MODIFICATION OF GLYCOPROTEINS BY N-ACETYLGLUCOSAMINYLTRANSFERASE-V ISGREATLY INFLUENCED BY ACCESSIBILITY OF THE ENZYME TO OLIGOSACCHARIDE ACCEPTORS

The formation of tri- and tetraantennary complex-type N-linked oligosa ccharides in animal glycoproteins is partly regulated by UDP-N-acetylg lucosamine:beta-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (EC 2.4.1.155) (GlcNAc-T V), which generates 2,6-branched mannose. In Chinese hamster ovary (CHO) cells we found that 2,6-branched mannosyl structures are preferentially contained on lysosome-associated membra ne proteins (LAMPs) and are generally low or absent in other cellular glycoproteins (Do, K.-P. and Cummings, R.D. (1993) J. Biol. Chem. 268, 22028-22035). To determine the mechanism by which GlcNAc-T V appears to preferentially recognize glycoproteins, we examined the activity of purified GlcNAc-T V toward a variety of glycoprotein accepters. Becau se GlcNAc-T V requires as accepters oligosaccharides lacking outer gal actosyl and sialyl residues, we utilized two classes of acceptor prepa rations. The first class of acceptor was enzymatically desialylated (D S) and degalactosylated (DG) preparations of bovine fetuin, human tran sferrin, and human fibrinogen. The second class was glycoproteins in e xtracts of the mutant CHO cell line, Lec8 CHO, which cannot add galact ose or sialic acid to N-linked oligosaccharides. GlcNAc-T V was highly active toward DSDG-fetuin, -transferrin, and -fibrinogen (K-m values ranged between 30 and 74 mu M), and the catalytic efficiencies (V-max/ K-m) of the enzyme toward different accepters were comparable. In the case of fetuin, each of its three sites for attachment of N-linked oli gosaccharides were shown to be utilized equally well by GlcNAc-T V. No tably, the enzyme exhibited a 2-3-fold higher rate of transfer toward DSDG-transferrin when it was further denatured by reduction and S-carb oxymethylation. When extracts of Lec8 CHO cells were used as accepters , GlcNAc-T V preferentially transferred to LAMPs, and only low level t ransfer was observed to other cell-derived glycoproteins, thus demonst rating specificity of GlcNAc-T V to ward native glycoprotein accepters . When the cell-derived glycoproteins were denatured by reduction and S-carboxymethylation prior to use as accepters for GlcNAc-T V, signifi cant transfer occurred to other glycoproteins. These results demonstra te that the mechanism of glycoprotein-specific branching by GlcNAc-T V is determined primarily by its accessibility to available bi/trianten nary oligosaccharides on glycoproteins and not by its recognition of p eptide determinants or conformation-specific determinants.