinen, Pirkko (2008) Myocardial infarction. Aspects relating to endogenous andexogenous melatonin and cardiac contractility
505. Xin, Weidong (2008) Continuum electrostatics of biomolecular systems
506. Pyhäjärvi, Tanja (2008) Roles of demography and natural selection in molecularevolution of trees, focus on Pinus sylvestris
507. Kinnunen, Aulis (2008) A Palaeoproterozoic high-sulphidation epithermal golddeposit at Orivesi, southern Finland
508. Alahuhta, Markus (2008) Protein crystallography of triosephosphate isomerases:functional and protein engineering studies
509. Reif, Vitali (2008) Birds of prey and grouse in Finland. Do avian predators limit orregulate their prey numbers?
A511etukansi.fm Page 2 Thursday, May 29, 2008 10:43 AM
U N I V E R S I TAT I S O U L U E N S I SACTAA
SCIENTIAE RERUMNATURALIUM
OULU 2008
A 511
Maija Risteli
SUBSTRATE SPECIFICITYOF LYSYL HYDROXYLASE ISOFORMS AND MULTI-FUNCTIONALITY OF LYSYL HYDROXYLASE 3
FACULTY OF SCIENCE,DEPARTMENT OF BIOCHEMISTRY,BIOCENTER OULU,UNIVERSITY OF OULU;FINNISH NATIONAL GLYCOSCIENCE GRADUATE SCHOOL
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UNIVERS ITY OF OULU P.O. Box 7500 F I -90014 UNIVERS ITY OF OULU F INLAND
A C T A U N I V E R S I T A T I S O U L U E N S I S
S E R I E S E D I T O R S
SCIENTIAE RERUM NATURALIUM
HUMANIORA
TECHNICA
MEDICA
SCIENTIAE RERUM SOCIALIUM
SCRIPTA ACADEMICA
OECONOMICA
EDITOR IN CHIEF
EDITORIAL SECRETARY
Professor Mikko Siponen
Professor Harri Mantila
Professor Hannu Heusala
Professor Olli Vuolteenaho
Senior Researcher Eila Estola
Information officer Tiina Pistokoski
Senior Lecturer Seppo Eriksson
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-951-42-8828-9 (Paperback)ISBN 978-951-42-8829-6 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)
A 511
ACTA
Maija R
isteliA511etukansi.fm Page 1 Thursday, May 29, 2008 10:43 AM