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1. An- Najah National University Faculty of Medicine and Health Sciences Division of Anatomy, Biochemistry and Genetics Introduction and Metabolic Biochemistry Chapter 14 Glycosaminoglycans , Proteoglycans and Glycoproteins Dr. Nihad Othman 1 3/6/2021 Dr. Nihad Othman

2. Glycosaminoglycans (GAGs): Are large complexes of negatively charged heteropolysaccharide chains. Associated with a small amount of protein (“ core protein ”), forming proteoglycans , which typically consist of up to 95% carbohydrate . I. OVERVIEW OF GLYCOSAMINOGLYCANS Proteoglycan core protein GAGs Proteoglycan = Glycosaminoglycan + Protein 3/6/2021 2 Dr. Nihad Othman

3. 3 Proteoglycans contain a greater amount of carbohydrate than do glycoproteins which consist primarily of protein with a variable (but typically small) amount of carbohydrate I. OVERVIEW OF GLYCOSAMINOGLYCANS Proteoglycan Glycoprotein 3/6/2021 Dr. Nihad Othman

4. 4 I. OVERVIEW OF GLYCOSAMINOGLYCANS GAGs have the special ability to bind large amounts of water , producing the gel-like matrix that forms the basis of the body’s ground substance, which, along with fibrous structural proteins such as collagen, elastin, and fibrillin-1, and adhesive proteins such as fibronectin, make up the extracellular matrix (ECM ). 3/6/2021 Dr. Nihad Othman

5. 5 I. OVERVIEW OF GLYCOSAMINOGLYCANS The hydrated GAGs serve as a flexible support for the ECM , interacting with the structural and adhesive proteins, as a molecular sieve , influencing movement of materials through the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of GAGs, which led to the original naming of these compounds as mucopolysaccharides . 3/6/2021 Dr. Nihad Othman

6. II. STRUCTURE OF GLYCOSAMINOGLYCANS C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. GAGs are associated with a small amount of protein (“ core protein ”) by an O-glycosidic bond, forming proteoglycans , which typically consist of up to 95% carbohydrate. Proteoglycans contain a greater amount of carbohydrate than do glycoproteins which consist primarily of protein with a variable (but typically small) amount of carbohydrate Proteoglycan = Glycosaminoglycan + Protein 3/6/2021 6 Dr. Nihad Othman

7. 3/6/2021 Dr. Nihad Othman 7 Proteoglycan aggregates: Proteoglycan monomers associate with a molecule of hyaluronic acid primarily through ionic interactions (not covalent) to form proteoglycan aggregates. The association is stabilized by additional small proteins called link proteins

8. 8 II. STRUCTURE OF GLYCOSAMINOGLYCANS GAGs are long, unbranched, heteropolysaccharide 1 chains composed of a repeating disaccharide unit [ acidic sugar – amino sugar ] n keratan sulfate is an exception: contains galactose instead of an acidic sugar 1 In general, heteropolysaccharides ( heteroglycans ) contain two or more different monosaccharide units 3/6/2021 Dr. Nihad Othman

9. 9 II. STRUCTURE OF GLYCOSAMINOGLYCANS The amino sugar is either: D-glucosamine or D- galactosamine , in which the amino group is usually acetylated, thus eliminating its positive charge . It may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. 3/6/2021 Dr. Nihad Othman

10. 10 II. STRUCTURE OF GLYCOSAMINOGLYCANS The acidic sugar is either: D-glucuronic acid Or L- iduronic acid (C-5 epimer of D-glucuronic acid) These acidic sugars contain carboxyl groups that are negatively charged at physiologic pH and, together with the sulfate groups , give GAGs their strongly negative nature. 3/6/2021 Dr. Nihad Othman

11. 11 II. STRUCTURE OF GLYCOSAMINOGLYCANS Relationship between glycosaminoglycan structure and function Being negatively charged GAG chains are extended in solution and repel each other and when brought together, they "slip" past each other This produces the "slippery" consistency of mucous secretions and synovial fluid 3/6/2021 Dr. Nihad Othman NH 2 NH N- acetylglucosamine

