GLYCOCONJUGATES

Structural analysis of proteins has shown that up to half of naturally occurring proteins are subject to post-translational modifications with the vast majority glyco-sylated. These covalent linkages involve several amino acids and have distinct structrual characteristics. In addition, a large number of lipids have covalently attached carbohydrate, necessary for their biological functions.

The glycoproteins may be classified into two broad categories: N-linked and O-linked.

A. W-Linked Glycoproteins

N-linked glycoproteins have carbohydrate covalently attached to asparagine residues that occur in the sequence Asn-X-Ser/Thr, where X is any residue except proline. This is a necessary but not sufficient key for glycosyla-tion since there are many examples of such sequences that are not glycosylated even when others on the same polypeptide are substituted with sugar. The linking sugar is invariably N-acetylglucosamine, which is the terminal saccharide of the attached unit (Fig. 25). The number of saccharides present in N-linked structures varies from about 7 to 20 or more; branching is universal with some structures having four separate branches (antennae). All of the saccharides have a common core structure: GlcNAc-GlcNAc-Man3. The first mannose is j-linked (unusual) and the other two mannoses are attached a-1-3 and a-1-6, thus forming the initial branch point. Unlike protein syn-thesis wherein the amino acid sequence is controlled by the genetic one, the final structure of saccharides is rarely so conserved. Thus, a given protein with several glycosy-lation loci is quite likely to have differing saccharide structures, even at the same amino acid siteā€”all of them will still contain the pentasaccharide core noted above. These various glycoforms give rise to a type of heterogeneity that is difficult to characterize completely and may have implications for function.


Repeating unit of hyaluronate. This polysaccharide is distributed throughout connective tissue and is the only mammalian polysaccharide not covalently attached to protein.

FIGURE 24 Repeating unit of hyaluronate. This polysaccharide is distributed throughout connective tissue and is the only mammalian polysaccharide not covalently attached to protein.

 Schematic of a typical W-linked oligosaccharide. Note the core structure, which contains two W-acetylglucosamine and three mannosyl residues. This is present in all units of this type.

FIGURE 25 Schematic of a typical W-linked oligosaccharide. Note the core structure, which contains two W-acetylglucosamine and three mannosyl residues. This is present in all units of this type.

The biosynthesis of these molecules is also unusual. The saccharide is preassembled, not as the final structure but as a common, 14-sugar, lipid-linked precursor that is transferred en bloc to the target asparagine in a cotranslational manner (Fig. 26). This saccharide unit (GlcNAc2-Man9-Glc3) is trimmed to a GlcNAc2-Man5 structure that is then modified by addition of either more mannosyl residues or by several sugars, including GlcNAc, Gal, NANA, and L-fucose. The latter category is generally termed complex as opposed to those which contain GlcNAc and Man only (high mannose).

B. OLinked Glycoconjugates

O-linked glycoconjugates have substantial diversity in that the saccharide units may be covalently attached to serine, threonine, tyrosine, hydroxylysine, or hydroxypro-line residues. In addition, the type of glycosyl substitution varies widely, from single sugars to extended polysac-charide chains. The following discussion highlights key features of these types but is not intended to provide full details.

One major category of O -linked glycosylation is termed mucin type. This is characterized by linkage of the sugar (N-acetylgalactosamine in the alpha configuration) to ser-ine or threonine hydroxyl groups (Fig. 27). There is no identifiable consensus amino acid sequence known which targets specific residues to be substituted. The saccha-ride units range from di- to intermediate size oligosaccha-rides (up to 10 sugars) and are very diverse. Additional sugars present include galactose, N-acetylglucosamine, L-fucose, and sialic acid; some of the saccharide units may be sulfated. Mannose is characteristically absent. These molecules are often found in epithelial secretions; the protein cores may be quite large with a single glyco-protein having an aggregate molecular weight of one million with a hundred or more saccharide units covalently attached.

Glycosylation of tyrosine residues is unusual but a key step in the biosynthesis of glycogen, the major storage glucan of liver and muscle. The core protein, glycogenin, is able to autoglucosylate and attaches a series of glucosyl residues to a single tyrosine in the protein. When the glucose chain has reached four (or more) units (all linked a-1-4), the resulting saccharide moiety is then recognized by glycogen synthase for continuation of glycogen formation. The final polysaccharide may have several thousand glucose residues.

