essentials of glycobiology lecture 21 may 4, 2004 richard d. cummings, ph.d
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ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 4, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma Center for Medical Glycobiology “GALECTINS”. Dr. Cummings. Outline. Historical Background - PowerPoint PPT PresentationTRANSCRIPT
ESSENTIALS OF GLYCOBIOLOGY
LECTURE 21
MAY 4, 2004
Richard D. Cummings, Ph.D.University of Oklahoma Health Sciences Center
College of MedicineOklahoma Center for Medical Glycobiology
“GALECTINS”
Dr. Cummings
Historical Background Definition of the Galectins and Sequence Motifs Different Subfamilies of Galectins
Prototype Galectins (Monomers)Chimeric GalectinsTandem Repeat Galectins
Tertiary/Quaternary Structures of Galectins Galectins in Diverse Species: From Early Metazoans
to Plants to Humans Biosynthesis and Secretion of Galectins Carbohydrate Ligands for Galectins Functions of Galectins
Intracellular FunctionsExtracellular Functions
Outline
Dr. Cummings
First discovered in electric organ of electric eel by Teichberg et al in 1975 as an agarose-binding protein capable of agglutinating trypsin-treated rabbit erythrocytes
Electrolectin required reducing conditions during storage for retention of activity
Protein with similar properties subsequently isolated from bovine and chicken organs (termed L14) now called galectin-1
A larger protein (termed CBP35 and L-29) now called galectin-3 isolated from murine fibroblasts
Other related proteins identified in many organisms, including C. elegans, zebrafish (Danio rerio), Drosophila, and sponges
All members share sequence termed S-type (thiol) carbohydrate-recognition domain (CRD) recognized by Drickamer in 1988
Group subsequently renamed galectins, since all members appear to recognize terminal, non-reducing galactose residues and have the ability to agglutinate cells
Prototypical Galectins: The Galactose-binding Lectins (Galectin-1 and -3)
Historical Background
Dr. Cummings
-WGTEQREAV--FPFQPGSVAEVCITFDQANLT---VKLPDGYEFKFPNRL-
-WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFSFPNRL-
Human Galectin-1
Chicken 14K Galectin
69
— H— —N— —R—X —V— —N— —X —W— —X— —X
FMLCVI
PLVAHI
5-10 4
CFRMNL
ST
GEK
EQ
70
— —X — — — —X— —X — — —3-6
RKE
PCTF
LIVMF
NQEGSKV
GH 3
DENKHS
LIVMFC
Conserved Carbohydrate-Recognition Domain (CRD) of Galectins
Definition of the Galectins and Sequence Motifs
Dr. Cummings
Different Subfamilies of Galectins
Dr. Cummings
Different Subfamilies of Galectins
Dr. Cummings
Crystal Structure (1.7 Å) of Dimeric Human Galectin-1 With Bound Lactose
Sideview Turned 90˚
Galectins have a highly conserved secondary structure with internally oriented hydrophobic residues in ß strands in the ß-sandwich of the galectin fold (Lobsanov et al., 1993; Liao et al., 1994; Leonidas et al., 1998).
Structures of Galectins
Dr. Cummings
NH2
NH2
COO-
COO-
Bovine Galectin-1 DimerCon A Dimer
Comparative Structures of a Leguminous Plant Lectin Con A (an L-type Lectin) and Human Galectin-1
Both -barrel proteins with no -helix
Structures of Galectins
Dr. Cummings
W69
H45
H2O
N47
R49
H53D55
R74
E72
N62
Amino Acids in Human Galectin-1 That Interact with Lactose
With Lactose Without Lactose
W69
H45
N47
R49
H53D55
R74
E72
N62
Structures of Galectins
Dr. Cummings
H2O H2O
Galectins in Diverse Species: From Early Metazoans to Plants to Humans
Dr. Cummings
From: The Website of Dr. Kurt Drickamer at http://ctld.glycob.ox.ac.uk/ctld/classes/Galectin2.html
Cytosol
Extracellular
mRNA
Kd ~1M
SecretionMechanism?
Dimer Inactive Forms
Dimer
Kd ~7M
3’
?
Monomer
GlycoproteinLigand
Monomer 5’
“Metastable Intermediate”
N
Biosynthesis and Secretion of Galectins
*
(Galectins lack a signal sequence)Dr. Cummings
Require reducing conditions for activity
Occur only as soluble proteins
Bind terminal Gal residues
Not post-translationallymodification, other than N-terminal acetylation
Can retain activity without reducing conditions in presence of ligands
Spliced forms may generate membrane-anchored proteins
Bind GalNAc, GlcNAc, and Gal at internal and terminal positions, and sialylated Gal(NAc)
Some galectins are phosphorylated, glutathionylated, or cross-linked by transglutaminase
New Info about GalectinsOld Galectin Dogma
Biosynthesis and Secretion of Galectins
Dr. Cummings
Carbohydrate Ligands for Galectins
Examples of Candidate Macromolecular Ligands
LamininFibronectinLysosome-associated membrane glycoproteins (LAMPs)CD7CD43CD45
Glycans
RelativeBinding
Affinity To Galectin-1
2 >20>2021 2
Dr. Cummings
Extracellular Galectin
CELL
CELL
Intracellular Galectin
Functions of Galectins
Extracellular Matrix
Dr. Cummings
Cell-cell adhesion(galectins can agglutinate cells)
Cell-matrix interaction(galectins can link cells to extracellular matrix (ECM) in anintegrin- and Ca2+-independent fashion)
RNA transport and splicing
Cytoskeletal organization
Many galectins are upregulated in tumor cells
Functions of Galectins
Dr. Cummings
Cell signalingGrowth arrest (galectins can arrest growth of embryonic
fibroblasts)Mitogenesis (galectins can stimulate growth of some cells, e.g.
