Biochemistry of Extracellular Matrix .pdf

Biochemistry of Extracellular Matrix Prof.Dr.Gülden Burçak 2023-2024 Extracellular matrix (ECM) Extracellular matrix (connective tissue) consists of fibrous proteins (collagen, elastin and fibrillin) and the adhesion proteins fibronectin and laminin nonfibrous ground substance: proteoglycans The c...

Biochemistry of Extracellular Matrix Prof.Dr.Gülden Burçak 2023-2024 Extracellular matrix (ECM) Extracellular matrix (connective tissue) consists of fibrous proteins (collagen, elastin and fibrillin) and the adhesion proteins fibronectin and laminin nonfibrous ground substance: proteoglycans The composition of the ECM varies in tissues depending on their functions ECM protects the organs ECM provides elasticity in blood vessels, lungs and skin ECM keeps cells, large molecules and microorganisms from moving to other locations ECM provides a porous pathway for the diffusion of nutrients and oxygen to individual cells : 2 Collagen The most abundant fibrous protein > 25% of the proteins of human body Skin, cartilage, bones, teeth, tendons, ligaments, cornea At least 28 distinct polypeptide chains, encoded by separate genes An elongated protein characterized by repetetive amino acid sequence regular secondary structure High degree of tensile strength 3 α-Chains of Collagen Three α-chains; may have different amino acid sequences Each α-chain has ~ 1000 aa, forms a left-handed helix, 3.3 residues/turn; (Gly-X-Y)n: X and Y can be any other amino acids X is often proline ~ 100 res; Y is often hydroxyproline ~ 100 res 35% glycine, 21% proline and hydroxyproline, 11% alanine; deficient in essential amino acids Only glycine at very tight junctions between α-chains Pyrrolidone rings permit sharp twisting and confer rigidity Hydroxyproline contributes to H-bonding between α-chains Hydroxylysine is the site of O-glycosidic linkages 4 Collagen triple helix (Tropocollagen) The three α-chains wind around each other in a unique conformation Rod-like molecule 300 nm long, 1.5 nm thick; MW: 300kDa α-chains are H-bonded between peptide NH (glycine) and peptide CO between hydroxyproline OH and peptide CO Very closely packed left-handed α-chains are wound into a right-handed super helix which resists to unwinding Superhelical cable, supramolecular assembly: stronger than steel! Stabilization by unusual covalent cross links both within and between the triple helical units Lys, HyLys or a few His residues contribute to these cross-links Synthesis and post-translational modification Intracellular (in fibroblasts) Preprocollagen, procollagen Hydroxylation of proline and lysine residues in procollagen (ER) Prolyl and lysyl hydroxylases (vitamin C) Glycosylation of some hydroxylysines in procollagen (ER) Glucosyl and galactosyltransferases Extension peptides with cysteine residues at both terminals -S-S-bonds assist self assembly into triple helices (coiled coils): tropocollagen No further hydroxylation or glycosylation after formation of the triple helix 6 Hydroxylation of proline and lysine residues 7 Extracellular Cleavage of N- and C- terminal extension peptides by procollagen N-proteinase and procollagen C-proteinase spontaneous assembly of the tropocollagen molecules into collagen fibrils Oxidative deamination of ɛ-amino groups of Lys, HyLys residues to aldehydes by lysyl oxidase (Cu) Formation of covalent cross-links both within and between the triple helices, such as dehydrohydroxylysinonorleucine After further chemical rearrangements, the stable covalent cross-links form which are important for the tensile strength Histidine may also be involved in certain cross-links 8 9  The triple helices (collagen molecules) assemble (self-assembly) into a quarter staggered alignment to form fibrils (10-300 nm in diameter) and cross-linked for strength  Areas with complete overlap of the molecules alternate with areas with a gap; mineralization begins in the gaps between successive collagen (type I) molecules  Fibrils associate into thicker fibers (1-20 μm in diameter); in tendons fibers associate into larger bundles 10 Fibronectin, secreted by fibroblasts, binds collagen fibers during aggregation and alters the kinetics of fiber formation Interaction of proteoglycans with collagen fibers may regulate the formation and determine the orientation of collagen fibers in tissues The main fibril forming collagens are types I and II, in skin, bone and cartilage Several types of collagen do not form fibrils They are characterized by interruptions of the triple helix with stretches