The E3-20.5K membrane protein of subgroup B human adenoviruses contains O-linked and complex N-linked oligosaccharides.
Subgroup B adenoviruses (Ad3, -7, -11, -35) contain two open reading frames (ORFs) in the early E3 transcription unit that are not present in subgroup C adenoviruses (Ad2, Ad5). The product of one of these ORFs, a 20,500-kDa (20.5K) protein, was shown previously to be expressed as two diffuse 22K and 36K bands on SDS-PAGE; the 22K appeared to be the precursor to the 36K species. As judged by its predicted sequence, 20.5K is a type I membrane glycoprotein with two potential sites for N-glycosylation and a transmembrane domain near its COOH-terminus. Here we show that when Ad3- or Ad7-infected cells were radiolabeled in the presence of tunicamycin, which prevents the addition of N-linked oligosaccharides, both the 22K and the 36K forms of 20.5K showed increased mobility in SDS-PAGE, indicating that both forms contain N-linked sugars. Both the 22K and the 36K forms were sensitive to digestion by endoglycosidase F and N-glycanase, again indicating that they both contain N-linked sugars. Only the 22K species was sensitive to endoglycosidase H, indicating that it contains high-mannose-type oligosaccharides and that the 36K species contains complex-type carbohydrates. The 36K form was sensitive to neuraminidase, indicating that its sugars contain terminal sialic acid. When digested with N-glycanase and neuraminidase, the 36K form was sensitive to O-glycanase, indicating that the 36K form has O-linked oligosaccharides. The 22K form was labeled with [3H]mannose and the 36K form was labeled with [3H]glucosamine and to a much lesser extent by [3H]mannose. Altogether these results indicate that the 20.5K protein is cotranslationally modified with N-linked high-mannose oligosaccharides, then the protein moves into the Golgi and trans-Golgi network where it acquires O-linked and complex N-linked oligosaccharides.
Hawkins
L K
LK
Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, Missouri 63104, USA.
Wold
W S
WS
D017503:Adenovirus E3 Proteins / Q000032:analysis / Q000737:chemistry*; D000260:Adenoviruses, Human / Q000737:chemistry*; D000581:Amidohydrolases; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases; D006801:Humans; D007624:KB Cells; D008358:Mannose / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D021382:Protein Sorting Signals / Q000235:genetics; D012794:Sialic Acids / Q000032:analysis; D014415:Tunicamycin
D017503:Adenovirus E3 Proteins; C094646:E3-20.5K membrane protein, Adenovirus; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D012794:Sialic Acids; D014415:Tunicamycin; D006596:Hexosaminidases; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid; D005944:Glucosamine; D008358:Mannose