laminin tuesday, feb. 15 group three: claire paulson, andrew kalas, jacquie fable & chris mckay
TRANSCRIPT
Laminin
Tuesday, Feb. 15
Group Three:
Claire Paulson, Andrew Kalas, Jacquie Fable & Chris McKay
Objectives
Predict what would occur in the absence of the basement membrane. (Level 2, Understand)
Compare and contrast, in both structure and function, Laminin with other important ECM proteins (Level 4, Analyze)
Design a potential treatment that could be applied to a patient with a laminin deficiency disease. (Level 6, Create)
Figure 19-43 Molecular Biology of the Cell (© Garland Science 2008)
Laminin in the ECM
Introduction to the Basal lamina Cell Signaling
Presents a microenvironment Tissue development, repair, normal homeostasis Errors can result in cancer, diabetes and
autoimmune complications Proteoglycans vs. glycoproteins?
Ratio of protein to carbohydrates present
Biological Structure of the Basal Lamina
The basement membrane is the earliest portion of the extracellular matrix formed during embryogenesis.
Provides signals to adjacent cells
Force-driven signals originating between the basal lamina components and linked cell adhesion receptors (integrins) is communicated to the interior of cells through a mechanotransduction system to influence cell polarity, metabolism, fate, and migration.
Overall function of basal lamina The basal lamina is a layer
of the ECM that is secreted by epithelial and mesenchymal cells.
Found on the basal side of polarized epithelial cell sheets
separates them from the underlying connective tissue
Surround individual muscle cells, fat cells, and cells lining peripheral nerve cell axons (as Schwann cells)
Mainly serves as a base on which cells can grow
Figure 19-40 Molecular Biology of the Cell (© Garland Science 2008)
The basement membrane proteins laminin, collagen IV, nidogen/entactin, fibulin-1, perlecan and SPARC all bind integrins. Various integrins all bind to the regions indicated for the various BM components. The arrows indicate the sites at which the various BM proteins bind to each other.
Interactions with the Basal Lamina Binds integrins to signal to other cells
Loss of these signals leads to lethality of mouse embryos at the pre-implantation stage
Collagen (type IV) Insoluble Gives basal lamina strength
Nidogens (nidogen-1) Bind laminin to collagen Formation of the laminin/nidogen-1 complex would be a key event during basement membrane deposition andepithelial differentiation
Heparin Sulfate Binds ligands involved in metastasis and blood coagulation
Formation during Embryogenesis Polymerization of laminin and collagen IV in
two separate networks associated together by nidogen
The mechanism of basal lamina formation and insolubilisation of the different components into a basement membrane, in vivo, is unknown
Integrins are first and primary way to mediate cell-cell interaction
Compartmentalization of Tissue with the Basal Lamina
Separates connective tissue from the epithelia, nerves, and muscle tissue.
Controls interactions which influence cell polarity, differentiation and migration
(A) Mucosal melanosis of the lip. Numerous melanocytes above the basal lamina appear as a brown ribbon
(B) pseudostratified respiratory mucosa of the nose with predominantly ciliated cells. Goblet cells have clear cytoplasm. The basal cells (arrow) are lying on thin basal lamina
Distribution of proteoglycans
In vitro testing Amount of laminin, collagen,
nidogen, and heparin sulfate proteoglycans varies based on tissue
Cannot be isolated as a complete structure or reconstituted in vitro. Some components can be purified Isolated molecules lack the molecular
interactions that constitute basement membranes
Isoform composition of the four major basement membrane components varies with tissue type
Activity 1: Pick a tissue!
Muscl
e!
Kidne
y!Ski
n!
Brain
!
Activity 1 (10 min)
Predict what would happen in your tissue if the basal lamina was absent.
