Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

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Abstract
Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2- 3 H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β- N -acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man 9 GlcNAc and Man 8 GlcNAc and small amounts of Man 7 GlcNAc and Man 6 GlcNAc. The pulse-chase sample had predominantly Man 8 GlcNAc and much less Man 9 GlcNAc, indicating that processing of the Man 9 GlcNAc to Man 8 GlcNAc had occurred during the chase period. Thus, Man 8 GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man 9 GlcNAc were identical to those of the lipid-linked Glc 3 Man 9 GlcNAc 2 donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein.