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Published on: December 28, 2015
Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells
M W Spellman1, C K Leonard, L J Basa
1Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Insights
This study details the N-linked oligosaccharide structures on recombinant CD4 (rCD4), a potential treatment for acquired immunodeficiency syndrome (AIDS). Researchers identified distinct complex and hybrid structures at glycosylation sites Asn-271 and Asn-300, crucial for rCD4 function.
Area of Science:
- Glycobiology
- Virology
- Biochemistry
Background:
- Human immunodeficiency virus (HIV) infects T-lymphocytes and macrophages by binding to the CD4 receptor.
- Soluble recombinant CD4 (rCD4) is a potential therapeutic agent for acquired immunodeficiency syndrome (AIDS).
- The N-linked oligosaccharides of rCD4 can influence its biological activity and therapeutic potential.
Purpose of the Study:
- To elucidate the structures of the Asn-linked oligosaccharides present on soluble recombinant CD4 (rCD4).
- To characterize the glycosylation patterns at the two potential N-glycosylation sites (Asn-271 and Asn-300) of rCD4.
- To provide insights into the structural heterogeneity of rCD4 oligosaccharides for potential therapeutic applications.
Main Methods:
- Purification of tryptic glycopeptides containing N-glycosylation sites using reversed-phase HPLC.
- Enzymatic release of oligosaccharides from purified glycopeptides.
- Structural determination using methylation analysis, high-pH anion-exchange chromatography, mass spectrometry (MS), and 1H NMR spectroscopy.
Main Results:
- Asn-271 primarily carries diantennary N-acetyllactosamine ('complex') oligosaccharides, with varying degrees of sialylation (8% asialo, 55% monosialyl, 37% disialyl).
- Approximately 18% of structures at Asn-271 contained fucose linked to the reducing GlcNAc residue.
- Asn-300 exhibits a mix of hybrid structures (34%) and diantennary N-acetyllactosamine structures (66%), with distinct sialylation and fucosylation patterns compared to Asn-271.
Conclusions:
- The study successfully characterized the complex and hybrid oligosaccharide structures at both N-glycosylation sites of rCD4.
- The elucidated structures provide critical information regarding the post-translational modifications of rCD4.
- Understanding these glycosylation patterns is essential for optimizing the therapeutic efficacy of rCD4 in AIDS treatment.
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.
