Fig. 2: Characterization of a gp120 glycotope presented by MHCII.

a–c MS/MS spectra of a gp120 variable-region (V2) glycopeptide (designated GpepIP) identified from MHCII-bound epitopes shows b- and y-ions defining peptide sequence KLDVVPIDNNN₁₈₇TSY in the non-protease group a, peptide sequence LDVVPIDNNN₁₈₇TSYR in the trypsin-treated group b, peptide sequence LDVVPIDNNN₁₈₇TSYR in the chymotrypsin-treated group c, and the N-glycosylation site at N₁₈₇ in all three groups. The @ symbol denotes that the conversion of an Asn residue to Asp with a heavy oxygen was detected at the indicated position. d, e Binding of the GpepIP epitope to MHCII molecule was verified. d Purified MHCII monomers (mouse allele I-A^d) were loaded with an equal amount of the indicated peptides. Peptide-loaded MHCII heterodimers were detected by running of complexes in IEF gel and immunoblotting with mouse MHCII antibody. Representative results are shown from one of three independent experiments performed. e Bands corresponding to GpepIP/MHCII, pepIP/MHCII, and OVA peptide/MHCII complexes were excised from IEF gels and subjected to LC–MS/MS analysis. Extracted ion chromatograms demonstrate the binding of three GpepIP glycoforms (M5N2 being the most abundant). PepIP and the positive control (OVA peptide) also bound and stabilized MHCII heterodimers. Source data are provided as a Source Data file.