12. 12 II. STRUCTURE OF GLYCOSAMINOGLYCANS When a solution of 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 synovial fluid and the vitreous humor of the eye 3/6/2021 Dr. Nihad Othman

13. 13 B. Classification of the glycosaminoglycans The six major types are divided according to monomeric composition, type of glycosidic linkages, and degree and location of sulfate All GAGs, except for hyaluronic acid, are sulfated and are found covalently attached to protein, forming proteoglycan monomers . II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

14. 14 B. Classification of the glycosaminoglycans II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

15. 15 II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

16. 16 II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

17. 17 II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

18. 18 II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 Dr. Nihad Othman

19. II. STRUCTURE OF GLYCOSAMINOGLYCANS 3/6/2021 19 Dr. Nihad Othman

20. II. STRUCTURE OF GLYCOSAMINOGLYCANS C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. Proteoglycan = Glycosaminoglycan + Protein 3/6/2021 20 Dr. Nihad Othman

21. II. STRUCTURE OF GLYCOSAMINOGLYCANS Structure of proteoglycan monomers: A proteoglycan monomer found in cartilage consists of: a core protein up to 100 linear chains of GAGs are covalently attached to the core protein Each GAG chain contain up to 200 disaccharide units In cartilage proteoglycan, the species of GAGs include chondroitin sulfate and keratan sulfate 3/6/2021 21 Dr. Nihad Othman

22. II. STRUCTURE OF GLYCOSAMINOGLYCANS Structure of proteoglycan monomers: Proteoglycan The resulting structure resembles a “bottle brush” Proteoglycans are grouped into gene families that encode core proteins with common structural features. Note: A number of proteoglycans have been characterized and named based on their structure and functional location. For example, syndecan is an integral membrane proteoglycan , versican and aggrecan are the predominant extracellular proteoglycans , and neurocan and cerebrocan are found primarily in the nervous system 3/6/2021 22 Dr. Nihad Othman

23. II. STRUCTURE OF GLYCOSAMINOGLYCANS 2. Linkage between the carbohydrate chain and the protein: linkage most commonly through a trihexoside (Gal-Gal-xylose) and a serine residue, respectively . An O-glycosidic bond is formed between the xylose and the hydroxyl group of the serine 3/6/2021 23 Dr. Nihad Othman

24. II. STRUCTURE OF GLYCOSAMINOGLYCANS 3. Proteoglycan aggregates: Proteoglycan monomers associate with a molecule of hyaluronic acid primarily through ionic interactions (not covalent) to form proteoglycan aggregates. The association is stabilized by additional small proteins called link proteins 3/6/2021 24 Dr. Nihad Othman

25. IV. DEGRADATION OF GLYCOSAMINOGLYCANS GAGs are degraded in lysosomes , Lysosomes contain hydrolytic enzymes that are most active at a pH of approximately 5. (a group, these enzymes are called acid hydrolases) The low pH optimum is a protective mechanism that prevents the enzymes from destroying the cell should leakage occur into the cytosol where the pH is neutral. 3/6/2021 25 Dr. Nihad Othman

26. IV. DEGRADATION OF GLYCOSAMINOGLYCANS The half-lives of GAGs vary from minutes to months and are influenced by the type of GAG and its location in the body. 3/6/2021 26 Dr. Nihad Othman

27. IV. DEGRADATION OF GLYCOSAMINOGLYCANS Phagocytosis of extracellular GAGs Because GAGs are extracellular or cell-surface compounds, they must first be engulfed by an invagination of the cell membrane (phagocytosis ), forming a vesicle inside of which the GAGs are to be degraded. This vesicle then fuses with a lysosome , forming a single digestive vesicle in which the GAGs are efficiently degraded This vesicle then fuses with a lysosome , forming a single digestive vesicle in which the GAGs are efficiently degraded by a large number of acid hydrolases for complete digestion 3/6/2021 27 Dr. Nihad Othman

