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
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
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DOI：
10.1021/pr5003643
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发表时间：
2014-09-01
影响因子：
4.4
通讯作者：
Desaire, Heather
中科院分区：
文献类型：
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作者：
Go, Eden P.;Hua, David;Desaire, Heather
HIV-1包膜蛋白（Env）介导病毒进入宿主细胞以引发感染，并且是基于抗体的疫苗开发的唯一靶标。已经对各种可溶形式的HIV Env免疫原的设计、工程化和表达进行了大量的努力，但是高度有效的免疫原仍然难以捉摸。开发有效的HIV疫苗的关键挑战之一是Env上存在一组复杂的翻译后修饰（PTM），即糖基化和二硫键，其影响蛋白质折叠、表位可及性和免疫原性。虽然这些PTM随表达系统而变化，但由于表达方法的变化而引起的Env PTM的变化尚未完全确定。在这项研究中，我们比较了进化枝C重组HIV-1 Env三聚体C97 ZA 012 gp 140的二硫键网络和糖基化特征，这些三聚体通过使用结合碰撞诱导解离（CID）和电子转移解离（ETD）的集成质谱工作流程的稳定和瞬时转染表达。位点特异性分析的N-和O-糖基化配置文件显示，C97 ZA 012 gp 140由两种转染方法产生的N-糖基化配置文件和网站占有率显示出高度的相似性，除了一个网站。相比之下，检测到不同的O-糖基化谱。对Env的二硫键网络的分析显示，两种转染方法产生的C97 ZA 012 gp 140采用了针对单体gp 120和gp 41鉴定的预期二硫键模式以及Cl、V1/V2和C2区域中的替代二硫键模式。这些蛋白质中二硫键始终存在异质性的发现也许是这些研究最重要的结果;这种二硫键异质性已在多种其他重组gp 140中报道过，并且它可能存在于大多数重组表达的Env免疫原中。
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 Cl, 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.