Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers
Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers
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DOI：
10.1074/jbc.m112.371898
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发表时间：
2012-07-13
影响因子：
4.8
通讯作者：
Moore, John P.
中科院分区：
文献类型：
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作者：
Depetris, Rafael S.;Julien, Jean-Philippe;Moore, John P.
三聚体包膜糖蛋白复合体(Env)是疫苗开发项目的重点，旨在产生对人类免疫缺陷病毒1型(HIV-1)的保护性体液反应。N-连接的葡聚糖几乎占外部Env结构域分子质量的一半，它产生了相当大的结构异质性，是结晶研究的主要障碍。此外，通过保护多肽骨架，当环境蛋白用作免疫原时，多糖可以阻止针对潜在表位的实质性子集产生中和抗体的尝试。在这里，我们描述了可溶的、裂解的重组Env三聚体的部分脱糖作用，方法是在Env生产过程中抑制复杂N-糖聚糖的合成，然后在保持Env三聚体完整性的条件下用糖苷酶处理。部分脱糖三聚体是稳定的，既不对蛋白水解性消化异常敏感，也不容易聚集。此外，去糖基化的三聚体保留或增加了它们结合CD4和针对构象表位的抗体的能力，包括CD4结合部位和V3区域。然而，正如预期的那样，它们不与糖依赖抗体2G12和PGT123或C型凝集素受体DC-SIGN反应。电子显微镜分析表明，部分去糖基化的三聚体具有类似于完全糖基化的三聚体的结构，表明糖链的去除不会实质上扰乱三聚体的结构完整性。Gly-can缺失的Env三聚体可用于结构和免疫原性研究。
The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The gly-can-depleted Env trimers should be useful for structural and immunogenicity studies.