Li Y, Luo L, Thomas D Y, Kang C Y
Siebens-Drake Research Institute, The University of Western Ontario, London, Ontario, N6G 2V4, Canada.
Virology. 2000 Jul 5;272(2):417-28. doi: 10.1006/viro.2000.0357.
Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide chain, during its transport, into the lumen of the endoplasmic reticulum (ER). We have analyzed the kinetics of the cleavage of the HIV-1 Env protein signal sequence from gp160 and gp120 in HeLa, BHK, and Jurkat cells. Furthermore, we have determined the effects of this cleavage on the association of the gp160 and gp120 glycoproteins with the ER protein calnexin and the effects of the signal sequence cleavage on protein folding. The cleavage of the HIV-1 Env protein signal sequence on both gp160 and gp120 occurred very slowly in all three cell lines with a t(1/2) of 45-60 min. The core glycosylated and signal-sequence-retained forms of gp160 and gp120 associated with calnexin while the signal-sequence-cleaved forms of gp160 and gp120 had disassociated from calnexin and correctly folded as determined by their ability to associate with the CD4 cellular receptor. Further analysis of the folding state of gp160 and gp120 in nonreducing SDS-PAGE revealed that the signal-sequence-retained and calnexin-associated forms of gp160 and gp120 migrated as broad, diffuse bands, whereas the signal-sequence-cleaved or CD4-associated forms of gp160 and gp120 migrated as single sharper bands. The cause of this retardation in the rate of folding and intracellular transport of HIV-1 glycoproteins was localized to their signal sequences by fusing the vesicular stomatitis virus G protein with the HIV-1 Env protein signal sequence and expressing this chimeric protein in mammalian cells. The HIV-1 Env protein signal sequence on the VSV-G protein also confers a reduced rate of cleavage and slow intracellular transport and folding of the chimeric G protein. These results provide direct evidence that in vivo the HIV-1 glycoprotein signal sequence inhibits the folding of HIV-1 Env protein. Our data also suggest a direct correlation between the rate of the signal sequence cleavage and protein folding.
分泌蛋白和大多数膜蛋白在合成时带有一个信号序列，该信号序列通常在新生多肽链向内质网（ER）腔转运的过程中从其上切割下来。我们分析了在HeLa细胞、BHK细胞和Jurkat细胞中HIV-1 Env蛋白信号序列从gp160和gp120上切割的动力学。此外，我们还确定了这种切割对gp160和gp120糖蛋白与ER蛋白钙连蛋白结合的影响，以及信号序列切割对蛋白质折叠的影响。在所有这三种细胞系中，gp160和gp120上HIV-1 Env蛋白信号序列的切割都非常缓慢，半衰期为45 - 60分钟。gp160和gp120的核心糖基化且保留信号序列的形式与钙连蛋白结合，而gp160和gp120的信号序列被切割的形式已与钙连蛋白解离，并通过它们与CD4细胞受体结合的能力确定已正确折叠。在非还原SDS-PAGE中对gp160和gp120折叠状态的进一步分析表明，gp160和gp120的保留信号序列且与钙连蛋白结合的形式迁移为宽的、弥散的条带，而gp160和gp120的信号序列被切割的或与CD4结合的形式迁移为单一的较清晰条带。通过将水泡性口炎病毒G蛋白与HIV-1 Env蛋白信号序列融合并在哺乳动物细胞中表达这种嵌合蛋白，将HIV-1糖蛋白折叠和细胞内转运速率延迟的原因定位到了它们的信号序列上。VSV-G蛋白上的HIV-1 Env蛋白信号序列也导致嵌合G蛋白的切割速率降低以及细胞内转运和折叠缓慢。这些结果提供了直接证据，证明在体内HIV-1糖蛋白信号序列会抑制HIV-1 Env蛋白的折叠。我们的数据还表明信号序列切割速率与蛋白质折叠之间存在直接相关性。
