The Glycosaminoglycans Found in Proteoglycans Are Built Up of Repeating Disaccharides

Proteoglycans, proteins that contain covalently linked glycosaminoglycans (GAGs), are a major component of the ECM. At least 30 have been characterized, for example, syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan, and fibromodulin. A proteoglycan consists of a core protein bound covalently to GAGs, and these units form large complexes with other components of the ECM, such as hyaluronic acid or collagen. Figures 1 and2 show the general structure of such complexes. They are very large with an overall structure resembling that of a bottlebrush. The example shown in Figure 2 contains a long strand of hyaluronic acid (one type of GAG) to which link proteins are attached noncovalently. In turn, the link proteins interact noncovalently with core protein molecules from which chains of other GAGs (eg, keratan sulfate and chondroitin sulfate) project. Proteoglycans vary in tissue distribution, nature of the core protein, attached GAGs, and their function. The amount of carbohydrate in a proteoglycan is usually much greater than that found in a glycoprotein, and may comprise up to 95% of its weight.

Fig1. Darkfield electron micrograph of a proteogly can aggregate. The proteoglycan subunits and filamentous back bone are particularly well extended in this image. (Reproduced with permission from Rosenberg L, Hellmann W, Kleinschmidt AK. Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage, J Biol Chem. 1975;250(5):1877–1883.)

Fig2. Schematic representation of a proteoglycan complex. In this example, proteoglycans are attached via noncovalent bonds to link proteins which, in turn, bond noncovalently to a long strand of the glycosaminoglycan (GAG), hyaluronic acid.

GAGs are unbranched polysaccharides made up of repeating disaccharides, one component of which is always an amino sugar(hence, the name GAG), either d-glucosamine or d-galactosamine. The other component of the repeating disaccharide (except in the case of keratan sulfate) is a uronic acid, either d-glucuronic acid (GlcUA) or its 5′-epimer, l-iduronic acid (IdUA). There are at least seven GAGs: hyaluronic acid (hyaluronan), chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate. With the exception of hyaluronic acid, all the GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate). Hyaluronic acid is also exceptional because it can exist as a polysaccharide in the ECM, with no covalent attachment to protein, as the definition of a proteoglycan given above specifies. Both GAGs and proteoglycans have proved difficult to work with, partly because of their complexity. However, since they are major components of the ECM and have a number of important biologic roles as well as being involved in a number of disease processes, interest in them has increased greatly in recent years.

Biosynthesis of Glycosaminoglycans Involves Attachment to Core Proteins, Chain Elongation, & Chain Termination

Attachment to Core Proteins

The linkage between GAGs and their core proteins is generally one of the following three types:

 1. AnO-glycosidic bond between xylose (Xyl) and a serine residue on the protein, a bond that is unique to proteo glycans. This linkage is formed by transfer of a Xyl resi due to serine from UDP-xylose. Two residues of galactose (Gal) are then added to the Xyl residue, forming a link trisaccharide, Gal-Gal-Xyl. Further chain growth of the GAG occurs on the terminal Gal.

2. An O-glycosidic bond between N-acetylgalactosamine (GalNAc) and serine (Ser) or threonine (Thr), present in keratan sulfate. This bond is formed by donation to Ser or Thr of a GalNAc residue, employing UDP-GalNAc as its donor.

3. An N-glycosylamine bond between N-acetylglucosamine (GlcNAc) and the amide nitrogen of asparagine (Asn), as found in N-linked glycoproteins. Its synthesis is believed to involve dolichol-PP oligosaccharide.

The synthesis of the core proteins occurs in theendoplasmic reticulum, and formation of at least some of the above link ages also occurs there. Most of the later steps in the biosynthe sis of GAG chains and their subsequent modifications occur in the Golgi apparatus.

Chain Elongation

 Appropriate nucleotide sugars and highly specific Golgi located glycosyltransferases are employed to synthesize the oligosaccharide chains of GAGs. The “one enzyme, one linkage” relationship appears to hold here, as in the case of certain types of linkages found in glycoproteins. The enzyme systems involved in chain elongation are capable of high-fidelity reproduction of complex GAGs.

Chain Termination

This appears to result from (1) sulfation, particularly at certain positions of the sugars, and (2) the progression of the growing GAG chain away from the membrane site where catalysis occurs.

