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Extracellular Matrix.pptx
- 2. Overview Introduction Composition of ECM (general) Proteoglycans: structure, function, metabolism Adhesion proteins Mucopolysaccharidosis Integrins Matrix metaloproteinases
- 3. Living tissue can be thought of as a dynamic meshwork of cells and liquid. Despite their close proximity to each other, the cells of a tissue are not simply tightly wound together. Instead, they are spaced out with the help of the extracellular meshwork. The matrix will act as a kind of filler that lies between the otherwise tightly packed cells in a tissue. Furthermore, not only is the matrix filling the gaps in between these cells but it is also retaining a level of water and homeostatic balance
- 4. Overview Basic components of ECM: - structural proteins (e.g., collagens) - proteoglycans (GAGs + protein backbone) - adhesion proteins (link components of matrix to each other and to cells) Collagens, elastin and laminin are the principal structural proteins of connective tissue
- 5. Composition of extracellular matrix Fibrous proteins: collagen, elastin, laminin Proteoglycans Adhesion proteins
- 6. Proteoglycans Form gel of ECM Found in interstitial connective tissue - synovial fluid, vitreous humor, arterial walls, bone, cartilage, cornea variety of proteins in the matrix: collagen, elastin, fibronectin, laminin
- 7. Proteoglycans Consist of polysaccharide called glycosaminoglycans (GAGs) linked to a core protein GAGs are composed of repeating units of disaccharides Proteoglycan may contain >100 GAG chains and consist of up to 95% carbohydrate by weight Interact with variety of proteins in the matrix: collagen, elastin, fibronectin, laminin
- 8. Glycosaminoglycans GAGs are long, unbranched, heteropolysaccharide composed of repeating disaccharide chains where one of the sugars is an N-acetylated amino sugar, either N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) and the other is an acidic sugar. A single exception is keratan sulfate, which contains galactose rather than an acidic sugar. The amino sugar is either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated, eliminating its positive charge The acidic sugar is either D-glucuronic acid or its C-5 epimer L-iduronic acid . These uronic sugars contain carboxyl groups that are negatively charged at physiologic pH and, together with the sulfate groups (−SO4 2−), give GAGs their strongly negative nature
- 10. Proteoglycans Negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules hydrated gel The Gel - provides a flexible mechanical support for ECM - acts as a filter: allows the diffusion of ions, H20, small molecules - slows diffusion of proteins and movement of cell - acts as a lubricant Hyaluronan – the only GAG occurring as a single long polysaccharide chain; the only GAG – not sulfated. glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues.
- 11. Because of the high concentration of negative charges, these repeating disaccharide chains tend to be extended in solution. They repel each other and are surrounded by a shell of water molecules When a solution containing GAGs is compressed, the water is squeezed out, and the GAGs are forced to occupy a smaller volume. When the compression is released, the GAGs spring back to their original, hydrated volume because of the repulsion of their negative charges. This property contributes to the resilience of cartilage, synovial fluid, and the vitreous humor of the eye
- 12. Proteoglycans - structure Contain many chains of GAGs (mucopolysaccharides) Function extracellularly Long, negatively charged glycosaminoglycan chains repel each other occupy a very large space and act as “molecular sieves”, determining which substances enter/leave cells GAGs differ in the monosaccharides present in their repeating disaccharide units: chondroitin sulfate, dermatan sulfate, heparin, heparin sulfate, hyaluronic acid, keratin sulfates I and II GAGs are linked to proteins (except for hyaluronic acid) - usually attached covalently to serine or threonine residues - keratin sulfate I is attached to asparagine
- 13. GAGs–protein linkage: GAGs attached to core protein via covalent linkage are most commonly through a trihexoside (galactose– galactose–xylose) and a serine residue in the protein. An Oglycosidic bond is formed between the xylose and the hydroxyl group of the serine.
- 14. Many proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent and occurs primarily through ionic interactions between the core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins
- 17. Proteoglycans - function determinate which substances enter/leave cells Give resilience and flexibility to substances (e.g., cartilage) – permitting compression and reexpansion of molecule In cartilage proteoglycans, chondroitin sulfate and keratin sulfate are the main types of GAGs.
