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Laminin Group Three: Claire Paulson, Andrew Kalas, Jacquie Fable & Chris McKay Tuesday, Feb. 15 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) Laminin in the ECM Figure 19-43 Molecular Biology of the Cell (© Garland Science 2008) 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 Collagen (type IV) Loss of these signals leads to lethality of mouse embryos at the pre-implantation stage 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 and epithelial 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! 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 noncovalent 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 • 6 Rough ER stabilizes & protects So how do these laminin molecules Association of and γ 5 form? • 2) 3) chains chain combines with dimer • 4) Terminal glycosylation • 5) Golgi Trimer secreted from cell • 6) rate limiting step Epithelial & mesenchymal cells Proteolytic processing 2 3 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 2 chain skin & epithelial cells 4 neuromuscular system 3 embryogenesis: epithelial cells adult: liver, kidney, & reproductive organs cells of mesenchymal origin vascular endothelial cells, cells derived from bone barrow 5 widely expressed epithelial, neuromuscular, vascular tissues embryogenesis Laminin Processing chains chains 3 3A γ chains γ2 Laminin Processing : Laminin-332 ! aka. Laminin 5 Skin, placental, mammary glands Hemidesmosomes, cellular migration Processing of 3 chain How is this possible? unprocessed – migration processed – hemidesmosomes alters intergrin binding Processing of γ2 chain DIII fragment binds erbB1 phosphorlyation of beta of 24 Marinkovich, 2007 Durbeej, 2010 Tzu & Markinkovich, 2008 Laminin-332 Where else is laminin-332 found? Cell migration wound healing What type of 3 chain? tumor invasion Margadant & Sonnenberg, 2010 Activity (min) Compare and contrast the major ECM proteins we’ve learn about so far: collagen, fibronectin, & laminin Collagen Structure Class of molecules Processing Connections within the ECM Function Importance in Embryogenesis Location within the body Associated diseases Fibronectin Laminin Collagen Structure Trimeric triple helix Gly-X-Y sequence Class of molecules proteins Processing Pro- chain, glycosylation, Pro-collagen, N & C terminal cleaved collagen Fibronectin Dimer with modules I, II, III, disulfide bonding, sheets glycoproteins Dimers secreted Laminin Trimeric, helical domain, globular ends, disulfide bonding glycoproteins Glycosylation, -γ dimer, addition chain, proteolytic cleavage Connections within the ECM Integrins, MMPs, laminin Cryptic sites Integrins, collagen, heparan , fibrin Collagen, integrins, dystroglycan, heparan sulfate proteinglycans Function Structure,/scaffolding signaling, movement, strength Cell adhesion, cell growth, inhibition of apoptosis, movement, differentiation Signaling, movement, tissue integrity, cellmatrix adhesion Importance in Embryogenesis Essential Essential -- Blastocoele formation Essential -- Embryonic epithelia, placenta Location within the body ECM everywhere ECM everywhere Blood plasma Chondroysplasias, osteoarthritis , osteoporosis, scurvy, lupus erythematosus Cancer progression Associated diseases Basal lamina – separates epithelial from connective tissue Muscular dystrophy, epidermolysis bullosa, cardiomyopathy, Pierson syndrome, cancer progression Laminin Mutations in Disease What kind of disease could result? Where is 2 chain expressed? Durbeej, 2010 Laminin Mutations in Disease What kind of disease could result? Where is 3 chain expressed? Durbeej, 2010 Laminin Mutations in Disease What kind of disease could result? Where is 4 chain expressed? Durbeej, 2010 Laminin Mutations in Disease Where were 1 and 5 chains important? Embroygenesis! Why are there no diseases associated with loss of these chains? Durbeej, 2010 Cylindromatosis Rare genetic disorder laminin-332 defective γ2 processing What was the γ2 involved in? Tzu & Marinkovich, 2008 Basement membrane thickens Multiple benign epithelial tumors Muscular Dystrophy : MDC1A LAMA2 gene mutation/deletion laminin-211 Where is this? muscle, heart, peripheral nerve, testis skeletal muscle no 2 chain C dystrophin glycoprotein complex (DGC) and ECM Phenotype: muscle weakness hypotonia joint stiffness inability to walk dysmyelinating peripheral neuropathy brain defects Durbeej, 2010 Reed, 2009 Pierson syndrome Mutation in 2 chain gene Glomerular defects renal failure Ocular abnormalities loss of vision Muscular and neurological defects early fatality Zenker, 2004 From Gene to Protein Transcription Translation From LAM to Laminin LAMA3 LAMB3 LAMC2 LAMA3 mRNA LAMB3 mRNA LAMC3 mRNA Laminin-332 Β3γ2 Complex 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 Misplaced tissue causes creation of auto-antibodies Auto-antibodies attack uterine tissue, causing infertility Inagaki et al. American Journal of Reproductive Immunology (2011). http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.2010.00956.x/abstract Laminin-332 and the Epithelial Basement Membrane LAMB 3 Correct Incorrect LAMB3LAMB3 Expression LAMA Expression 3 LAMC 2 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) References Durbeej 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.