Further Modifications

 After formation of the GAG chain, numerous chemical modifications occur, such as the introduction of sulfate groups onto GalNAc and other moieties and the epimerization of GlcUA to IdUA residues. The enzymes catalyzing sulfation are designated sulfotransferases and use 3′-phosphoadenosine 5′-phosphosulfate (PAPS, also called active sulfate) as the sulfate donor. These Golgi-located enzymes are highly specific, and distinct enzymes catalyze sulfation at different positions (eg, carbons 2, 3, 4, and 6) on the acceptor sugars. An epimerase catalyzes conversions of glucuronyl to iduronyl residues.

Proteoglycans Are Important in the Structural Organization of the Extracellular Matrix

 Proteoglycans are found in every tissue of the body, mainly in the ECM or ground substance, transparent, colorless material of the ECM which fills spaces between cells and largely consists of proteoglycans. Here they are associated with each other and also with the other major structural components of the matrix, collagen, and elastin, in specific ways. Some proteoglycans bind to collagen and others to elastin. These interactions are important in determining the structural organization of the matrix. Some proteoglycans (eg, decorin) can also bind growth factors such as TGF-β, modulating their effects on cells. In addition, some of them interact with certain adhesive proteins such as fibronectin and laminin (see earlier), also found in the matrix. The GAGs present in the proteoglycans are polyanions and hence bind polycations and cations such as Na+ and K+. This latter ability attracts water by osmotic pressure into the ECM and contributes to its turgor. GAGs also gel at relatively low concentrations. Because of the long, extended nature of the polysaccharide chains of GAGs and their ability to gel, the proteoglycans can act as sieves, restricting the passage of large macromolecules into the ECM but allowing relatively free diffusion of small molecules. Again, because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the matrix relative to proteins.

Various Glycosaminoglycans Exhibit Differences in Structure & Have Characteristic Distributions and Diverse Functions

The seven GAGs named above differ from each other in a number of the following properties: amino sugar composition, uronic acid composition, linkages between these components, chain length of the disaccharides, the presence or absence of sulfate groups and their positions of attachment to the constituent sugars, the nature of the core proteins to which they are attached, the nature of the linkage to core protein, their tissue and subcellular distribution, and their biologic functions.

The structure (Figure 3), distribution, and functions of each of the GAGs will now be briefly discussed. The major features of the seven GAGs are summarized in Table1.

Fig3. Structures of glycosaminoglycans and their attachments to core proteins. (Ac, acetyl; Asn, l-asparagine; Gal,-galactose; GalN, d-galactosamine; GalNAc, N-acetyl d-galactosamine; GlcN, d-glucosamine; GlcNAc, N-acetyl d-glucosamine; GlcUA,-glucuronic acid; IdUA, l-iduronic acid; Man, d-mannose; NeuAc, N-acetylneuraminic acid; Ser, l-serine; Thr, l-threonine; Xyl, l-xylose.) The sum mary structures are qualitative representations only and do not reflect, for example, the uronic acid composition of hybrid glycosaminoglycans such as heparin and dermatan sulfate, which contain both l-iduronic and d-glucuronic acid. Hyaluronic acid has no covalent attachment to protein. Chondroitin sulfates, heparin, heparan sulfate, and dermatan sulfate attach to a Ser on the core protein via the Gal-Gal-Xyl link trisaccharide. Keratan sulfate I links to a core protein Asn via GlcNAc and Keratan sulfate II to a Ser (or Thr) via GalNAc.

Table1. Properties of Glycosaminoglycans

Hyaluronic Acid

Hyaluronic acid consists of an unbranched chain of repeating disaccharide units containing glucuronic acid (GlcUA) and GlcNAc. It is present in bacteria and is found in the ECM of nearly all animal tissues, but is especially high in concentration in highly hydrated types such as skin and umbilical cord, and in bone, cartilage, joints (synovial fluid) and in vitreous humor in the eye, as well as in embryonic tissues. It is thought to play an important role in permitting cell migration during morphogenesis and wound repair. Its ability to attract water into the ECM triggers loosening of the matrix, aiding this process. The high concentrations of hyaluronic acid together with chondroitin sulfates present in cartilage contribute to its com pressibility.

