Cargando…

Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation

[Image: see text] The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen,...

Descripción completa

Detalles Bibliográficos
Autores principales: Go, Eden P., Hua, David, Desaire, Heather
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156237/
https://www.ncbi.nlm.nih.gov/pubmed/25026075
http://dx.doi.org/10.1021/pr5003643
_version_ 1782333698208694272
author Go, Eden P.
Hua, David
Desaire, Heather
author_facet Go, Eden P.
Hua, David
Desaire, Heather
author_sort Go, Eden P.
collection PubMed
description [Image: see text] The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env’s PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens.
format Online
Article
Text
id pubmed-4156237
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher American Chemical Society
record_format MEDLINE/PubMed
spelling pubmed-41562372015-07-15 Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation Go, Eden P. Hua, David Desaire, Heather J Proteome Res [Image: see text] The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env’s PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens. American Chemical Society 2014-07-15 2014-09-05 /pmc/articles/PMC4156237/ /pubmed/25026075 http://dx.doi.org/10.1021/pr5003643 Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)
spellingShingle Go, Eden P.
Hua, David
Desaire, Heather
Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title_full Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title_fullStr Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title_full_unstemmed Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title_short Glycosylation and Disulfide Bond Analysis of Transiently and Stably Expressed Clade C HIV-1 gp140 Trimers in 293T Cells Identifies Disulfide Heterogeneity Present in Both Proteins and Differences in O-Linked Glycosylation
title_sort glycosylation and disulfide bond analysis of transiently and stably expressed clade c hiv-1 gp140 trimers in 293t cells identifies disulfide heterogeneity present in both proteins and differences in o-linked glycosylation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156237/
https://www.ncbi.nlm.nih.gov/pubmed/25026075
http://dx.doi.org/10.1021/pr5003643
work_keys_str_mv AT goedenp glycosylationanddisulfidebondanalysisoftransientlyandstablyexpressedcladechiv1gp140trimersin293tcellsidentifiesdisulfideheterogeneitypresentinbothproteinsanddifferencesinolinkedglycosylation
AT huadavid glycosylationanddisulfidebondanalysisoftransientlyandstablyexpressedcladechiv1gp140trimersin293tcellsidentifiesdisulfideheterogeneitypresentinbothproteinsanddifferencesinolinkedglycosylation
AT desaireheather glycosylationanddisulfidebondanalysisoftransientlyandstablyexpressedcladechiv1gp140trimersin293tcellsidentifiesdisulfideheterogeneitypresentinbothproteinsanddifferencesinolinkedglycosylation