28. IV. DEGRADATION OF GLYCOSAMINOGLYCANS B . Lysosomal degradation of GAGs The lysosomal degradation of GAGs requires a large number of acid hydrolases for complete digestion. polysaccharide chains are cleaved by endoglycosidases , producing sulfated oligosaccharides. Further degradation of these oligosaccharides occurs sequentially from the nonreducing end of each chain 3/6/2021 28 Dr. Nihad Othman

29. IV. DEGRADATION OF GLYCOSAMINOGLYCANS B. Lysosomal degradation of GAGs the last group (sulfate or sugar) added during synthesis being the first group removed (by sulfatases or exoglycosidases ). 3/6/2021 29 Dr. Nihad Othman

30. IV. DEGRADATION OF GLYCOSAMINOGLYCANS B. Lysosomal degradation of GAGs Endo- and exoglycosidases are also involved in the lysosomal degradation of glycoproteins and glycolipids. Deficiencies in these enzymes result in the accumulation of partially degraded carbohydrates, resulting in tissue damage. 3/6/2021 30 Dr. Nihad Othman

31. V. MUCOPOLYSACCHARIDOSES hereditary diseases (1:25,000 live births) that are clinically progressive 1 They are characterized by accumulation of glycosaminoglycans in various tissues, causing varied symptoms, such as skeletal and extracellular matrix deformities, and mental retardation caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of heparan sulfate and/or dermatan sulfate This results in the presence of oligosaccharides in the urine, because of incomplete lysosomal degradation of glycosaminoglycans 3/6/2021 31 Dr. Nihad Othman 1 Progressive disease or progressive illness is a disease or physical ailment whose course in most cases is the worsening, growth, or spread of the disease

32. V. MUCOPOLYSACCHARIDOSES characterized by lysosomal accumulation of GAGs in various tissues, Incomplete lysosomal degradation of GAGs causing a range of symptoms, such as skeletal and extracellular matrix deformities, and intellectual disability. All are autosomal recessive disorders except Hunter syndrome, which is X linked. 3/6/2021 32 Dr. Nihad Othman

33. V. MUCOPOLYSACCHARIDOSES Progressive 1 disorders: Children who are homozygous for any one of these diseases are apparently normal at birth and then gradually deteriorate. In severe cases, death occurs in childhood. There currently is no cure. These fragments can be used to diagnose the specific mucopolysaccharidosis, namely by identifying the structure present on the nonreducing end of the oligosaccharide. That residue would have been the substrate for the missing enzyme. Some of the lysosomal enzymes required for the degradation of glycosaminoglycans also participate in the degradation of glycolipids and glycoproteins 1 Progressive disease or progressive illness is a disease or physical ailment whose course in most cases is the worsening, growth, or spread of the disease 3/6/2021 33 Dr. Nihad Othman

34. V. MUCOPOLYSACCHARIDOSES Diagnosis Incomplete degradation of GAGs cause the appearance of oligosaccharides in the urine. fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end (that residue would have been the substrate for the missing enzyme) Diagnosis confirmation: by measuring the patient’s cellular level of the lysosomal hydrolases. 3/6/2021 34 Dr. Nihad Othman

35. V. MUCOPOLYSACCHARIDOSES Treatment: 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. 3/6/2021 35 Dr. Nihad Othman

36. V. MUCOPOLYSACCHARIDOSES Note: Deficiencies in the degradation of keratan sulfate result in Morquio syndrome, A and B. Deficiencies in the degradation of dermatan sulfate result in Maroteaux-Lamy syndrome.] 3/6/2021 36 Dr. Nihad Othman

37. V. MUCOPOLYSACCHARIDOSES 3/6/2021 37 Dr. Nihad Othman

38. 3/6/2021 Dr. Nihad Othman 38 Glycoproteins are proteins to which oligosaccharides are covalently attached. They differ from the proteoglycans (which might be considered a special case of glycoproteins) in that the length of the glycoprotein's carbohydrate chain is relatively short (usually 2–10 sugar residues in length, although they can be longer), whereas it can be very long in the glycosaminoglycans. In addition, whereas glycosaminoglycans have diglucosyl repeat units, the carbohydrates of glycoproteins do not have serial repeats. The glycoprotein carbohydrate chains are often branched instead of linear, and may or may not be negatively charged. OVERVIEW OF GLYCOPROTEINS

