Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes

Glycoproteins isolated from hen oviduct contain highly branched asparagine-linked oligosaccharides (N-glycans). Six N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V, and VI) are involved in initiating the synthesis of these branches, as indicated below:[Formula: see text]where R is GlcNAcβ1—4(+/−Fucα1—6)GlcNAcAsn-X. HPLC has been used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct. Oligosaccharides with free reducing GlcNAc termini were prepared from various glycoproteins by hydrazinolysis–re-N-acetylation and used as GlcNAc-T substrates and HPLC standards. Enzyme assay components were separated on AG1 × 8, followed by HPLC on amine-bonded silica columns eluted with acetonitrile–water mixtures. Absorbance at 195 nm and radioactivity of eluted compounds were monitored. Substrates and products were identified by comparison of their retention times with those of oligosaccharides with known structures. Enzyme assay by HPLC is more rapid and convenient than previous GlcNAc-T assays using lectin columns or electrophoresis. Since some substrates yielded multiple products, these could be used to assay more than one GlcNAc-T in the same incubation. GlcNAc-T VI was shown to act on both bisected and nonbisected GlcNAc-terminating tetraantennary oligosaccharide substrates; GlcNAc-T II, IV, and V acted poorly or not at all on bisected substrates. GlcNAc-T V was the only enzyme among the six transferases studied that could be assayed in the absence of Mn2+.

Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(β 1-4)GlcNAc terminus by the Gal(β 1-4)GlcNAc(NeuAc-Gal) (α 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.