- Tissue structure- Boundaries- Abnormalities or benefits
Laminin’s Composition heterotrimeric
protein composed of various α, β, and γ subunits
most subunits contain similar LE (or EGF like) and LN domains
only the alpha subunit has LG domains
Structure
each subunit contains multiple 7 long amino acid sequences that enable the subunits to come together
the structure of the coiled coil maximizes the number of non-covalent bonds formed between the subunits and confers structural stability to the completed timer
once the coiled coil is formed it is covalently strengthened by disulfide bonds
Domains of the , , and γ chains LG domain can bind
various ECM molecules such as heparin, α dystroglycan , sulfatides, integrins, nidogen, and fibulin
LG domains are often found to be present in tandem which increases the efficiency of ligand binding
LN domains are primarily involved in binding to other laminins
Shapes different subunit types
create the diverse number of laminins (presently 18 are known)
not all the combinations of the three chains have been found to occur in living organisms
Some Biochemistry
LE Domain
-left, the classical look of disulfide connectivity based on X ray structures of the laminin γ1 chain (includes nidogen binding site)
-right, through mass spectometry they determined a different disulfide bonding patter of LE domains 1-4
this suggests that LE domains differing in function also differ in their disulfide patters
LG domain-Integrin Interactions the C terminus of the
β, and γ chains fine tunes the conformation of the LG domains so that they can interact with integrin
the dotted circle shows the integrin binding site
an aspartic acid residue found at the C terminus of the γ chain helps coordinate the divalent metal ion
Heterotrimer Formation
1) Glycosylation of subunits• Rough ER• stabilizes & protects
2) Association of and γ chains
3) chain combines with dimer• rate limiting step
4) Terminal glycosylation• Golgi
5) Trimer secreted from cell• Epithelial &
mesenchymal cells
6) Proteolytic processing
2
3
6
5So how do these laminin molecules form?
Discovery of Laminin Synthesis
the β and γ chains associate with one another first, followed by the α chain which combines with the dimeric complex
the α chains, the only chain which can be secreted free, drives secretion of paired βγ dimers
thus, the coiled coil domain of the long arms are crucial for assembly of the three chains of laminin
Polymerization Laminin-111 has been
the most thoroughly studied laminin and has helped understand how laminin contributes to the basement membrane architecture
it was determined that laminins interact with each other between the LN domains of the short arms thus forming a meshwork polymer
Receptor Mediated Laminin Assembly this model suggests that
laminin serves as an integrating receptor during its assembly into the basement membrane
by binding to integrin and dystroglycan, laminin can polymerize through short arm interactions and thus reorganize the receptors
this laminin/receptor network represents a widespread regulatory mechanism
Laminin Tissue Distribution 1 chain
embryogenesis: epithelial cells adult: liver, kidney, & reproductive organs
2 chain neuromuscular system
3 skin & epithelial cells
4 cells of mesenchymal origin vascular endothelial cells, cells derived from bone barrow
5 widely expressed epithelial, neuromuscular, vascular tissues embryogenesis
Laminin Processing
chains chains
γ chains
3A3
γ2
aka. Laminin 5 Skin, placental, mammary glands Hemidesmosomes, cellular migration
Processing of 3 chain unprocessed – migration processed – hemidesmosomes alters intergrin binding
Processing of γ2 chain DIII fragment binds erbB1 phosphorlyation of beta of 24
Marinkovich, 2007
Durbeej, 2010
Laminin Processing : Laminin-332
How is this possible?
!
Tzu & Markinkovich, 2008
Laminin-332 Where else is laminin-332
found? Cell migration
wound healing
What type of 3 chain?
Margadant & Sonnenberg, 2010
tumor invasion
Activity (min) Compare and contrast the major ECM proteins
we’ve learn about so far: collagen, fibronectin, & laminin
Collagen Fibronectin Laminin
Structure
Class of molecules
Processing
Connections within the ECM
Function
Importance in Embryogenesis
Location within the body
Associated diseases
Collagen Fibronectin Laminin
Structure
Class of molecules
Processing
Connections within the ECM
Function
Importance in Embryogenesis
Location within the body
Associated diseases
Trimeric triple helixGly-X-Y sequence
Trimeric, helical domain, globular ends, disulfide bonding
Dimer with modules I, II, III, disulfide bonding, sheets
Essential Essential -- Embryonic epithelia, placenta
Essential -- Blastocoele formation
proteins glycoproteinsglycoproteins
Pro- chain, glycosylation, Pro-collagen, N & C terminal cleaved collagen
Glycosylation, -γ dimer, addition chain, proteolytic cleavage
Dimers secreted
Integrins, MMPs, laminin
Collagen, integrins, dystroglycan, heparan sulfate proteinglycans
Cryptic sitesIntegrins, collagen, heparan , fibrin
Structure,/scaffolding signaling, movement, strength
Signaling, movement, tissue integrity, cell-matrix adhesion
Cell adhesion, cell growth, inhibition of apoptosis, movement, differentiation
ECM everywhere Basal lamina – separates epithelial from connective tissue
ECM everywhereBlood plasma
Chondroysplasias, osteoarthritis , osteoporosis, scurvy, lupus erythematosus
Muscular dystrophy, epidermolysis bullosa, cardiomyopathy, Pierson syndrome, cancer progression
Cancer progression
Laminin Mutations in Disease
Durbeej, 2010
Where is 2 chain expressed?