Currently about 20 proteins have been identified as col-lagens. Criteria for this classification include the presence of a triple helical domain ("collagen helix") and the presence of hydroxyproline and hydroxylysine residues. The latter may also be glycosylated with either a single galactose residue or a disaccharide (glucopyranosyl 1-4 galactosyl-hydroxylysine). The extent of glycosyla-tion varies from as little as 1 per 1000 amino acids to 8 or more. It is suggested that addition of carbohydrate causes local disruption in the collagen helix, thus opening the protein structure into a more mesh-like conformation.

 Biosynthesis of the 14-sugar, lipid-linked oligosaccharide, the universal precursor for W-linked glycosylation.

FIGURE 26 Biosynthesis of the 14-sugar, lipid-linked oligosaccharide, the universal precursor for W-linked glycosylation.

Typical O-linked, mucin-type oligosaccharide.

FIGURE 27 Typical O-linked, mucin-type oligosaccharide.

Glycosylation on hydroxyproline residues is rare but has been reported in both plants and fungi. Interestingly, this does not occur in collagen where hydroxyproline is a prominent residue.

An unusual form of O -glycosylation has recently been described wherein a single N-acetylglucosaminyl residue is attached to either a serine or threonine residue in target proteins. In contrast to other O-linked glycoproteins, the entities involved are cytosolic and not secreted. It has been suggested that this modification is reciprocal with phos-phorylation, a common form of regulatory substitution.

A major class of O-linked glycoconjugates is the pro-teoglycans, key components of the extracellular matrix of animals. These glycoconjugates are distinguished by the linking sugar (D-xylose, attached to serine), a linear core saccharide (Gal-Gal-Xyl; Fig. 28), and continuation of the saccharide chain as a linear polysaccharide, which contains alternating residues of an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and a uronic acid (D-glucuronic or L-iduronic acid) (Fig. 29). The saccharides are generally sulfated (may include N-sulfation instead of N-acetylation of the glucosaminyl residues) giving rise to chains of considerable structural diversity. Examples include heparin, a natural anticoagulant, and the chondroitin sulfate proteoglycans. In the case of heparin, it has been established that the antithrom-bin activity resides in a specific pentasaccharide sequence within the structure with a defined pattern of sulfation and sugar components. This type of "information" is known to be present in other complex saccharides and broadens the function of these molecules beyond that of space occupancy and water and electrolyte management. It is interesting to note that the biosynthesis of the iduronosyl moiety in heparin and related heparan sulfate chains occurs at the polymer level by inversion of configuration at C-5 of already incorporated glucuronosyl residues. Extracellular proteoglycans such as those of the chondroitin and dermatan sulfate families are associated with organization of the fibrillar elements of connective tissues (primarily collagens), bone deposition and maintenance of tissue hydration.

Glycolipids represent another diverse class of glycoconjugates. In this case, the saccharides are assembled on a nitrogenous lipid (ceramide) derived from a C-18 amino alcohol (sphingosine) by fatty acylation of the amino group (Fig. 30). The primary hydroxyl group at C-1 is the site of sugar attachment. The saccharides range up to 10 sugars and are extremely diverse. They are found on cell surfaces and function as receptors and immunologic determinants. The blood group ABO system is defined by specific sugars present on erythrocyte glycolipids; thus, type A is characterized by alpha-linked N-acetylgalactosamine, type B by alpha-linked galactose, etc. Glycosphingolipids that contain sialic acid are termed gangliosides (originally isolated from neural tissue) and are involved in development, especially in the nervous system.

Core saccharide of proteoglycans.

FIGURE 28 Core saccharide of proteoglycans.

Structure of the repeating unit of chondroitin 4-sulfate. Other glycosaminoglycan chains presents in proteoglycans include dermatan sulfate (L-iduronic acid replacing d-glucuronic acid), variants with sulfate in the 6-position, and the heparin-heparan sulfate family, which contains both uronic acids, glucosamine, and both N- and O-sulfate esters.

FIGURE 29 Structure of the repeating unit of chondroitin 4-sulfate. Other glycosaminoglycan chains presents in proteoglycans include dermatan sulfate (L-iduronic acid replacing d-glucuronic acid), variants with sulfate in the 6-position, and the heparin-heparan sulfate family, which contains both uronic acids, glucosamine, and both N- and O-sulfate esters.

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