The Glycosaminoglycans Found in Proteoglycans Are Built Up of Repeating Disaccharides
Proteoglycans are proteins that contain covalently linked glycosaminoglycans (GAGs) (see Chapter 15). At least 30 have been characterized and given names such as syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan, and fibromodulin. The proteins bound covalently to glycosaminoglycans are called “core proteins.” Proteoglycans vary in tissue distribution, nature of the core protein, attached glycosaminoglycans, and their function; they have proved difficult to isolate and characterize, but the use of recombinant DNA technology is beginning to yield important information about their structures. 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. Figures 50–8 and 50–9 show the general structure of one particular proteoglycan, aggrecan, the major type found in cartilage. It is very large (about 2 × 103 kDa), with its overall structure resembling that of a bottlebrush. It contains a long strand of hyaluronic acid (one type of GAG) (see Chapter 15) to which link proteins are attached noncovalently. In turn, the link proteins interact noncovalently with core protein molecules from which chains of other GAGs (keratan sulfate and chondroitin sulfate in this case) project. More details on this macromolecule are given when cartilage is discussed below.
Darkfield electron micrograph of a proteoglycan aggregate. The proteoglycan subunits and filamentous backbone are particularly well extended in this image.(Reproduced, with permission, from Rosenberg L, Hellman W, Kleinschmidt AK: Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J Biol Chem 1975;250:1877.)
Schematic representation of the proteoglycan aggrecan. (Reproduced, with permission, from Lennarz WJ: The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980. Reproduced with kind permission from Springer Science and Business Media.)
There are at least seven GAGs: hyaluronic acid (hyaluronan), chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate. 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 l-glucuronic acid (GlcUA) or its 5′-epimer, l-iduronic acid (IdUA). 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 appears to 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 three types.
An O-glycosidic bond between xylose (Xyl) and Ser, a bond that is unique to proteoglycans. This linkage is formed by transfer of a Xyl residue to Ser from UDP-xylose. Two residues of Gal are then added to the Xyl residue, forming a link trisaccharide, Gal-Gal-Xyl-Ser. Further chain growth of the GAG occurs on the terminal Gal.
An O-glycosidic bond forms between GalNAc (N-acetylgalactosamine) and Ser (Thr) (see Figure 46–1A), present in keratan sulfate II. This bond is formed by donation to Ser (or Thr) of a GalNAc residue, employing UDP-GalNAc as its donor.
An N-glycosylamine bond between GlcNAc (N-acetylglucosamine) and the amide nitrogen of Asn, as found in N-linked glycoproteins (see Figure 46–1B). Its synthesis is believed to involve dolichol-PP oligosaccharide.
The synthesis of the core proteins occurs in the endoplasmic reticulum, and formation of at least some of the above linkages also occurs there. Most of the later steps in the biosynthesis of GAG chains and their subsequent modifications occur in the Golgi apparatus.
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.
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.
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; active sulfate] (see Chapter 32) 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.” There 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 above), 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 extracellular matrix 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 50–10) distribution and functions of each of the GAGs will now be briefly discussed. The major features of the seven GAGs are summarized in Table 50–6.
Structures of glycosaminoglycans and their attachments to core proteins. (Ac, acetyl; Asn, l-asparagine; Gal, d-galactose; GalN, d-galactosamine; GlcN, d-glucosamine; GlcUA, d-glucuronic acid; IdUA, l-iduronic acid; Man, d-mannose; NeuAc, N-acetylneuraminic acid; Ser, l-serine; Thr, l-threonine; Xyl, l-xylose.) The summary 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. Neither should it be assumed that the indicated substituents are always present, for example, whereas most iduronic acid residues in heparin carry a 2′-sulfate group, a much smaller proportion of these residues are sulfated in dermatan sulfate. The presence of link trisaccharides (Gal-GalXyl) in the chondroitin sulfates, heparin, and heparin, and dermatan sulfates is shown. (Slightly modified and reproduced, with permission, from Lennarz WJ: The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980. Reproduced with kind permission from Springer Science and Business Media.)
TABLE 50–6Properties of Glycosaminoglycans ||Download (.pdf) TABLE 50–6 Properties of Glycosaminoglycans
|GAG ||Sugars ||Sulfatea ||Protein Linkage ||Location |
|Hyaluronic acid ||GlcNAc, GLcUA ||- ||None ||Skin, synovial fluid, bone, cartilage, vitreous humor, embryonic tissues |
|Chondroitin sulfate ||GalNAc, GlcUA ||GalNAc ||Xyl-Ser; associated with HA via link proteins ||Cartilage, bone, CNS |
|Keratan sulfate I and II ||GlcNAc, Gal ||GlcNAc || |
GlcNAc-Asn (KS I)
GalNAc-Thr (KS II)
|Cornea, cartilage, loose connective tissue |
|Heparin ||Gln, IdUA || |
|Ser ||Mast cells, liver, lung, skin |
|Heparan sulfate ||GlcN, GlcUA ||GlcN ||Xyl-Ser ||Skin, kidney basement membrane |
|Dermatan sulfate ||GalNAc, IdUA, (GlcUA) || |
|Xyl-Ser ||Skin, wide distribution |
Hyaluronic acid consists of an unbranched chain of repeating disaccharide units containing 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 compressibility (see below).
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 (see below). 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 and are a major component of cartilage. They are 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.
As shown in Figure 50–10, 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. However, the two GAGs differ in the structural links to the core proteins, and since it is now known that the distribution of the two types is not tissue specific the classification is based on the different protein linkage. 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.
