Diversity of Oligosaccharide Structures on the Envelope Glycoprotein gp120 of Human Immunodeficiency Virus 1 from the Lymphoblastoid Cell Line H9 PRESENCE OF COMPLEX-TYPE OLIGOSACCHARIDES WITH BISECTING N-ACETYLGLUCOSAMINE RESIDUES*

The N-linked oligosaccharide structures on the en- velope glycoprotein gp120 of human immunodefi- ciency virus 1 derived from chronically infected lymphoblastoid (H9) cells have been investigated by enzy- matic microsequencing after release from protein by hydrazinolysis, labeling with NaB3H4, and chromatog- raphy on adsorbent columns of Phase&s vulgaris erythrophytohemagglutinin and Ricinus communis ag- glutinin (iV& 120,000) and on Bio-Gel P-4.

of human immunodeficiency virus 1 (HIV-l)' is the surface component with a key role in viral adhesion and the initiation of infection through interaction with the CD4 glycoprotein of T lymphocytes (l-4). gpl20 is richly glycosylated, with approximately half the molecular mass consisting of carbohydrate distributed on some 20 N-glycosylation sites (5). Detailed structural characterization by methylation analyses and enzymatic microsequencing of NaB3H4-labeled oligosaccharides after release by hydrazinolysis from recombinant gp120 produced in Chinese hamster ovary cells (rgp120) has been reported (6, 7). A diversity of structures were identified including high mannose-type (Mans to Man9 structures amounting to -33%) and hybrid-type (4%) chains as well as four categories of complex-type chains (mono-, bi-, tri-, and tetraantennary) with or without N-acetyllactosamine repeats and with or without core region fucose residues among which digalactosyl biantennary structures predominated (34%). Altogether, 29 structures were identified after desialylation. The actual number of oligosaccharides is much greater (estimated in excess of 100) since before desialylation, there was evidence that among the hybrid-and complex-type chains, all but 6% contained sialic acid at C-3 in terminal galactose residues, and partially sialylated forms of the bi-and multiantennary chains were present. In another study (8) gp120 produced by human T cells (H9) persistently infected with the HTLV-IIIB strain of HIV-l were investigated. In that study, the oligosaccharides had been enzymatically released with endo-/3-N-acetylglucosaminidase H and peptide N-glycosidase F from two preparations of gp120 derived (a) from cell-free culture supernatants that had been metabolically labeled with n-[6-3H]glucosamine and (b) from cell lysates metabolically labeled with D-[2-3H]mannose. With both radioactive labels, high mannose-type oligosaccharides (Man7 to Mans) were identified.
These accounted for almost 50% of the labeled oligosaccharides detected in the cell-free preparation in which additional complex-type structures (bitri-, and tetraantennary sialo-oligosaccharides) were identiked. With the cell-associated preparation, Man7 to Man9 structures were the predominant labeled species detected (almost 80%), and the remaining oligosaccharides were not identified. In this study, the oligosaccharide structures of cellassociated gp120 (cgpl20) isolated from H9 cells chronically infected with the HTLV-IIIB strain of HIV-l have been investigated by enzymatic microsequencing of the chains released by hydrazinolysis and radiolabeled with NaB3H4. We describe here observations of an even greater diversity of oligosaccharide structures than hitherto reported. Among these are complex-type chains with bisecting N-acetylglucosamine residues. EXPERIMENTAL PROCEDURES AND RESULTS'