of protein lacking Gly-X-Y repeat sequences They have areas of globular structure interspersed in the triple helical structure; type IV collagen form networks in basement membranes 11 Classification of collagens according to their structures FACITs: Fibril-associated collagen with interrupted triple helices Multiplexins: Multiple triple helix domains and interruptions 12 Once formed, collagen is relatively metabolically stable Collagen has little nutritional value as a protein, because it is extremely low in many amino acids that are essential in the human diet Its breakdown is increased during starvation and various inflammatory states Excessive production of collagen occurs in a number of conditions, for example, hepatic cirrhosis Aging connective tissue has an increasingly rigid and brittle character results from chemically accumulated covalent cross-links in collagen fibrils 13 Genetic disorders of collagen biosynthesis Osteogenesis imperfecta (brittle bones): In the most frequent type, glycine replaced with a larger R group abnormal pro-chains assemble into abnormal fibrils abnormal fragility of bones, scleras often thin and translucent, appearing blue due to a deficiency of ECM Ehlers-Danlos syndrome: Mutations that affect specific collagen genes (lysl hydroxylase, procollagen N-proteinase) hyperextensibility of the skin, abnormal tissue fragility, increased joint mobility, spontaneous arterial/ocular rupture the variable clinical picture reflects genetic heterogeneity 14 Defective synthesis of collagen Scurvy: Deficiency of ascorbic acid reduction in post-translational modifications by prolyl/lysyl hydroxylases which give collagen rigidity bleeding gums, subcutaneous hemorrhages, poor wound healing Menkes disease: Deficiency of copper and decreased activity of copperdependent enzyme lysyl oxidase defective cross-linking of collagen and elastin kinky hair and growth retardation 15 Elastin A connective tissue protein responsible for extensibility and elastic recoil in large amounts, particularly in lung, large arterial blood vessels and some elastic ligaments smaller quantities in skin, ear cartilage, several other tissues A highly cross-linked, insoluble, amorphous protein with random coil conformations Tetrafunctional cross-links (desmosine) unique to elastin Three lysine-derived aldehydes condense with an unmodified lysine Not as widespread as collagen 16 Synthesized as a soluble monomer tropoelastin (~70 kDa) On secretion from the cell, cross-linking by lysyl oxidase generates a fibrous mesh that encircles the cells The mature cross-linked elastin is highly insoluble, extremely stable, has a very low-turnover rate (a half-life of up to 70 yrs) In pulmonary emphysema, cutis laxa and aging of skin: fragmentation or a decrease of elastin 17 Fibrillin Fibrillins are large glycoproteins (350 kDa), 10 -12 nm in diameter major component of insoluble microfibrils Fibrillin microfibrils provide a scaffold for deposition of elastin in elastic fibers and in elastin-free bundles in the eye, kidney, and tendons Major Differences Between Collagen and Elastin Collagen Elastin Many different genetic types One genetic type Triple helix No triple helix; random coil conformation permitting stretching (Gly-X-Y)n repeating structure No (Gly-X-Y)n repeating structure Presence of hydroxylysine No hydroxylysine Carbohydrate-containing No carbohydrate Intramolecular aldol crosslinks Intramolecular desmosine cross-links Presence of extension No extension peptides present during peptides during biosynthesis biosynthesis 19 The α-chains of collagen molecules and the collagen molecules of fibrils are cross-linked by unusual types of covalent bonds Elastin exhibits a variety of random coil conformations that permit the protein to stretch and subsequently recoil 20 Genetic defects in elastin and fibrillin Williams-Beuren syndrome: Deletions in the elastin gene in ~ 90% of subjects a developmental disorder affecting ECM and CNS, a causative role in supravalvular aortic stenosis Marfan syndrome: Due to mutations in the gene for fibrillin-1 a relatively prevalent inherited disease of connective tissue abnormal fibrillin and/or lower amounts deposited in the ECM affects eyes, skeletal system and cardiovascular system decrease in binding of TGF-β, which is a ubiquitous multifunctional cytokine, essential for survival (growth, development, inflammation, repair, host immunity) 21 Fibronectin: An adhesive protein A major glycoprotein in ECM, also found in a soluble form in plasma Two identical subunits, joined