39. OVERVIEW OF GLYCOPROTEINS Glycoprotein carbohydrate GAGs of proteoglycans Chain length Shorter (usually 2 to 10 sugars, they can be longer) very long Repeating units do not have serial repeats repeating disaccharide units Branching Branched Linear Negative Charge may or may not Highly negatively charged Differences between Glycoprotein carbohydrate chains and GAGs of proteoglycans 3/6/2021 39 Dr. Nihad Othman

40. OVERVIEW OF GLYCOPROTEINS Glycoproteins contain highly variable amounts of carbohydrate but typically less than that in GAGs. For example: IgG contains less than 4% of its mass as carbohydrate, human gastric glycoprotein (mucin) contains more than 80% carbohydrate. Nonenzymatic glycosylation of proteins is known as glycation 3/6/2021 40 Dr. Nihad Othman

41. OVERVIEW OF GLYCOPROTEINS Membrane-bound glycoproteins participate in a broad range of cellular phenomena including: cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens), as components of the ECM protective biologic lubricants of the mucins of the urogenital and gastrointestinal tracts, 3/6/2021 41 Dr. Nihad Othman

42. LYSOSOMAL DEGRADATION OF GLYCOPROTEINS similar to that of the GAGs lysosomal acid hydrolases generally specific for the removal of one component of the glycoprotein. They are primarily exo -enzymes that remove their respective groups in the reverse order of their incorporation (“last on, first off”). If any one degradative enzyme is missing, degradation by the other exoenzymes stops . 3/6/2021 42 Dr. Nihad Othman

43. IX. LYSOSOMAL DEGRADATION OF GLYCOPROTEINS Glycoprotein storage diseases ( oligosaccharidoses ) A group of autosomal recessive diseases very rare, caused by a deficiency of any one of the degradative enzymes, results in accumulation of partially degraded structures in the lysosomes. After cell death, the oligosaccharide fragments appear in the urine. [Note: These disorders are very often directly associated with the same enzyme deficiencies involved in mucopolysaccharidoses and the inability to degrade glycolipids.] 3/6/2021 43 Dr. Nihad Othman

44. VIII. SYNTHESIS OF GLYCOPROTEINS 3. Enzymes destined for lysosomes: I-cell disease (inclusion-cell disease): a rare lysosomal storage disease in which lysosomal acid hydrolases are absent, resulting in an accumulation of substrates normally degraded by these enzymes. Note: I-cell disease is so-named because of the large inclusion bodies seen in cells of patients with this disease (hence the name) 3/6/2021 44 Dr. Nihad Othman

45. VIII. SYNTHESIS OF GLYCOPROTEINS 3. Enzymes destined for lysosomes: I-cell disease (inclusion-cell disease) (cont.): high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes rather than the synthetic pathway of these enzymes is deficient . 3/6/2021 45 Dr. Nihad Othman

46. VIII. SYNTHESIS OF GLYCOPROTEINS 3. Enzymes destined for lysosomes: I-cell disease (inclusion-cell disease) (cont.): Deficiency of the phosphotransferase needed to phosphorylate the mannose residues of potential lysosomal enzymes, causes the enzymes to be secreted (by default), rather than being targeted to lysosomal vesicles. 3/6/2021 46 Dr. Nihad Othman

47. VIII. SYNTHESIS OF GLYCOPROTEINS 3. Enzymes for lysosomes: I-cell (inclusion-cell disease) (con.): characterized by: skeletal abnormalities restricted joint movement coarse (dysmorphic) facial features and severe psychomotor impairment Death usually occurs by eight years of age. [Note: I-cell disease is considered to be a glycoprotein storage disease. 3/6/2021 47 Dr. Nihad Othman