Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase
Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase
复制标题
DOI：
10.1074/jbc.m109.066670
复制
发表时间：
2010-04-30
影响因子：
4.8
通讯作者：
Fenouillet, Emmanuel
中科院分区：
文献类型：
--
作者：
Papandreou, Marie-Jeanne;Barbouche, Rym;Fenouillet, Emmanuel
细胞催化剂Calnexin(CNX)和蛋白质二硫键异构酶(PDI)协同作用建立HIV包膜(Env)糖蛋白的二硫键。在HIV与淋巴细胞结合后，细胞表面的PDI也会降低Env以诱导融合构象。我们试图定义Env和这些催化剂之间的接触点，以阐明它们作为治疗靶点的潜力。在表达Env的细胞裂解产物中，15%的gp160前体与CNX共沉淀，而gp120不与CNX共沉淀，而只有0.25%的gp160和gp120与PDI共沉淀。在模拟病毒/细胞接触过程中的Env/PDI相互作用的体外条件下，PDI很容易与Env结合。然后用CNX或PDI封闭结合部位的抗Env抗体确定Env在细胞内与CNX或体外与PDI相互作用的结构域。针对V1/V2、C2和V3 C末端的抗体不与CNX相关的Env结合，而针对C1、V1/V2和CD4结合域的抗体不与PDI相关的Env反应。此外，后一种抗体的混合物干扰了PDI介导的Env减少。因此，Env通过离散的、基本上非重叠的区域与细胞内的CNX和细胞外的PDI相互作用。相互作用位点解释了针对这两种催化剂的化合物的作用模式，并可能使进一步设计新的竞争性试剂成为可能。
The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonover-lapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.