THE EXTRACELLULAR MATRIX

THE EXTRACELLULAR MATRIX • Four lectures to cover the following topics: • What is ECM and where can you find it? • Building blocks of ECM: Did you know that 25% of total protein in your body is collagen. • More ECM components: Laminin, Fibronectin etc. (and what the heck is a heparan-sulphate proteoglycan?) • ECM functions: it is not only to keep cells in place WHAT IS THE EXTRACELLULAR MATRIX • Complex arrangements of molecules filling in spaces between the cells. • Not an amorphous jelly or glue but highly organised structure. • Mostly found in connective tissues, such as tendon, cartilage, bone or dermis of the skin. • Diverse structures created by different amounts and organisation of ECM components SOME FUNCTIONS OF ECM MAJOR TYPES OF ECM MOLECULES • Glycosaminoglycans: polysaccharide chains usually found attached to proteins to form proteoglycans • Fibrillar proteins such as collagens (mainly structural role) or fibronectin (adhesive glycoprotein) CONNECTIVE TISSUES EXAMPLES OF ORGANISATION OF ECM - DERMIS EXAMPLES OF ORGANISATION OF ECM - CARTILAGE EXAMPLES OF ORGANISATION OF ECM - BASEMENT MEMBRANE THE COLLAGEN FAMILY • Triple helical domain • Repeated Gly - X - Y amino acid sequence, where X is often proline and Y hydroxyproline • 19 different collagen types (+ possibly 4 more) containing polypeptides encoded by at least 38 genes COLLAGENS -examples out 19 different types Collagens assemble to diverse structures COMMON THEMES IN ECM SYNTHESIS • Extensive post-translational modification • Route: ER - Golgi - Secretory vesicles • During this journey protein are glycosylated or decorated with long GAG chains • Amino acid recidues can be modified (in collagens proline -> hydroxyproline Collagen Biosynthesis intracellular steps COLLAGEN Collagen Biosynthesis extracellular steps Collagen Biosynthesis extracellular assembly Collagen Biosynthesis extracellular assembly Non-collagenous domains • Triple-helical collagen rods are not the only functional domains • Example: Type XVIII collagen that is found in many tissues associated with basal lamina. – Endostatin is a 22kDa polypeptide that is proteolytically cleaved from the C-terminus of type XVIII collagen – Endostatin found in blood vessel walls and basement membranes Collagens in disease • Inherited diseases with mutations in collagen genes • Osteogenesis Imperfecta • Fibrotic diseases with accumulation of ECM • Liver Chirrosis • Lung Fibrosis Collagens in disease • Osteogenesis Imperfecta - Brittle bone disease (not to be confused with osteoporosis) • Variable from mild to embryonic lethal • Often a point mutation in one of type I collagen genes can cause disease • Glycine substitutions to another amino acid more severe than mutations of X or Y in Gly - X - Y triplet. Why? • Dominant negative effect of some Collagens in disease • Fibrotic diseases such as liver chirrosis are characterised by accumulation of ECM • Collagen synthesis is mainly regulated by the level of gene activity. • Some growth factors such as TGF-b signal to increase collagen synthesis. • Enzymes in the collagen synthesis are investigated as drug targets to treat fibrotic diseases Collagens: a summary • All collagens contain a repeating Gly-X-Y sequence and fold into a characteristic triple-helical structure • Collagens assemble to fibrils or networks • Procollagen chains are modified in ER where they also assemble into a triple helix • Type I collagen is the most abundant type; it is a major structural protein of bone, tendon and dermis Fibronectin • Large extracellular glycoprotein • Name = fibro + nectere (to bind) • Multiple domains with different binding sites for other ECM proteins or for receptors on cell surface • Present in tissues and in blood plasma Fibronectin structure Fibronectin structure: domains and interactions Plasma Fibronectin Fibronectin binds cell surfaces by an RGD sequence Fibronectin is essential for embryonic development • Gene targeting => complete lack of fibronectin • Embryonic lethal. • Gross malformations, notchord and somites missing, heart malformation • Problems in cell adhesion, migration and Proteoglycans have long sugar chains attached to a core protein Proteoglycan biosynthesis • Signal peptide directs the nascent polypeptide to ER • Protein modifications starts in late ER. GAG side chains elongation and modification takes place in Golgi. • Several specific enzymes to add disaccharide units and to modify them (e.g. sulphation). For example over 30 Cell-surface proteoglycans Syndecans and glypicans • Syndecans are transmembrane proteins. Four family members. Short cytoplasmic tail contains highly conserved sequences that bind to adaptor proteins. Variable part of syndecan-4 cytoplasmic domain binds protein kinase C and affect cell signalling • Glypicans (6 known family members) are lipid anchored to plasma membrane. GPI= glycosylphosphatidylinositol. • Both families: individual family members Sugar sequence in GAG chains is functionally important Sugar sequence in GAG chains is functionally important Proteoglycans modulate growth factor activity Proteoglycans modulate growth factor activity • Matrix associated PG: sequestration • Membrane-bound PG: presentation • Certain sugar sequences promote FGF signalling and others inhibit • Membrane-bound PGs can be cleaved from cell surface into matrix • Sugar chains can be cleaved by heparanase enzymes to oligosaccharides. Heparin binding proteins • Certain growth factors, especially Fibroblast Growth Factor family (FGF) • Enzymes and their inhibitors, e.g. proteases • Blood coagulation factors • ECM proteins • Note: proteoglycans can bind several proteins at the same time Proteoglycans can modulate cell adhesion to ECM proteins • Serendipitous discovery in transgenic mice over-expressing syndecan-1 under a viral promoter => maturity-onset obesity. • Heparan-sulphate sugar cahins potentiate signalling in hypothalamus that induces over-eating. • In normal mice syndecan-3 is present in hypothalamus (in addition to other neural tissues). Food deprivation induces syndecan-3 expression several fold and More functions for proteoglycans - syndecan-3 regulates appetite Aggrecan: Example of Matrix Proteoglycans Proteoglycans in human diseases Hyaluronic acid Hyaluronic acid CD44 • Adhesive glycoprotein • Numerous isoforms from alternative splicing • Originally found as a „homing receptor‟ in T-lymphocytes • Some splice isoforms suggested to play a role in tumour metastasis • Cytoplasmic tail of CD44 binds to ERM proteins (ezrin-radixin-moiesin family) that can regulate dynamics of actin Basement membrane • Also known as basal lamina • Thin sheetlike network • Epithelial, endothelial, muscle and Schwann cells • Physical support, developmental control, filtering functions • Major constituents: laminins, collagen type IV, perlecan (a proteoglycan) Basement membrane Laminins Laminins • Molecular composition of basement membranes is tissue-specific • Laminins: at least 11 heterotrimers – Five alternative alpha chains, – Three alternative beta chains – Two alternative gamma chains – For example: in skin in the BM between epidermis and dermis, Laminin-5 (a3b3g2) is the predominant laminin isoform. Interactions of laminins Type IV collagen More Basal Lamina Proteins • Perlecan: a large heparan sulfate proteoglycan. HS chains bind other BM components and contribute to filtering functions • Entactin: interacts with laminin and type IV collagen • In some epithelia: epidermis, bladder, trachea, breast and amnion • Shares some ultrastructural features with desmosomes: both display dense, membrane-associated cytoplasmic plaques that are connected to intermediate fialments. But molecular composition is different. • Transmembrane glycoproteins connect basement membrane to intracellular Hemidesmosome: a cell basement membrane adhesion site Hemidesmosomes and basement membraneultrastructural view Hemidesmosomes and basement membranemolecular composition Basal lamina functions -structural support Basal lamina functions -filter Basal lamina functions -developmental guidance • Early embryo: keeps 4 and 8 cell stages together • Differentiation of epithelial organs; epithelial - mesenchymal interactions • Neurite outgrowth: guidance of axon growth by ECM containing laminin sububits Basal lamina functions -developmental guidance ECM Turnover - MMPs • Matrix metalloproteinases are enzymes that cleave components of ECM • Over 20 different enzymes with differenrt specificities. • Common theme: expressed as an inactive proenzyme • Also other substrates than ECM proteins • TIMPs = tissue inhibitors of MMPs MMPs - some examples • “Old names” collagenases, gelatinases and stromelysisn replaced by numbers (e.g. MMP-1) • MMP-1 (collagenase-1) cuts triple helical collagens • MMP-9, (Gelatinase-B) chops e.g. type IV collagen and laminins • MT-MMPs are membrane-bound enzymes MMPs - some functions • Regulate amount of ECM - degradation and remodelling • Cell migration, wound healing, angiogenesis • Activate other MMPs • Release or activate growth factors and other bioactive molecules MMPs in diseases • Extensive matrix degradation in e.g. in periodontitis, rheumatoid arthritis • Tumour cell invasion and metastasis: – Carcinoma breaks basement membrane and invades surrounding stroma. • MMP inhibitors tested for therapeutic use Integrins Integrins • At least 24 different heterodimers from 9 b subunits and 18 a subunits. • Variable pairing: b1 integrin can have 11 different a partners. • Overlapping ECM binding: e.g. 8 different integrins can bind fibronectin • An integrin can bind one or several ECM proteins Integrins and cell behaviour • Clustering of integrins (“velcro effect” in adhesion) • Responses to cell adhesion include spreading, cytoskeletal re-organisation, polarisation, migration, proliferation, activation of specific genes • Cell survival: epithelial cells that are detached commit suicide (this type of apoptosis is called anoikis). • Also, inside out signalling: integrins can Integrins - variety in functions SUMMARY • Collagens: Triple helical rod and noncollagenous domains. Important structural role. Extensive post-translational modifications • Fibronectin: adhesive glycoprotein in matrix and plasma • Proteoglycans: GAG-chains attached to core protein. • Laminins: major components of basement membranes SUMMARY:FUNCTIONS OF ECM