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.

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

“…The mass accuracy for the smallest signal labeled, Hyb 4, is better than 0.05%. This mass accuracy combined with previously obtained oligosaccharide structural information is sufficient to infer the structures shown in Table II and III (Spellman et al, 1989). All structures reported by Spellman et al (1989) are observed in the two mass spectra (Figures 4 and 5).…”

“…This mass accuracy combined with previously obtained oligosaccharide structural information is sufficient to infer the structures shown in Table II and III (Spellman et al, 1989). All structures reported by Spellman et al (1989) are observed in the two mass spectra (Figures 4 and 5). No ions corresponding to losses of H 2 O are observed in Figure 4 indicating that lactone formation of sialylated oligosaccharides does not occur during isolation.…”

“…Furthermore, the 96-well format offers the potential for automation of the assay. We have chosen rt-PA as the model glycoprotein for development of this assay, because rt-PA contains high mannose, complex, and hybrid oligosaccharides (Spellman et al, 1989) When using this method as a high-throughput assay, 50 µg of each glycoprotein sample is typically loaded into an individual well. According to the manufacturer, the capacity of an individual well is 25 µg for bovine serum albumin, and 47 µg for an immunoglobulin.…”

“…Structures and average molecular weights of the neutral N-linked oligosaccharides from rt-PA *The abbreviation used for the structures are: Gal, galactose; GLcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose. The structures are based upon the known structures of rt-PA determined by 1 H-NMR (Spellman et al, 1989).…”

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain

“…A d 8 4 is only partially glycosylated leading to glycoprotein variants with two or three oligosaccharide chains. Carbohydrate structure analyses of tPA from Bowes melanoma cells (Pohl et al, 1987;Parekh et al, 1989a;Chan et al, 1991) or human colon fibroblasts (Parekh et al, 1989a) as well as recombinant tPA from Chinese hamster ovary cells (Spellman et al, 1989;Chan et al, 1991) or C127 cells (Pfeiffer et al, 1989) demonstrated a site-specific glycosylation of this enzyme in so far as Asnll7 carries almost exclusively high-mannosetype glycans, whereas Asnl84 and Asn448 bear preponderantly complex-type sugar chains. Furthermore, melanomaderived tPA as well as recombinant tPA expressed in Chinese hamster ovary or human embryonic kidney cells were found to carry an O-glycosidically linked fucose residue in their domain which resembles epidermal-growth-factor (EGF) (Harris et al, 1991).…”