Document Detail


Characterization of host-cell line specific glycosylation profiles of early transmitted/founder HIV-1 gp120 envelope proteins.
MedLine Citation:
PMID:  23339644     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Eden P Go; Hua-Xin Liao; S Munir Alam; David Hua; Barton F Haynes; Heather Desaire
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-02-20
Journal Detail:
Title:  Journal of proteome research     Volume:  12     ISSN:  1535-3907     ISO Abbreviation:  J. Proteome Res.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-01     Completed Date:  2013-08-29     Revised Date:  2014-03-06    
Medline Journal Info:
Nlm Unique ID:  101128775     Medline TA:  J Proteome Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1223-34     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
CHO Cells
Chromatography, Liquid
Cricetinae
Cricetulus
Glycosylation
HEK293 Cells
HIV Envelope Protein gp120 / chemistry,  metabolism*
Humans
Mass Spectrometry
Molecular Sequence Data
Sequence Homology, Amino Acid
Surface Plasmon Resonance
Grant Support
ID/Acronym/Agency:
1R01AI094797/AI/NIAID NIH HHS; P01AI61734/AI/NIAID NIH HHS; R01 AI094797/AI/NIAID NIH HHS; R01 GM103547/GM/NIGMS NIH HHS; R01 RR026061/RR/NCRR NIH HHS; R01RR026061/RR/NCRR NIH HHS; U19AI067854/AI/NIAID NIH HHS; UM1 AI100645/AI/NIAID NIH HHS; UM1AI100645/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/HIV Envelope Protein gp120
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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