Chondroitin Sulfates (Chondroitin 4-Sulfate & Chondroitin 6-Sulfate)

Proteoglycans linked to chondroitin sulfate by the Xyl-Ser O-glycosidic bond are prominent components of cartilage. The repeating disaccharide is similar to that found in hyaluronic acid, containing GlcUA but with GalNAc replacing GlcNAc. The GalNAc is substituted with sulfate at either its 4′ or its 6′ position, with approximately one sulfate being present per disaccharide unit. Chondroitin sulfates have an important role in maintaining the structure of the ECM. They are located at sites of calcification in endochondral bone as well as in cartilage. They are also found in high amounts in the ECM of the central nervous system and, in addition to their structural function, are thought to act as signaling molecules in the prevention of the repair of nerve endings after injury.

Keratan Sulfates I & II

As shown in Figure 3, the keratan sulfates consist of repeating Gal-GlcNAc disaccharide units containing sulfate attached to the 6′ position of GlcNAc or occasionally of Gal. Keratan sulfate I was originally isolated from the cornea, while keratan sulfate II came from cartilage. The two GAGs I or II, are classified based on their different linkages to the core protein (see Figure 3). In the eye, they lie between collagen fibrils and play a critical role in corneal transparency. Changes in proteoglycan composition found in corneal scars disappear when the cornea heals.

Heparin

The repeating disaccharide heparin contains glucosamine (GlcN) and either of the two uronic acids (GlcUA or iduronic acid [IdUA]) (Figure 4). Most of the amino groups of the GlcN residues are N-sulfated, but a few are acetylated (GlcNAc). The GlcN also carries a sulfate attached to carbon 6.

Fig4. Structure of heparin.Structural features typical of heparin are shown. Each repeating disaccharide contains glucosamine (GlcN) and either d-glucuronic (GlcUA) or l-iduronic acid (IdUA). A few GlcN residues are acetylated (GlcNAc). The sequence of variously substituted repeating disaccharide units has been arbitrarily selected. Non-O-sulfated or 3-O-sulfated glucosamine residues may also occur.

The vast majority of the uronic acid residues are IdUA. Initially, all of the uronic acids are GlcUA, but a 5′-epimerase converts approximately 90% of the GlcUA residues to IdUA after the polysaccharide chain is formed. The protein molecule of the heparin proteoglycan is unique, consisting exclusively of serine and glycine residues. Approximately two-thirds of the serine residues contain GAG chains, usually of 5 to 15 kDa but occasionally much larger. Heparin is found in the granules of mast cells and also in liver, lung, and skin. It is an important anticoagulant. It is released into the blood from capillary walls by the action of lipoprotein lipase and it binds with factors IX and XI, but its most important interaction is with plasma anti thrombin.

Heparan Sulfate

This molecule is present in a proteoglycan found on many extracellular cell surfaces. It contains GlcN with fewer N-sulfates than heparin, and, unlike heparin, its predominant uronic acid is GlcUA. Heparan sulfates are associated with the plasma membrane of cells, with their core proteins actually spanning that membrane. In this, they may act as receptors and may also participate in the mediation of the cell growth and cell-cell communication. The attachment of cells to their substratum in culture is mediated at least in part by heparan sulfate. This proteoglycan is also found in the basement membrane of the kidney along with type IV collagen and laminin, where it plays a major role in determining the charge selectiveness of glomerular filtration.

Dermatan Sulfate

This substance is widely distributed in animal tissues. Its structure is similar to that of chondroitin sulfate, except that in place of a GlcUA in β-1,3 linkage to GalNAc it contains an IdUA in an α-1,3 linkage to GalNAc. Formation of the IdUA occurs, as in heparin and heparan sulfate, by 5′-epimerization of GlcUA. Because this is regulated by the degree of sulfation and because sulfation is incomplete, dermatan sulfate contains both IdUA-GalNAc and GlcUA-GalNAc disaccharides. Dermatan sulfate has a widespread distribution in tissues, and is the main GAG in skin. Evidence suggests it may play a part in blood coagulation, wound repair, and resistance to infection.

Proteoglycans are also found in intracellular locations such as the nucleus where they are thought to have a regulatory role in functions such as cell proliferation and transport of molecules between the nucleus and the cytosol. The various functions of GAGs are summarized in Table2.

Table2. Some Functions of Glycosaminoglycans & Proteoglycans

Deficiencies of Enzymes That Degrade Glycosaminoglycans Result in Mucopolysaccharidoses

Both exo- and endoglycosidases degrade GAGs. Like most other biomolecules, GAGs are subject to turnover, being both synthesized and degraded. In adult tissues, GAGs generally exhibit relatively slow turnover, their half-lives being days to weeks.

