The synthesis of complex-type oligosaccharides. II. Characterization of the processing intermediates in the synthesis of the complex oligosaccharide units of the vesicular stomatitis virus G protein.

The synthesis of the complex-type oligosaccharide units of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer, the oligosaccharide precursor, a branched molecule containing 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues (Li, E., Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 7762-7770) is processed to give rise to the mature complex-type oligosaccharide. To elucidate the sequence of processing, pulse-chase experiments were performed with virus-infected Chinese hamster ovary cells using labeled mannose, glucosamine, and galactose. The processing intermediates were isolated and characterized. At 20 to 30 min after labeling, the major processing intermediates are (Glc)l(Man)e(GlcNAc)z and (Man)s(GlcNAc)z. Small amounts of (Man)s(GlcNAc)z and (Man)T(GlcNAc)z were detected along with trace amounts of (Glc)z(Man)s(GlcNAc)z and (Man)s(GlcNAc)z. In addition, by 20 to 30 min completely processed oligosaccharides containing only 3 mannose residues were also present. These data demonstrate that processing is initiated by the rapid removal of 2 of the 3 glucose residues of the precursor oligosaccharide. At 20 to 30 min the last glucose residue is removed and processing proceeds rapidly with the ultimate removal of 6 of the 9 mannose residues and the addition of the outer branch sugars. Clone 15B cells, a line deficient in UDP-GlcNAc: glycoprotein N-acetylglucosaminyltransferase I, accumulate an intermediate with the structure Mancvl+ 3 (Manal--+ 6) Manal--+ 6 (Mancul -+ 3) Ma@1 += 4 GlcNAc fil+ 4 GlcNAc. This indicates that normal processing requires the transfer of a Nacetylglucosamine residue to the mannose residue linked cwl + 3 to the P-linked core mannose. Following transfer of the N-acetylglucosamine residue, an Ly-mannosidase removes the 2 mannose residues linked to the mannose which is linked cul + 6 to the core mannose. Direct evidence for this reaction is presented in the following paper (Tabas, I., and Kornfeld, S. (1978) J. Biol. Chern. 253, 7779-7786). Complex oligosaccharide biosynthesis is then completed by the addition of the final outer Wacetylglucosamine residues and the galactose, sialic acid, and fucose residues.