Glycoproteins - Hughes R. 1985

Biosynthesis
Assembly of N-glycans
Termination reactions

So far, we have discussed the assembly of high-mannose type N-glycans containing up to nine mannose residues. How is the other major Class of N-glycans—those containing peripheral sialic acid and galactose residues along with additional N-acetylglucosamine residues—synthesized? How is The addition of fucose regulated? Interestingly, fucose linked to the chitobiose core sequence is absent in oligomannose glycans. Furthermore, fucose is frequently found in the galß1→4glcNAc structures at the non-reducing termini of complex N-glycans, much like sialic acid. Structures in which the monosaccharide sequence is substituted with both sialic acid and fucose have not been observed.

Fig. 3.8. Biosynthesis of galactose-containing N-glycans, Tr — transferase (for Abbreviations, see Table 1.1).

The starting point for the assembly of complex N-glycans is the Structure containing five mannose residues (Fig. 3.8). Harry Schachter and his coworkers identified a specific ß-N-acetylglucosaminyltransferase that utilizes this substrate by transferring a monosaccharide residue from UDP-N-acetylglucosamine to the a-mannose unit, forming an (a1→3)-glycosidic bond in the core region [35]. This enzyme is known as N-acetylglucosaminyltransferase I, or glcNAc-TrI. Experiments using Cell-free systems or purified Enzymes have demonstrated that the five-mannose structure is a viable substrate for this enzyme, although other high-mannose glycopeptides and Glycoproteins can also serve as acceptors in this reaction. The addition of the N-acetylglucosamine residue triggers further Processing of the oligosaccharide generated by the action of a-mannosidase. This a-mannosidase (or a-mannosidases) has been obtained in purified form. It has been shown to hydrolyze (a1→3)- and (a1→6)-bonds, whereas mannosyl-(a1→2)-bonds are not cleaved by this enzyme, which clearly differs from the enzymes involved in earlier processing reactions (Fig. 3.6). The product of a-mannosidase action is an asymmetric sequence containing two a-mannosyl residues linked to the core region. This structure serves as a substrate for another specific N-acetylglucosaminyltransferase, namely II, or glcNAc-TrII. Subsequent reactions involve The transfer of a galactose residue from UDP-galactose to the N-acetylglucosamine residues at C-4, forming N-acetyllactosamines typical of many complex N-glycans. A galactosyltransferase capable of synthesizing the galß1→3glcNAc sequence at these same sites of the monosaccharide chains has been recently described [36], as such a sequence has been detected in certain N-glycans. Thus, glcNAc-transferase I is the key enzyme in converting high-mannose N-glycans into complex chains.

Fig. 3.9. Carbohydrate chains of bovine rhodopsin. R — glcNAcß1→4glcNAc.Asn.

Another type of control in N-glycan assembly is found in The biosynthesis of rhodopsin, the major glycoprotein comprising the disc membranes of the rod outer segments in the retina. The Cells responsible for the biosynthesis of this protein apparently exhibit low a-mannosidase activity, which is reflected in The structure of the rhodopsin N-glycan (Fig. 3.9). Although it is evident that the product of N-acetylglucosaminyltransferase I is formed in these cells and processing occurs, the second N-acetylglucosamine is absent.

The reaction catalyzed by N-acetylglucosaminyltransferase I appears to govern another important Modification of the glycan. Fucosyltransferase activity has been detected in cell extracts, whereby a fucose residue is attached via an (a1→6)-linkage to the N-acetylglucosamine residue linked to asparagine [11, 35]. This transferase can act on the Man₃glcNAc2.Asn core structure only when at least one N-acetylglucosamine residue is attached to the a-mannose residue. In other words, N-acetylglucosaminyltransferase I must act before the corresponding sequence becomes accessible to the action of the specific fucosyltransferase (Fig. 3.8). This accounts for the observation that such a linkage is absent in mannose-only N-glycans.

Fig. 3.10. Regulation of terminal reactions in N-glycan biosynthesis (for R, see Fig. 3.9).

The final stages of complex N-glycan assembly involve the addition of sialic acids and fucose using CMP-sialic acid and GDP-fucose, respectively. Various sialic acid derivatives are known to reside at the non-reducing termini of N-glycans, linked glycosidically to C-3 or C-6 of the preceding galactose residues. Different sialyltransferases presumably catalyze The formation of these glycosidic bonds. Further structural diversity is subsequently generated by modifications of the sialic acids—for instance, through The oxidation of N-acetyl groups to N-glycolyl groups, or via the addition of O-acetyl groups, which may occur after the transfer of sialic acids to the glycan chain.

Studies on The activity of a purified sialyltransferase that forms (a2→6)-glycosidic bonds with galactose revealed [38] a competitive interaction with fucosyltransferase (Fig. 3.10). Terminal galactosyl-ß1→4N-acetylglucosamine-type sequences can serve as acceptors for either sialic acid or fucose. These two reactions are mutually exclusive, shedding light on the recently elucidated N-glycan structures in salivary a-amylase (Fig. 3.11). Fucal→3GlcNAc is also present in the N-glycan of orosomucoid (Fig. 2.1) and in other glycoproteins.

Several major challenges remain unresolved. First, no information has yet been obtained regarding the N-acetylglucosaminyltransferase that adds residues to the ß-mannose units in hybrid structures, or to C-4 and C-6 of the a-mannose residue in highly branched complex N-glycans, such as orosomucoid (Fig. 2.11). However, it is highly likely that these are distinct enzymatic activities, different from transferases I and II. Second, we still do not understand how processing and termination are regulated relative to THE POSITION OF the asparagine residue within the polypeptide chain.

Fig. 3.11. Structure of N-glycans from parotid gland a-amylase [39].