What kind of disease could result?
Laminin Mutations in Disease
Durbeej, 2010
What kind of disease could result? Where is 3 chain
expressed?
Laminin Mutations in Disease
Durbeej, 2010
Where is 4 chain expressed?
What kind of disease could result?
Laminin Mutations in Disease
Durbeej, 2010
Where were 1 and 5 chains important?
Why are there no diseases associated with loss of these chains?
Embroygenesis!
Cylindromatosis Rare genetic disorder
laminin-332 defective γ2 processing
Basement membrane thickens Multiple benign epithelial tumors
Tzu & Marinkovich, 2008
What was the γ2 involved in?
Muscular Dystrophy : MDC1A
LAMA2 gene mutation/deletion laminin-211 Where is this?
muscle, heart, peripheral nerve, testis skeletal muscle
dystrophin glycoprotein complex (DGC) and ECM
Phenotype: muscle weakness hypotonia joint stiffness inability to walk dysmyelinating peripheral neuropathy brain defects
Durbeej, 2010
no 2 chain C
Reed, 2009
Pierson syndrome Mutation in 2 chain gene
Glomerular defects
Ocular abnormalities
Muscular and neurological defects
Zenker, 2004
renal failure
loss of vision
early fatality
From Gene to Protein
Transcription
Translation
From LAM to Laminin
LAMA3 LAMC2LAMB3
LAMA3 mRNA LAMC3 mRNALAMB3 mRNA
Β3γ2 Complex
Laminin-332
Activity 3 (10 Min) You have a patient that is exhibiting
symptoms of a disease cause by a defect in correct Laminin expression
To determine a course of treatment, you must first determine the origin of the defect
Activity 3 (10 Min) Genetic defect in the gene itself Deletion of the gene itself Malfunction in RNA Polymerase Ribosomal Defect Defects in processing (subunit goes
unprocessed) Auto-Immune
Endometriosis: Auto-Immune Laminin Disease?
Laminin 111 and 511 critical in embryonic development
Inagaki et al. American Journal of Reproductive Immunology (2011). http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.2010.00956.x/abstract
Misplaced tissue causes creation of auto-antibodies
Auto-antibodies attack uterine tissue, causing infertility
Laminin-332 and the Epithelial Basement Membrane
LAMA3
LAMC2
LAMB3
Correct LAMB3 Expression
Incorrect LAMB3 Expression
Epidermolysis Bullosa (EB)
Severe skin disorder characterized by hemidesmosome dysfunction
Disconnect between Epidermis and Dermis
Characterized by severe blistering and loss of skin
Sawamura et al. Journal of Dermatology (2010); 37: 214-219
Junctional Epiermolysis Bullosa
Buchroithner et al. Laboratory Investigation (2004); 84: 1279-1288
Activity 4 (20 Minutes) You have a patient who is exhibiting blisters
on their skin and you suspect EB.
Explore ways to confirm JEB and the possible ways in which it could be treated.
Possible Answers Slide Diagnose
Electron Microscopy DNA Sequencing ELISA
Treatment Gene Therapy Stem Cells Artificial Grafts Artificial Protein Therapy
Objectives
Predict what would occur in the absence of the basement membrane. (Level 2, Understand)
Compare and contrast, in both structure and function, Laminin with other important ECM proteins (Level 4, Analyze)
Design a potential treatment that could be applied to a patient with a laminin deficiency disease. (Level 6, Create)
ReferencesDurbeej M (2010) Laminins. Cell Tissues Res. 339:
259-268.
Marinkovich MP (2007) Laminin 332 in squamous-cell carcinoma. Nature Reviews Cancer. 7: 370-380.
Reed UC (2009) Congenital muscular dystrophy Part I: a review of phenotypical and diagnostic aspects. Arq Neuropsiquiatr. 67(1): 144-168.
Tzu J & Marinkovich MP (2007) Bridging structure with function: Structural, regulatory, and developmental role of laminins. IJBCB. 40: 199-214.