by two disulfide bridges Large multidomain protein able to bind/interact with collagen, proteoglycans, heparin, fibrin and integrin (cell surfaces) involved in cell adhesion and migration helps cells to find their way through the ECM Loss of adhesion can lead to either physiologic or abnormal cell movement sharply reduced around many transformed cells causing faulty interaction with the ECM 22 Integrins: A transmembrane receptor protein Integrins are heterodimers (various types of α- and β-chains) which interact directly with collagen, fibronectin and laminin indirectly with actin microfilaments in the cytosol The interaction of fibronectin with integrin is a means of communication of the outside of the cell with the inside Fibronectin contains an Arg-Gly-Asp (RGD) sequence that binds to integrin 23 Interaction of cells through its integrins with major proteins of the ECM a and b indicate and α- and β-polypeptide chains of integrins Interaction of fibronectin with actin Attachment proteins (talin, vinculin, α-actinin and paxillin) form focal adhesions which anchor cells in the ECM 24 Interactions between cells and the ECM Structure of the Basal Lamina: Laminin Basal laminas act as a supportive tissue for layers of epithelial cells, muscle cells, adipose cells, peripheral nerves The most abundant protein in basal lamina is laminin Laminin is a glycoprotein with α, β, γ- chains, ~ 850 kD, 70 nm Laminin anchors the basal lamina to the cells by interacting with integrins or dystroglycans and is attached to collagen by entactin and perlecan (heparan sulfate proteoglycan) 26 Structural defects in laminin Junctional epidermolysis bullosa (JEB): Defects in laminin 5 and 6 defective cohesion of the dermis and epidermis severe spontaneous blistering of the skin and mucous membranes epithelial blistering of the respiratory, digestive and genito-urinary systems results in death Congenital muscular dystrophy (CMD): Defect in laminin 2 defective linking of the muscle cell cytoskeleton to the ECM muscle cell apoptosis results in weakened muscles 27 Glomerular basement membrane (GBM) The GBM is an amorphous, 300- to 350-nm-thick extracellular structure acting as a size- and charge-selective macromolecular filter The normal GBM is composed of laminin, type IV collagen, nidogen and heparin sulfate proteoglycan Podocytes are considered the primary source of the GBM components Glomerular membrane has pores large enough to allow molecules up to ~ 8 nm albumin is smaller than this pore size the negative charges of heparan sulfate and of certain sialic acid containing glycoproteins repel the negatively charged albumin and most plasma proteins 28 The normal structure of the glomerulus may be severely damaged in certain types of glomerulonephritis massive amounts of albumin and of certain other plasma proteins can pass into urine, resulting in severe albuminuria Accumulation of advanced glycation end products (AGEs) on GBM in diabetes mellitus cause a decrease in binding of heparan sulfate and thus a decrease in the negatively charged state of GBM electrostatic repulsion of albumin decreases and permeability of basement membrane increases 29 Glycosaminoglycans (GAGs) Glycosaminoglycans or mucopolysaccharides are unbranched polysaccharides made up of repeating disaccharides hyaluronic acid, chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, dermatan sulfate GAGs are huge aggregates that have extended structures: occupy a larger volume than the proteins in ECM GAGs are polyanions that bind to cations or polycations: attract water by osmotic pressure GAGs contribute to turgor of the tissue GAGs form gel at low concn: act as sieves 30 In GAGs one component is always D-glucosamine or D-galactosamine Except keratan sulfate, the other component is a uronic acid, L-glucuronic acid or its 5′-epimer, L-iduronic acid Except hyaluronic acid, all contain sulfate groups, O-sulfates (heparin) or Nsulfates (heparan sulfate) Except hyaluronic acid, all of the GAGs are bound to a core protein A tetrasaccharide linker connects the GAG to a Ser residue in the core protein 31 Hyaluronic acid is not covalently attached to a protein (hyaluronan) and forms a clear, highly viscous solution in synovial fluid: lubricate skeletal joints in vitreous body of the eye: jelly like consistency, glassy appearance in cartilage and loose connective tissues: tensile strength and elasticity hyaluronidase in some pathogenic bacteria and sperm Chondroitin 4-sulfate or 6-sulfate: in cartilage, tendons, ligaments and aorta Keratan sulfate: in cornea (transparency) and cartilage Heparan sulfate: On many cell surfaces and extracellular the sulfated segments of the chain allow it to interact with ECM components, growth factors, various enzymes and factors present in plasma the charge selectiveness of glomerular filtration barrier Heparin: A highly sulfated, intracellular form of heparan sulfate produced primarily by mast cells; in liver, lung and skin the highest negative charge density of any known biological macromolecule purified heparin is used as a therapeutic agent to inhibit coagulation of blood through its capacity to bind antithrombin Proteoglycans Proteoglycans are proteins with covalently linked GAGs Proteoglycans provide the ground/packing substance of connective tissue; associated with each other, with collagen, elastin, fibronectin and laminin Proteoglycans are found in all tissues mainly in the ECM but some as integral membrane components Tissue distribution, nature of the core protein and attached GAGs vary among the proteoglycans At least 30 types of proteoglycans have been characterized syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan and fibromodulin 35 Proteoglycans function as signaling molecules and influence cell behavior Proteoglycans are important in determining the structural organization of ECM some (eg, decorin) can bind growth factors such as TGF-β, modulating their effects on cells Syndecans and glypicans in basal lamina are membrane heparan sulfate proteoglycans that can be shed into the extracellular space Proteoglycan shedding is highly regulated and provides a way for a cell to change its surface features quickly Proteoglycan shedding is involved in cell-cell recognition, adhesion, proliferation and differentiation of cells Shedding is activated in cancer cells 36 Interactions of heparan sulfate with proteins Glycoconjugates Mucopolysaccharidoses GAGs are subject to turnover; slow turnover in adults Deficiencies in GAG degrading lysosomal hydrolases, endoglycosidases, exoglycosidases and sulfatases None is common; usually autosomal recessive Chronic and progressive, affecting multiple organs organomegaly (eg.,hepatomegaly, splenomegaly) severe abnormalities in the development of cartilage, bone abnormal facial appearance mental retardation defects in hearing, vision and the cardiovascular system 40 Proteoglycans play a part in diverse diseases Proteoglycans play a part in fibrosis, cardiovascular disease and cancer Dermatan sulfate is synthesized in high amounts in arterial SMCs that proliferate in atherosclerotic lesions dermatan sulfate binds plasma LDL and may play an important role in development of the atherosclerotic plaque Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM tumor cells can induce fibroblasts to synthesize great amounts of hyaluronic acid to facilitate their own spread some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of their adhesiveness 41 Association of proteoglycans with aging and major diseases On aging, the amounts of certain GAGs in the skin change characteristically In the development of degenerative joint disease as in osteoarthritis age-dependent decrease in chondroitin sulfate in cartilage age-dependent increase in keratan sulfate and hyaluronic acid increased activity of aggrecanase Proteoglycans may act as autoantigens in various types of arthritis: autoimmune destruction of articular and periarticular tissues as in rheumatoid arthritis 42 Matrix metalloproteinases (MMPs) At least 23 different types of human MMPs exist Zn-containing proteases cleave all of the ECM proteins Zn positions water appropriately to participate in proteolysis The propeptide of MMPs contains a cysteine residue which binds to Zn and thus prevents the proteolysis Once activated, certain MMPs can activate other MMP forms. MMP activity is regulated by transcriptional control proteolytic activation inhibition by α2-macroglobulin and tissue inhibitors of metalloproteinases, TIMPs 43 Coordinated expression of the MMPs and TIMPs Coordinated expression of the MMPs and TIMPs is required for appropriate growth and cell movement Destruction of the ECM by the MMPs releases the bound growth factors, thereby allowing them to bind to cell surface receptors to initiate growth of tissues For cell movement within the ECM, remodeling of the various components of the matrix is required Dysregulation may facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis Cancer cells that metastize require extensive ECM remodeling and use MMP activity to spread throughout the body 44