Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.
Infection of T-lymphocytes and macrophages by human immunodeficiency virus (HIV) is mediated by the binding of the HIV envelope glycoprotein to the cell-surface receptor glycoprotein CD4. A soluble, recombinant CD4 molecule (rCD4), produced by expression of a truncated CD4 gene in Chinese hamster ovary (CHO) cells [Smith et al. (1987) Science 238, 1704-1707], is in clinical trials as a potential therapeutic agent in the treatment of acquired immunodeficiency syndrome (AIDS). In the present study, the structures of the Asn-linked oligosaccharides of soluble rCD4 have been elucidated. The rCD4 molecule has two potential sites for N-glycosylation, Asn-271 and Asn-300. Tryptic glycopeptides containing either of the sites were purified by reversed-phase HPLC, and their oligosaccharides were released enzymatically. The structures of the oligosaccharides were determined by methylation analysis, high-pH anion-exchange chromatography, fast-atom bombardment mass spectrometry, and 1H NMR spectroscopy at 500 MHz. Asn-271 was found to carry diantennary N-acetyllactosamine-type ("complex") oligosaccharides, of which 8% were asialo, 55% were monosialyl, and 37% were disialyl. Approximately 18% of these structures contained fucose alpha(1-->6) linked to the reducing GlcNAc residue. Two different hybrid structures were found to account for 34% of the oligosaccharides attached to Asn-300. The remainder of the oligosaccharides attached to Asn-300 were diantennary N-acetyllactosamine-type, of which 10% were asialo, 61% were monosialyl, and 29% were disialyl. Approximately 9% of the hybrid structures and 40% of the N-acetyllactosamine structures at Asn-300 were found to contain fucose alpha(1-->6) linked to the innermost GlcNAc residue.
Spellman
M W
MW
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Leonard
C K
CK
Basa
L J
LJ
Gelineo
I
I
van Halbeek
H
H
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006020:Glycopeptides / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010053:Ovary; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D014357:Trypsin
D015704:CD4 Antigens; D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase