The spectrum of N‐linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells

Abstract

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S. A., Hemling, M. E., Folena-Wasserman, G., Sweet, R. W., Anumula, K., Barr, J. R., Huddleston, M. J. & Taylor, P. (1989) J . B i d . Chem. 264,21286-21 2951. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows : fFucal Gal@l-4GlcNAcpl-2Manal

Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant

“…Had this cell line been secreting a glycoprotein, extracellular degradation by sialidase would have been insignificant under these conditions. Differential exposure to extracellular glycosidases could explain, a t least in part, why the same recombinant glycoprotein produced in CHO cell culture by different groups can have strikingly different oligosaccharide structures-for example, the oligosaccharide structures of t-PA produced by Monsanto and analyzed by the Oxford University group (Parekh et al, 1989) versus t-PA produced and analyzed by Genentech (Spellman et al, 1989) and soluble CD4 produced by Invitron and analyzed at SmithKline (Yuen et al, 1990) versus CD4 produced and analyzed by Genentech (Spellman et al, 1991). For t-PA and CD4, the oligosaccharide structures of the glycoproteins produced by Monsanto and Invitron show decreased monosaccharide content (less sialic acid, less galactose, less fucose, etc.)…”

“…Some of these deficiencies could be attributable to differences in biosynthesis, purification protocol, or even the method of oligosaccharide analysis. However, some of the oligosaccharide structures of the Invitronproduced CD4, including a monoantennary structure (see fraction 11-E in Figure 5 of Yuen et al (1990)), are difficult to explain on the basis of any known biosynthetic route (reviewed in Kornfeld and Kornfeld (1985)), suggesting that extracellular degradation by glycosidases may have occurred in this case.…”

“…Heterogeneity of oligosaccharide structure is typically observed at each N-linked or O-linked glycosylation site, leading to the concept of "g1ycoforms"-that is, a set of molecules with identical amino acid sequences but distinct oligosaccharide structures (Rademacher et al, 1988). Much of the heterogeneity that has been observed among glycoforms of Chinese hamster ovary (CHO) produced glycoproteins such as tissue plasminogen activator (t-PA) (Spellman et al, 1989;Parekhet al, 1989), erythropoietin (EPO) (Sasaki et al, 1988), CD4 (Spellman et al, 1991;Yuen et al, 1990), and interferon-61 (Kagawa et al, 1988) is due to variability in the presence of sialic acid and fucose.…”

Host-Soluble Galectin-1 Promotes HIV-1 Replication through a Direct Interaction with Glycans of Viral gp120 and Host CD4

“…Since Gal-1 rapidly stabilizes HIV-1 attachment onto susceptible target cells (40), it is possible that Gal-1 also binds to glycans attached to the primary cellular receptor of gp120, the CD4 molecule. CD4 carries two N-linked glycans, both of which are CX biantennary chains (64). Thus, to test this possibility, Gal-1 or Gal-3 protein was subjected to CD4 affinity chromatography.…”

“…Despite high genetic variability among different groups of HIV-1, the N-glycosylation sites of gp120 are spatially conserved. Two types of N-linked glycans are found on gp120, namely, oligomannose-type (OM) glycans, which are rich in mannose residues, and complex-type (CX) glycans, which carry 2 to 6 ␤-galactoside residues (i.e., lactosamine residue [LacNAc]) (25,26,41,64). Glycosylation of gp120 exhibits two unique features that distinguish it from glycosylation patterns normally found on host membrane proteins (53).…”