DISCUSSION
The salient conclusion from this study is that there is an enormous diversity of oligosaccharide structures among gp120 molecules produced by H9 cells that are chronically infected by HIV-l (Fig. 7). The neutral components, which could be readily separated from the acidic components by paper electrophoresis, constituted -60% of the oligosaccharides released by hydrazinolysis and labeled with NaB3H4. These were identified as a series of high mannose-type oligosaccharides (Man,GlcNAcz to MansGlcNAc2). The acidic components were identified as an array of sialylated oligosaccharides which were all rendered neutral after sialidase treatment. Sequential chromatography on lectin affinity columns (Phu.seolus vulgaris erythrophytohemagglutinin and Ricinus communk agglutinin (M, 120,000)) and on Bio-Gel P-4 revealed that the diversity of oligosaccharides was far greater than was observed with oligosaccharides derived from recombinant gp120 produced in Chinese hamster ovary cells (rgpl20) investigated similarly (6, 7). Biantennary complex-type oligosaccharides which were major components (34%) in Chinese hamster ovary cell-derived rgpl20 were minor components (2%) in cgpl20. In addition to hybrid-type (1.8% in cgpl20) and tri-and tetraantennary complex-type (3.6%) structures common to rgpl20 and cgpl20, oligosaccharides with bisecting N-acetylglucosamine residues were detected in cgpl20. These included bi-, tri-, and tetraantennary complex-type structures with and without core region fucose residues and amounted to 16% of the total oligosaccharides from cgpl20. Just over 80% of the cgpl20 oligosaccharides could be identified after desialylation in this study. The remaining oligosaccharides (a heterogeneous array of complex-type chains having one or more outer chains with galactose residues) could not be further characterized due to the small amounts available. The resistance of some of these oligosaccharides to further digestion with mixtures of @-galactosidase and &N-acetylglucosaminidase suggests the presence of other substituents on the N-acetylglucosamine or galactose residues of their outer chains.
The detection of a higher proportion (80%) of high mannose-type oligosaccharides in a preparation of gpl20 from H9 cells in an earlier study (8) may well reflect the different labeling procedure used, i.e. metabolic labeling of the mannose residues with a resultant bias in favor of high mannose-type structures. The inclusion of a lentil lectin affinity step in the purification of the glycoprotein in this study may, on the contrary, have favored the enrichment of glycosylation variants with complex-type chains. Another difference in procedure that may account for the under-representation of complex-type oligosaccharides in the earlier study is the method of oligosaccharide release from peptide: the enzymatic release in the earlier study uers'sus the more exhaustive chemical release in this study. A further consideration is a possible divergence in the glycosylation patterns of the repeatedly subcultured H9 cells. Nevertheless, the overall conclusion that can be drawn from this study and previous structural studies is that there is a great diversity of oligosaccharides present and that alternative structures are likely to occur on at least some of the glycosylation sites of gp120. Hence, many glycosylation variants are likely to exist in the glycoprotein even when produced in a single cell line. Viral glycosylation is the product of host cell glycosyltransferases. Therefore, in infected individuals, innumerable glycosylation variants of gpl20 would be predicted to arise from the heterogeneous cell populations harboring the virus.
The functional significance of the extensive glycosylation and of the diversity of structures on the envelope glycoprotein is an important subject for investigation. Whereas there is evidence (9, 27) that glycosylation of gp120 is a prerequisite for binding to the host cell glycoprotein CD4 receptor and that deglycosylation procedures abolish (9) or impair (28) binding, the precise roles of the oligosaccharides in this and other recognitive interactions in HIV infection are not yet known. As discussed earlier (6), the various oligosaccharide structures are potential ligands for carbohydrate-binding proteins of the host (endogenous lectins). Carbohydrate-mediated reactivities of g-p120 with two proteins of the host have been documented thus far. The first is with the serum lectin known as mannose-binding protein (29, 30), where the involvement of high mannose-type oligosaccharides of both rgpl20 and cgpl20 has been demonstrated (30). It has been suggested that such interaction on the virus particle is a potential inhibitor of HIV-l infection of CD4' cells (29) and a potential means of viral entry into CD4-cells (30). The second carbohydrate-mediated interaction, shown with gp120 (30), is with the endocytosis receptor of human macrophage membranes. Here it has been suggested (30) that high affinity binding (which would be predicted to occur with glycosylation variants of the viral envelope that are rich in the accessible mannose, N-acetylglucosamine, or fucose residues recognized (31, 32) by this receptor) may lead to viral uptake by macrophages irrespective of the presence of the membrane-associated CD4 receptor. Oligosaccharides of the Envelope Glycoproteingp120 of HIV-l