Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.

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.

• Authors: M W Spellman, C K Leonard, L J Basa, I Gelineo, H van Halbeek
• Journal: Biochemistry (Biochemistry) Vol. 30 Issue 9 Pg. 2395-406 (Mar 05 1991) ISSN: 0006-2960 [Print] United States
• PMID: 2001369 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• CD4 Antigens
• Glycopeptides
• Oligosaccharides
• Recombinant Proteins
• Trypsin
• Amidohydrolases
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

Topics

• Amidohydrolases
• Amino Acid Sequence
• Animals
• CD4 Antigens (genetics)
• Carbohydrate Conformation
• Carbohydrate Sequence
• Cell Line
• Cricetinae
• Cricetulus
• Female
• Glycopeptides (isolation & purification)
• Humans
• Magnetic Resonance Spectroscopy
• Molecular Sequence Data
• Oligosaccharides (chemistry, isolation & purification)
• Ovary
• Peptide Mapping
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
• Recombinant Proteins (chemistry)
• Transfection
• Trypsin