Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant.
Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant.
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DOI：
10.1016/s0021-9258(19)74548-7
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发表时间：
1993-11
期刊：
影响因子：
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通讯作者：
Eric David Anderson;L. Thomas;J. Hayflick;G. Thomas
中科院分区：
文献类型：
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作者：
Eric David Anderson;L. Thomas;J. Hayflick;G. Thomas
弗林蛋白酶是一种膜结合的钙依赖性丝氨酸内切蛋白酶，其在共有序列-Arg-X-Lys/Arg-Arg-的羧基侧切割前蛋白。使用定点诱变，构建变体α 1-抗胰蛋白酶（α 1-AT），其在其反应性位点-Arg-X-X-Arg-中含有弗林蛋白酶有效加工所需的最小序列（Molloy，S.美国，Bresnahan，P. A.，Leppla，S. H、克林佩尔，K. R.，和托马斯，G.（1992）J.Biol.Chem.267，16396-16402）。这种α 1-AT变体，[Arg 355 Arg 358] α 1-AT（α 1-PDX），在体外抑制弗林蛋白酶方面比[Arg 358] α 1-AT（α 1-AT匹兹堡，α 1-PIT）有效3，000倍以上（K0.5 = 0.03微克/ml）。此外，与α 1-PIT（其含有P4 Ala）相比，α 1-PDX中的P4 Arg大大减弱了该丝氨酸蛋白酶抑制剂的凝血酶抑制性质（> 300倍），从而增加了α 1-PDX对弗林蛋白酶的选择性。表达研究表明，α 1-PDX，而不是α 1-PIT，阻断两个弗林蛋白酶底物，前β-神经生长因子和人类免疫缺陷病毒（HIV）-1 gp 160在转染细胞的加工。此外，合胞体测定显示α 1-PDX阻断HIV-1 gp 160的膜融合特性。潜在的使用α 1-PDX在操纵激活的前蛋白在组织和时间特异性的方式进行了讨论。
Furin is a membrane-associated calcium-dependent serine endoprotease that cleaves proproteins on the carboxyl side of the consensus sequence -Arg-X-Lys/Arg-Arg-. Using site-directed mutagenesis, a variant alpha 1-antitrypsin (alpha 1-AT) was constructed which contains in its reactive site -Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402). This alpha 1-AT variant, [Arg355 Arg358]alpha 1-AT (alpha 1-PDX), is greater than 3,000-fold more effective than [Arg358]alpha 1-AT (alpha 1-AT Pittsburgh, alpha 1-PIT) at inhibiting furin in vitro (K0.5 = 0.03 microgram/ml). Furthermore, the P4 Arg in alpha 1-PDX greatly attenuates the thrombin inhibitory properties of this serpin (> 300-fold) compared with alpha 1-PIT (which contains a P4 Ala), thus increasing the selectivity of alpha 1-PDX for furin. Expression studies show that alpha 1-PDX, and not alpha 1-PIT, blocks the processing of two furin substrates, pro-beta-nerve growth factor and human immunodeficiency virus (HIV)-1 gp160 in transfected cells. In addition, a syncytium assay shows that alpha 1-PDX blocks the membrane fusogenic properties of HIV-1 gp160. The potential use of alpha 1-PDX in manipulating the activation of proproteins in a tissue- and time-specific manner is discussed.