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Purification of processing protease for retroviral envelope glycoprotein and role of it in the process of retroviral infection

逆转录病毒包膜糖蛋白加工蛋白酶的纯化及其在逆转录病毒感染过程中的作用

基本信息

批准号：
03454162
负责人：
KIDO Hiroshi
金额：
$ 3.9万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

A processing protease for the HIV-1 envelope glycoprotein gp160 precursor has been purified to homogeneity from the post-nuclear membrane fraction of human T4^+ lymphocyte clone. Most of the processing activity was found to be present in the fractions of endoplasmic reticulum and Golgi appratus of the cells. The purified enzyme has a monomeric structure with a molecular mass of 26*3kDa, as judged by gel-permeation liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The purified enzyme converted gp160 to gp120 and gp41, showing pH optimum of 6.5-7.0. Direct amino acid sequence of the amino terminus of the product gp41 revealed that the cleavage site of gp160 was between Arg^<511> and Ala^<512>. The enzyme activity was inhibited by trypsin-type protease inhibitors and by SH reagents, such as dithiothreitol, cysteine and N-acetyl L-cystein, but was not affected by CaCl2, MgCl2 or chelating agents. The properties of the purified enzyme are clearly distinct from those of processing proteases reported previously. Judging from its cleavage specificity and subcellular localization, this endopeptidase appears to be a processing enzyme for the HIV-1 gp160 precursor protein in human T cells. In cell culture system, low molecular weight protease inhibitors, such as diisopropylfluorophosphate, cystein and N-acetyl L-cystein, inhibited the processing of gp160 and the transportation of gp120 to the plasma membrane.

HIV-1包膜糖蛋白gp 160前体的一种加工蛋白酶已从人T4^+淋巴细胞克隆的核膜后组分中纯化至均一。大部分的加工活动被发现存在于内质网和高尔基体的细胞的组分。通过凝胶渗透液相色谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳在还原和非还原条件下判断，纯化的酶具有分子量为26* 3 kDa的单体结构。纯化的酶将gp 160转化为gp 120和gp 41，显示最适pH为6.5-7.0。对产物gp 41的氨基端进行直接氨基酸序列测定，结果表明gp 160的切割位点位于Arg-1<511>和Ala-1之间<512>。胰蛋白酶型蛋白酶抑制剂和SH试剂，如二硫苏糖醇，半胱氨酸和N-乙酰L-半胱氨酸抑制酶的活性，但不受CaCl 2，MgCl 2或螯合剂。纯化的酶的性质明显不同于先前报道的加工蛋白酶的性质。从其切割特异性和亚细胞定位判断，这种内肽酶似乎是人类T细胞中HIV-1 gp 160前体蛋白的加工酶。在细胞培养体系中，低分子量蛋白酶抑制剂如二异丙基氟磷酸、半胱氨酸和N-乙酰L-半胱氨酸等抑制gp 160的加工和gp 120向质膜的转运。