CONTROL OF EXPRESSION, GLYCOSYLATION, AND SECRETION OF HIV-1 GP120 BY HOMOLOGOUS AND HETEROLOGOUS SIGNAL SEQUENCES
CONTROL OF EXPRESSION, GLYCOSYLATION, AND SECRETION OF HIV-1 GP120 BY HOMOLOGOUS AND HETEROLOGOUS SIGNAL SEQUENCES
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DOI：
10.1006/viro.1994.1531
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发表时间：
1994-10-01
期刊：
影响因子：
3.7
通讯作者：
KANG, CY
中科院分区：
文献类型：
--
作者：
LI, Y;LUO, LZ;KANG, CY
30个氨基酸的HIV-1 gp 120信号序列比大多数糖蛋白信号序列长，平均含有5个带正电荷的氨基酸。HIV-1 gp 120基因及其天然信号序列在任何原核或真核表达系统中表达均显示极低水平的合成和分泌。然而，HIV-1 gp 120信号序列的缺失导致在草地贪夜蛾细胞中产生大量的非糖基化形式的gp 120。用来自蜜蜂蜂毒肽或鼠白细胞介素3的信号序列取代gp 120天然信号序列促进糖基化形式的gp 120的高水平表达和有效分泌。这些异源信号序列含有一个（蜂毒肽）或不含（IL-3）带正电荷的氨基酸，并使我们研究了HIV-1 gp 120信号序列中带正电荷的氨基酸的作用。通过定点突变构建了HIV-1 gp 120信号序列的四种电荷改变形式，其中带正电荷的氨基酸依次被中性氨基酸取代。这些实验的结果表明，gp 120的表达和分泌通过逐步消除带正电荷的氨基酸而逐渐增加。然而，所有带正电荷的氨基酸的取代导致非糖基化的gp 120在细胞内的积累，糖基化形式的gp 120的量减少。这些结果表明，HIV-1 gp 120信号序列中带正电荷的氨基酸是决定其表达和分泌不良的关键因素。对gp 120的细胞内转运和折叠的分析进一步表明，高电荷、未切割的信号序列的存在是限制gp 120从粗糙ER转运到高尔基体的重要因素，（C）1994 Academic Press，Inc.
The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. However, deletion of the HIV-1 gp120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells. Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120. Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion. However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120. These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion. Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus, (C) 1994 Academic Press, Inc.