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 ov...

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Bibliographic Details
Published in:Biochemistry (Easton) 1991-03, Vol.30 (9), p.2395-2406
Main Authors: Spellman, Michael W, Leonard, Cordelia K, Basa, Louisette J, Gelineo, Ivana, Van Halbeek, Herman
Format: Article
Language:English
Subjects:
AIDS/HIV
Amidohydrolases
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Carbohydrate Conformation
Carbohydrate Sequence
CD4 Antigens - genetics
Cell Line
Cricetinae
Cricetulus
Female
Fundamental and applied biological sciences. Psychology
Glycopeptides - isolation & purification
Glycoproteins
Humans
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Oligosaccharides - chemistry
Oligosaccharides - isolation & purification
Ovary
Peptide Mapping
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Proteins
Recombinant Proteins - chemistry
Transfection
Trypsin
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00223a015