- 18. Clinical correlate ECM not only holds the cells together, but also serves to keep cells from moving to other locations and prevents large molecules and particles (e.g., microorganisms) from reaching contiguous and distant cells Infections spread, in part, because the infectious agent alters the “containing” capacity of the ECM Cancer cells that metastasize can do so only by altering the integrity of matrix Disease like rheumatoid arthritis and osteoarthritis involve damage to the functional capacity of the matrix Alterations in the matrix of the renal glomerulus allows proteins to be excreted into urine Genetic defects structurally and functionally abnormal matrix connective tissue disorders (e.g., Ehlers- Danlos syndrome, Marfan syndrome) Deficiencies of lysosomal enzymes involved in normal degradation of matrix molecules mucopolysaccharidoses
- 19. Clinical correlate Functional properties of a normal joint depend, in part, on the presence of a soft, well-lubricated, deformable, and compressible layer of cartilaginous tissue - covers the ends of the long bones that constitute joint
- 20. Proteoglycans - synthesis Protein component of proteoglycans – synthesized on the ER Enters the lumen of ER, where the initial glycosylations occur Proteoglycans are synthesized in the ER and Golgi complex - Synthesis of the proteoglycans starts with the attachment of a sugar to a serine, threonine, or asparagine residue of the protein - Additional sugars, donated by UDP-sugar precursors, add sequentially to the nonreducing end of the molecule - Glycosylation occurs initially in the lumen of the ER and subsequently in the Golgi complex Glycosyltransferases – enzymes that add sugars to the chain - specific for the sugar being added, the type of linkage that is formed, and the sugars already present in the chain Once the initial sugars are attached to the protein, the alternating action of 2 glycosyltransferases adds the sugars of repeating disaccharide to the GAG chain Sulfation occurs after addition of sugar - 3’-phosphoadenosine 5’-phosphosulfate (PAPS) = active sulfate: provides sulfate groups Epimerase converts glucuronic acid residues to iduronic acid residues
- 21. Bottlebrush structure of a proteoglycan, with a magnified segment • After synthesis, proteoglycan is secreted from the cell • Final structure resembles a bottlebrush, with many GAG chains extending from the core protein
- 22. Proteoglycan aggregate Many proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent and occurs primarily through ionic interactions between the core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins
- 23. Interactions between the cell membrane and the components of ECM The proteoglycans interact with adhesion protein, fibronectin, which is attached to the cell membrane protein integrin - cross-linked fibers of collagen also associate with this complex ECM
- 24. Proteoglycans in cartilage Long polysaccharide side chains of proteoglycans in cartilage contain many anionic groups High concentration of negative charges attracts cations that create a high osmotic pressure within cartilage, drawing water into this specialized connective tissue and placing collagen network under tension At equilibrium, tension balances the swelling pressure caused by proteoglycans The complementary roles of this macromolecular organization give cartilage its resilience Cartilage can withstand the compressive load of weight bearing and then re-expand to its previous dimensions when that load is relieved
- 25. Synthesis of chondroitin sulfate Sugars are added to the protein one at a time - UDP-sugars serve as precursors A xylose residue is added to a serine in the protein Then 2 galactose residues are added, followed by glucuronic acid (GlcUA) and an N0acetylglucosamine (GalNac) Subeqquent additions occur by the alternating action of 2 enzymes that produce the repeating disaccharide units
- 26. Proteoglycans - degradation Lysosomal enzymes degrade proteoglycans, glycoproteins, and glycolipids - brought into the cell by endocytosis Lysosomes fuse with the endocytic vesicles Lysosomal proteases digest the protein component Carbohydrate component is degraded by lysosomal glycosidases - endoglycosidases – cleave chains into shorter oligosaccharides - exoglycosidases – specific for each type of linkage, removes the sugar residues, one at a time, from reducing ends
- 27. Mycopolysaccharidoses An inability to degrade proteoglycans mucopolysaccharidoses Deficiencies of lysosomal glycosidases Deficiencises of lysosomal glycosidases partially degraded carbohydrates from proteoglycans, glycoproteins, glycolipids accumulate within membrane-enclosed vesicles inside cells enlargement of organ with impairment of it function Skeleton deformities, developmental delay, impaired cognitive abilities
- 28. Mucopolysaccharidoses are hereditary diseases (approximately 1:25,000 live births) caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of heparan sulfate, dermatan sulfate, and/or keratin sulfate They are progressive disorders characterized by lysosomal accumulation of GAGs in various tissues, causing a range of symptoms, such as skeletal and ECM deformities, and intellectual disability. All are autosomal- recessive disorders except Hunter syndrome, which has X-linked inheritance. . Diagnosis is confirmed by measuring the patient’s cellular level of the lysosomal hydrolases. Bone marrow and cord blood transplants, in which transplanted macrophages produce the enzymes that degrade GAGs, have been used to treat Hurler and Hunter syndromes, with limited success. Enzyme replacement therapy is available for both syndromes but does not prevent neurologic damage
- 31. I-Cell Disease I-Cell disease is a rare lysosomal storage disease named for large inclusion bodies seen in cells of patients with the disease. GlcNAc phosphotransferase is deficient and mannose 6-phosophate is not generated on proteins destined for lysosomes. Lack of M6P on amino acid residues causes precursor acid hydrolases to traffic to the plasma membrane and be secreted constitutively, instead of trafficking to lysosomes. Consequently, the acid hydrolases are absent from lysosomes, and the macromolecule substrates for these digestive enzymes accumulate within the lysosomes, generating the inclusion bodies that define the disorder.
- 32. In addition, high concentrations of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes is deficient. I-Cell disease is characterized by skeletal abnormalities, restricted joint movement, coarse (dysmorphic) facial features, and severe psychomotor impairment. Because I-cell disease has features in common with the mucopolysaccharidoses and sphingolipidoses, it is termed a mucolipidosis (ML II). Currently, there is no cure, and death from cardiopulmonary complications usually occurs in early childhood. Pseudo-Hurler polydystrophy (ML III) is a less severe mucolipidosis form of I-cell disease, in which the phosphotransferase maintains some residual enzymatic activity, and it symptomatically resembles a mild form of Hurler syndrome.
- 34. integrins Integrins – major cellular receptors for ECM proteins Provide a link between the internal cytoskeleton of cells (primarily actin) and extracellular proteins (eg, fibronectin, collagen, laminin Involved in signaling options Consist of alpha and beta subunit 24 unique alpha/beta dimers have been discovered Can be activated by “inside-out” or “outside-in” mechanisms Certain integrins, such as those associated WBCs, are normally inactive, and get activated during infection - activation allows them to bind to vascular endothelial cells (leukocyte adhesion) at the site of infection Leukocyte adhesion deficiency (LAD) is a genetic disorder that results from mutations in the beta2 integrin - leukocytes can not be recruited to the sites of infection drugs have been developed to block either beta2 or alpha 4 integrins (on lymphocytes) to treat inflammatory and autoimmune disorders by interfering with the normal WBC response to cytokines
- 35. Adhesion proteins Members: fibronectin, laminin Extracellular glycoproteins Link integrins to ECM components Contain separate distinct binding domains for proteoglycans, collagen , and fibrin - allow to bind the various components of the ECM Contain specific binding domains – for cell surface receptors – integrins - integrins bind to fibronectin on the external surface, span the plasma membrane and adhere to proteins, which, in turn, bind to the intracellular actin filaments of the cytoskeleton - also provide a mechanism for signaling between cells via both internal and external signals
- 36. Key concepts Extracellular matrix IECM) consists of fibrous structural proteins, proteoglycans, and adhesion proteins ECM provides support to the tissues and restricts movement of cells Proteoglycans consist of polysaccharides (glycosaminoglycans) bound to a core protein - polysaccharides are usually a repeating disaccharide unit, containing negative charges - because of charge repulsion, the proteoglycans form a hydrated gel that provides flexible mechanical support to the ECM Integrins are cellular membrane receptors for ECM proteins and they link the cellular cytoskeleton to extracellular protein Integrins are also signaling proteins when they are bound to appropriate compounds Adhesion proteins link the integrins to ECM components Matrix metalloproteinasesare the only proteases that can degrade ECM components, and they are carefully regulated by the tissue inhibitors of matrix metalloproteinases (TIMPS)
- 37. Main literature Biochemistry (Lippincott’s illustrated review) –8th edition chapter chapter 14