Important note: Information in this article was accurate in 1991. The state of the art may have changed since the publication date.
Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.
Biochemistry. 1991 Mar 5;30(9):2395-406. Unique Identifier : AIDSLINE MED/91159419 Spellman MW; Leonard CK; Basa LJ; Gelineo I; van Halbeek H; Department of Medicinal and Analytical Chemistry, Genentech,; Inc., South San Francisco, California 94080.
Abstract:
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.
Keywords: Amino Acid Sequence Animal *Antigens, CD4/GENETICS Carbohydrate Conformation Carbohydrate Sequence Cell Line Cricetulus Female Glycopeptides/ISOLATION & PURIF Glycoside Hydrolases Hamsters Human Molecular Sequence Data Nuclear Magnetic Resonance Oligosaccharides/*CHEMISTRY/ISOLATION & PURIF Ovary Peptide Mapping Recombinant Proteins/CHEMISTRY Support, U.S. Gov't, P.H.S. Transfection Trypsin JOURNAL ARTICLE 910630
M9160349
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Important note: Information in this article was accurate in 1991. The state of the art may have changed since the publication date.