The repeating disaccharide heparin contains glucosamine (GlcN) and either of the two uronic acids (Figure 50–11). Most of the amino groups of the GlcN residues are N-sulfated, but a few are acetylated. The GlcN also carries a sulfate attached to carbon 6.
Structure of heparin. The polymer section illustrates structural features typical of heparin; however, the sequence of variously substituted repeating disaccharide units has been arbitrarily selected. In addition, non-O-sulfated or 3-O-sulfated glucosamine residues may also occur. (Modified, redrawn, and reproduced, with permission, from Lindahl U, et al: Structure and biosynthesis of heparin-like polysaccharides. Fed Proc 1977;36:19.)
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 binds with factors IX and XI, but its most important interaction is with plasma antithrombin (discussed in Chapter 55). Heparin can also bind specifically to lipoprotein lipase present in capillary walls, causing a release of this enzyme into the circulation.
This molecule is present on many cell surfaces as a proteoglycan and is extracellular. It contains GlcN with fewer N-sulfates than heparin, and, unlike heparin, its predominant uronic acid is GlcUA. Heparan sulfate is 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 (see above), where it plays a major role in determining the charge selectiveness of glomerular filtration.
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; their function in this organelle has not been elucidated. They are present in some storage or secretory granules, such as the chromaffin granules of the adrenal medulla. It has been postulated that they play a role in release of the contents of such granules. The various functions of GAGs are summarized in Table 50–7.
TABLE 50–7Some Functions of Glycosaminoglycans and Proteoglycans ||Download (.pdf) TABLE 50–7 Some Functions of Glycosaminoglycans and Proteoglycans
Act as structural components Components of the ECM
Have specific interactions with collagen, elastin, fibronectin, laminin, and other proteins such as growth factors
As polyanions, bind polycations and cations
Contribute to the characteristic turgor of various tissues
Act as sieves in the ECM
Facilitate cell migration (HA)
Have role in compressibility of cartilage in weight-bearing (HA,CS)
Play role in corneal transparency (KS I and DS)
Have structural role in sclera (DS)
Act as anticoagulant (heparin)
Are components of plasma membranes, where they may act as receptors and participate in cell adhesion and cell-cell interactions (eg, HS)
Determine charge selectiveness of renal glomerulus (HS)
Are components of synaptic and other vesicles (eg, HS)
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 (see Chapter 46) and glycosphingolipids (see Chapter 24), 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 (Table 50–8).
TABLE 50–8The Mucopolysaccharidoses ||Download (.pdf) TABLE 50–8 The Mucopolysaccharidoses
|Disease Name ||Abbreviationa ||Enzyme Defective ||GAG(s) Affected ||Symptoms |
|Hurler-, Scheie- Hurler-Scheie syndrome ||MPS I ||α-l-Iduronidase ||Dermatan sulfate, heparan sulfate ||Mental retardation, coarse facial features, hepatosplenomegaly, cloudy cornea |
|Hunter syndrome ||MPS II ||Iduronate sulfatase ||Dermatan sulfate, heparan sulfate ||Mental retardation |
|Sanfilippo syndrome A ||MPS IIIA ||Heparan sulfate-N-sulfataseb ||Heparan sulfate ||Delay in development, motor dysfunction |
|Sanfilippo syndrome B ||MPS IIIB ||α-N-Acetylglucosaminidase ||Heparan sulfate ||As MPS IIIA |
|Sanfilippo syndrome C ||MPS IIIC ||α-Glucosaminide N-acetyltransferase ||Heparan sulfate ||As MPS IIIA |
|Sanfilippo syndrome D ||MPS IIID ||N-Acetylglucosamine 6-sulfatase ||Heparan sulfate ||As MPS IIIA |
|Morquio syndrome A ||MPS IVA ||Galactosamine 6-sulfatase ||Keratan sulfate, chondroitin 6-sulfate ||Skeletal dysplasia, short stature |
|Morquio syndrome B ||MPS IVB ||β-Galactosidase ||Keratan sulfate ||As MPS IVA |
|Maroteaux-Lamy syndrome ||MPS VI ||N-Acetylgalactosamine 4-sulfatasec ||Dermatan sulfate ||Curvature of the spine, short stature, skeletal dysplasia, cardiac defects |
|Sly syndrome ||MPS VII ||β-Glucuronidase ||Dermatan sulfate, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate ||Skeletal dysplasia, short stature, hepatomegaly, cloudy cornea |
|Natowicz syndrome ||MPS IX ||Hyaluronidase ||Hyaluronic acid ||Joint pain, short stature |
Degradation of GAGs is carried out by a battery of lysosomal hydrolases. These include endoglycosidases, exoglycosidases, and sulfatases, generally acting in sequence to degrade the various GAGs. A number of them are indicated in Table 50–8.
The mucopolysaccharidoses (MPSs) (Table 50–8) share a common mechanism of causation, as illustrated in Figure 50–12. They 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.
Simplified scheme of causation of a mucopolysaccharidosis, such as the Hurler syndrome. Marked accumulation of the GAGs in the tissues mentioned in the figure could cause hepatomegaly, splenomegaly, disturbances of growth, coarse facial features, and mental retardation.
The term “mucolipidosis” was introduced to denote diseases that combined features common to both mucopolysaccharidoses and sphingolipidoses (see Chapter 24). 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, mannosidosis, 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 will generate 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, a lysosomal storage disorder in which hyaluronic acid accumulates in the joints.
Proteoglycans Are Associated With Major Diseases & With Aging
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 perhaps 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 it is these cells that 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 contribute 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 are also observed with aging and help to account for the characteristic changes noted in this organ in the elderly.
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.