Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry

Abstract

Structural characterization of the HIV-1 envelope protein gp120 is very important for providing an understanding of the protein's immunogenicity and its binding to cell receptors. So far, the crystallographic structure of gp120 with an intact V3 loop (in the absence of a CD4 coreceptor or antibody) has not been determined. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for coreceptor binding and entry of the virus into T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated the glycosylation occupancy of gp120-OD8; 11 sites from 15 glycosylation motifs were determined as having high-mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information about protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4-gp120-OD8 crystal structure revealed a flexible V3 loop structure in which the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure.

Figure 1

SPR kinetic analysis of binding of mAb IgG1b12 to gp120-OD8. mAb IgG1b12 was immobilized on a CM-5 chip, and gp120-OD8 at various concentrations was passed over the chip surface.

Figure 2

4%-20% SDS-PAGE gel of gp120-OD8 (Lane 1), Endo H treated gp120-OD8 (Lane 2) and PNGase F treated gp120-OD8 (bottom band in Lane 3). Upper band in Lane 3 is PNGase F (Mw: 36 kDa)

Figure 3

Identification of glycosylation sites using Endo H treatment. 3a is MS/MS spectrum of the glycopeptide precursor ion at m/z of 931.9750 Da. 3b is MS/MS spectrum of unmodified peptide of the same sequence (RSTQLLLNGSLAEEE [1-15]).

Figure 3

Identification of glycosylation sites using Endo H treatment. 3a is MS/MS spectrum of the glycopeptide precursor ion at m/z of 931.9750 Da. 3b is MS/MS spectrum of unmodified peptide of the same sequence (RSTQLLLNGSLAEEE [1-15]).

Figure 4

Quantiation of glycosylation occupany of Asn 8 in peptide 1-15 (RSTQLLLNGSLAEEE) by use of Endo H treatment. Top: Chromtography peak of unmodified peptide 1-15. Peak area was calculated m/z ranging from 830.4260-830.4360. Bottom: Chromtography peak of glycopeptide 1-15. Peak area was calculated as m/z ranging from 931.9700-931.9800.

Figure 5

Quantitative glycosylation occupancy of gp120-OD8. Glycosylation of 11 sites vary from 0.8% to 99.2%. N191 might contain two types of glycosylation, N136, N142 and N148 were potential glycosylation sites but not identified in Endo H treatment. No glycosylation on N 203 was detected.

Figure 6

Identification of glycopeptides by combination of CID and ECD MS. The peptide sequence was identified as ⁵¹KSIHIGPGQAFYATGEIIGDIRQAHCNISR⁸⁰, Asn 77 was identified as the glycosylation site containing 2 N-acetylgucosamine and ⁹Mannose. 4a is a typical CID MS/MS of the high mannose glycopeptides with many abundant neutral loss peaks and 528 Da (2 Mannose + 1 GlcNac) signature peak. 4b is ECD MS/MS of the glycopeptide providing information of peptide sequence by fragmenting peptides to C ions and Z ions.

Figure 6

Identification of glycopeptides by combination of CID and ECD MS. The peptide sequence was identified as ⁵¹KSIHIGPGQAFYATGEIIGDIRQAHCNISR⁸⁰, Asn 77 was identified as the glycosylation site containing 2 N-acetylgucosamine and ⁹Mannose. 4a is a typical CID MS/MS of the high mannose glycopeptides with many abundant neutral loss peaks and 528 Da (2 Mannose + 1 GlcNac) signature peak. 4b is ECD MS/MS of the glycopeptide providing information of peptide sequence by fragmenting peptides to C ions and Z ions.

Figure 7

Homology Model of the gp120-OD8 including V3, V4 loops and the bridging sheet. Glycosylation sites are identified experimentally by mass spectrometry. Red: high mannose glycosylation sites. Green: oxidized residues in synchrotron radiolysis. Yellow: oxidized residues in N-terminal of V3 loop in synchrotron radiolysis which should be more exposed than other residues including those on V3 tip according to footprinting data. Blue: disulfide bond in V3 loop.