Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility
Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility
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DOI：
10.1021/pr7006957
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发表时间：
2008-04-01
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.;Irungu, Janet;Desaire, Heather
HIV-1包膜（Env）是介导病毒进入和融合宿主细胞的关键决定因素，也是HIV疫苗开发的主要靶点。虽然Env通常是约50质量%的聚糖，但已知糖基化位点会进化，其中一些糖基化谱可能比其他糖基化谱更有效地促进中和逃逸。因此，表征Env和天然病毒体的糖基化模式并将糖基化谱与感染性和Env免疫原性相关联是设计有效免疫原的必要的第一步。在此，我们描述了一种基于质谱的策略来确定HIV-1 Env糖基化模式，并比较了两种哺乳动物细胞表达的重组Env免疫原，一种是有限的免疫原，另一种是诱导交叉交叉中和抗体。我们已经使用了基于糖肽的质量作图方法，通过阐明利用哪些位点以及每个糖基化位点存在何种类型的聚糖基序来鉴定和表征Env的糖基化模式。我们的研究结果表明，免疫原显示不同程度的糖基化，以及不同的特征集的聚糖基序。因此，这些技术可用于（1）确定重组Env蛋白和成熟病毒体上Env的糖基化谱，（2）确定每个糖基化位点的特定碳水化合物部分，和（3）确定某些碳水化合物在HIV-1感染性和调节Env免疫原性中的作用。
The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others.' Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces crossclade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.