Understanding of the degradative pathways for GAGs, as in the case of glycoproteins and glycosphingolipids, has been greatly aided by elucidation of the specific enzyme deficiencies that occur in certain inborn errors of metabolism. When GAGs are involved, these inborn errors are called mucopolysaccharidoses (MPSs) (Table 3).

Table3. The Mucopolysaccharidoses

Degradation of GAGs is carried out by a battery of lysosomal hydrolases. These include endoglycosidases, exo glycosidases, and sulfatases, generally acting in sequence. The MPSs (see Table 3) share a common mechanism of causation involving a mutation in a gene encoding a lysosomal hydroxylase responsible for the degradation of one or more GAGs. This leads to a defect in the enzyme and the accumulation of the substrate GAGs in various tissues, including the liver, spleen, bone, skin, and the central nervous system. The diseases are usually inherited in an autosomal recessive manner, with Hurler and Hunter syndromes being perhaps the most widely studied. None is common. In general, these conditions are chronic and progressive and affect multiple organs. Many patients exhibit organomegaly (eg, hepato- and splenomegaly); severe abnormalities in the development of cartilage and bone; abnormal facial appearance; and mental retardation. In addition, defects in hearing, vision, and the cardiovascular system may be present. Diagnostic tests include analysis of GAGs in urine or tissue biopsy samples; assay of suspected defective enzymes in white blood cells, fibroblasts or serum; and test for specific genes. Prenatal diagnosis is now sometimes possible using amniotic fluid cells or chorionic villus biopsy samples. In some cases, a family history of a mucopolysaccharidosis is obtained.

The term “mucolipidosis” was introduced to denote dis eases that combined features common to both mucopolysac charidoses and sphingolipidoses. In sialidosis (mucolipidosis I, ML-I), various oligosaccharides derived from glycoproteins and certain gangliosides accumulate in tissues. I-cell disease (ML-II) and pseudo-Hurler polydystrophy (MLIII) are described in Chapter 46. The term “mucolipidosis” is retained because it is still in relatively widespread clinical usage, but it is not appropriate for these two latter diseases since the mechanism of their causation involves mislocation of certain lysosomal enzymes. Genetic defects of the catabolism of the oligosaccharide chains of glycoproteins (eg, manno sidosis, fucosidosis) are also described in Chapter 46. Most of these defects are characterized by increased excretion of various fragments of glycoproteins in the urine, which accumulate because of the metabolic block, as in the case of the mucolipidoses.

Hyaluronidase is one important enzyme involved in the catabolism of both hyaluronic acid and chondroitin sulfate. It is a widely distributed endoglycosidase that cleaves hexosaminidic linkages. From hyaluronic acid, the enzyme generates a tetrasaccharide with the structure (GlcUAβ-1, 3-GlcNAc-β-1,4)2 , which can be degraded further by a β-glucuronidase and β-N-acetylhexosaminidase. A genetic defect in hyaluronidase causes MPS IX (Natowicz syndrome), a lysosomal storage disorder in which hyaluronic acid accumulates in the joints.

Proteoglycans Are Associated With Major Diseases & With Aging

stores.com Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Tumor cells can induce fibroblasts to synthesize greatly increased amounts of this GAG, thereby facilitating their own spread. Some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of adhesiveness that these cells display.

The intima of the arterial wall contains hyaluronic acid and chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans. Of these proteoglycans, dermatan sulfate binds plasma low-density lipoproteins. In addition, dermatan sulfate appears to be the major GAG synthesized by arterial smooth muscle cells. Because these cells proliferate in atherosclerotic lesions in arteries, dermatan sulfate may play an important role in development of the atherosclerotic plaque.

In various types of arthritis, proteoglycans may act as autoantigens, thus contributing to the pathologic features of these conditions. The amount of chondroitin sulfate in cartilage diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase. These changes may con tribute to the development of osteoarthritis, as may increased activity of the enzyme aggrecanase, which acts to degrade aggrecan. Changes in the amounts of certain GAGs in the skin help to account for its characteristic alterations with aging.

In the past few years, it has become clear that in addition to their structural role in the ECM, proteoglycans function as signaling molecules which influence cell behavior, and they are now believed to play a part in diverse diseases such as fibrosis, cardiovascular disease, and cancer.

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