lymphocytes)Apoptosis
Galectins can induce death of some cells (in some cases this appears to be by apoptotic
processes) Galectins can also suppress apoptosis in some cells Galectins can also induce exposure of
phosphatidylserine (PS)on cell surfaces - often used as a marker of apoptosis)Phagocytosis and Clearance (galectin induction of PS
can lead to recognition and phagocytosis of cells by professional phagocytes
Functions of Galectins
Dr. Cummings
Potential Involvement of Galectins in Immune Regulation and Cancer
Galectin-1 may be a negative regulator of immune responseinduces death of T- cellsinhibits cell growthInhibits cell-matrix interactions
In experimental animal models of inflammation galectin-1 reduces tissue injury
Galectin-1, -7, -8, -9, and -12 all are reported have apoptosis-inducing activity to certain types of cells
By contrast, Galectin-3 has proinflammatory properties.inhibits T cell deathmay interact with bcl-2 to promote anti-apoptotic activity (Gal-3 contains the antideath Asp-Trp-Gly- Arg (NWGR) motif that is conserved in the Bcl-2 homology domain (BH1) of the Bcl-2 family)
promotes T cell-matrix interactionsis chemoattractantpromotes leukocyte activation
Functions of Galectins
Dr. Cummings
Potential Involvement of Galectins in Immune Regulation and Cancer
Galectin-3 null mice have reduced leukocyte infiltration in experimental models of inflammation (peritoneal inflammation)
Macrophages from Galectin-3 null mice have reduced phagocytic activity, associated with reduced intracellular Galectin-3 levels
Functions of Galectins
Dr. Cummings
(LEFT FIGURE) Reduced phagocytosis of apoptotic thymocytes by gal3–/– macrophages. (a) In vitro phagocytosis of apoptotic thymocytes by macrophages was performed and the phagocytic index was calculated following 20-minute incubation of these cells. Data shown represent the mean ± SD from six experiments (P < 0.001). (b) Scatter plots of the percentage of macrophages containing phagocytosed apoptotic thymocytes and/or apoptotic bodies 30 minutes after injection of the cells into the peritoneum of wild-type and gal3–/– mice with sterile peritonitis, shown as mean (heavy horizontal lines) ± SD. Each data point represents the result from one mouse; seven mice of each genotype were used (P < 0.01). (RICHT FIGURE) Distribution of galectin-3 (upper panels; green) and F-actin (middle panels; red) inside wild-type macrophages. An overlay of these images demonstrates colocalization of F-actin and galectin-3 (lower panels; yellow). Panels on the left show wild-type macrophages before stimulation and those on the right show 1 minute after stimulation with opsonized srbc’s.(From Sano et al (2003) J Clin Invest. 2003 Aug;112(3):389-97)
Potential Involvement of Galectins in Immune Regulation and Cancer
Galectin-3 null mice have abnormalities in chondrocyte proliferation and extracellular matrix in the the hypertrophic zone of long bones, associated with decreased chondrocyte survival/turnover
Galectin-3 overexpression correlated with enhanced metastatic potential - implicated in many aspects of tumor biology
While Galectin-4 is normally expressed primarily in the alimentary tract, it shows strong expression of galectin-4 in cancers from other tissues including breast and liver
Galectin-9 (also called ecalectin) was identified as a T cell-derived potent eosinophil chemoattractant
Functions of Galectins
Dr. Cummings
Galectin Cross-Linking and Signal Transduction
Monomeric Mutants of Galectin Lack Signaling Functions!
Functions of Galectins
Dr. Cummings
Annexin V-FITCStaining
Hoechst StainingFor DNA
Uniform Exposure of Phosphatidylserine (PS) in Apoptotic Neutrophils
Example of a Merged Image Single Cell
Functions of Galectins
Dr. Cummings
From: Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93
Annexin V is a 35 kDa protein with a high affinity for PS (Kd estimated at 5 x 10-10 M )
Polarized Exposure of PS in Galectin-1-Treated Activated Neutrophils
Annexin V-FITCStaining
Hoechst Staining
Example of a Merged Image Single Cell
Functions of Galectins
Dr. Cummings
From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93
0 10 20 30 40
Resting Neutrophils
Activated Neutrophils
Activated Neutrophils + dGal-1
Activated Neutrophils + dGal-1 + Lactose
Activated Neutrophils + mGal-1
Aged Neutrophils
% Phagocytosis
0 10 20 30 40
Untreated dsHL-60
dsHL-60 + dGal-1
dsHL-60 + dGal-1 + Lactose
dsHL-60 + mGal-1
HL-60 + Camptothecin
Aged Neutrophils
A
B
Praeparesis of Human Leukocytes Induced with Dimeric, but not Monomeric, Human Galectin-1 Enhances Their Phagocytosis by Activated Macrophages
Human Neutrophils
HL-60 Cells
% Phagocytosis
Untreated
+ dGal-1
+ dGal-1 + Lactose
+ mGal-1+ Camptothecin
Aged Neutrophils
Resting Cells
Activated CellsActivated Cells + dGal-1
Activated Cells + dGal-1 + Lactose
Activated Cells + mGal-1Aged Neutrophils
dGal-1 = Dimeric Gal-1
mGal-1 = Monomeric Gal-1
Background
From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93
Functions of Galectins
Functions of Galectins
Galectins and Their Effects on Cellular Homeostasis
From: Hsu and Liu (2004) Glycoconjugate J. 19, 507–515
Dr. Cummings
Functions of Galectins