Journal
JOURNAL OF PROTEOME RESEARCH
Volume 12, Issue 3, Pages 1223-1234Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr300870t
Keywords
glycopeptide; mass spectrometry; gp120; HIV; O-linked glycan; glycosylation site; ETD; CID; immunology
Categories
Funding
- NIH [R01RR026061, 1R01A1094797, PO1A161734]
- Center for HIV/AIDS Vaccine Immunology [U19AI067854]
- Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery grant from the Division of AIDS, NIAID, NIH [UM1AI100645]
- Vaccine Discovery Center of the Collaboration for AIDS Vaccine Development Program grant from the Bill and Melinda Gates Foundation [OPP1033098s]
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Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study with the inclusion of a fragmentation technique, electron transfer dissociation (ETD), to complement collision-induced dissociation. The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation, two sites (N386 and N392) in the V4 region were populated with high mannose glycans in the CHO cell derived 1086.C gp120, while these sites had a mixture of high mannose and processed glycans in the 293T cell derived 1086.C gp120. Compositional analysis of O-linked glycans revealed that 293T cell derived 1086.C gp120 consisted of core 1, 2, and 4 type O-linked glycans, while CHO cell derived 1086.C exclusively consisted of core 1 type O-linked glycans. Overall, glycosylation site occupancy of the CHO and 293T cell derived 1086.C gp120 showed a high degree of similarity except for one site at N88 in the C1 region. This site was partially occupied in 293T-gp120 but fully occupied in CHO-gp120. Site-specific glycopeptide analysis of transmitted/founder 1086.C gp120 expressed in CHO cells revealed the presence of phosphorylated glycans, while 293T cell-produced 1086.C gp120 glycans were not phosphorylated. While the influence of phosphorylated glycans on immunogenicity is unclear, distinguishing host-cell specific variations in glycosylation profiles provide insights into the similarity (or difference) in recombinant vaccine products. While these differences had minimal effect on envelope antigenicity, they may be important in considering immunogenicity and functional capacities of recombinant envelope proteins produced in different expression systems.
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