Analysis of essential regions for biological activities of the potent anti-HIV protein actinohivin that binds to high-mannose sugar chains of gp 120

与 gp 120 高甘露糖链结合的强效抗 HIV 蛋白放线菌素生物活性的重要区域分析

• 批准号: 14572064
• 负责人: INOKOSHI Junji
• 金额: $ 1.41万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572064/
• 关键词: Actinohivin; anti-HIV; recombinant protein; syncytium formation; production; actinomycete; Eschelichia coli; entry inhibitor; アクチノヒビン; 組換えたん白質; 糖鎖結合モジュール; 高マンノース型糖鎖

Actinohivin (AH) which is a novel anti-HIV protein isolated from the culture broth of Longispora albida gen. novo., sp. novo., consists of 114 amino acid residues. The proposes of this project were to clarify the structure-activity relationship of AH and to obtain the basic evidence for the creation of new low moleculer weight anti-HIV compounds.AH gene coloned from an AH producing strain was introduced into E. coli expression vector pET30 Xa/LIC to construct the AH expression plasmid. The truncated AHs were amplified by PCR using full length AH encoding gene cloned in pBluescript II SK+ as a template. Site-directed mutagenesis on pBluescript II SK+::2K3A was performed using appropriate primers. The mutant constructs were expressed in E. coli and the recombinant proteins obtained were subjected to syncytium formation assay using env-expressing HeLa cells and CD4/CXCR4expressing HeLa cells.AH consists of highly conserved triple tandem segments. Six kinds of the truncate variations which consist of one or two segments of AH were constructed and tested for the syncytium formation inhibitory activity. It was considered that three segments of AH are essential for the syncytium formation inhibitory activity. Since the mutant AH in which two cysteine residues in a segment 2 were replaced by serine also had a similar inhibitory activity to AH, which indicates that the cysteine residues do not affect the activity of AH. Then, we identified that six amino acid residues, ^<15>D, ^<23>Y, ^<25>L, ^<28>N, ^<32>Y and ^<33>Q between LD and QXW consensus sequence of segment 1 were essential for binding to the sugar chain. Furthermore, the mutant AHs in which the LD-QXW regions of three segments were replaced with that of segment 1(Seg 1 trimer) had increased activities. These results suggest that the region between LD and QXW is important for binding to the sugar chain and the other regions are needed for suitable arrangement of each segment.

放线菌素（Actinohivin，AH）是从白长孢霉（Longispora albida gen. novo.）新种，由114个氨基酸残基组成。本课题的目的是阐明AH的构效关系，为开发新的低分子量抗HIV化合物提供基础依据。coli表达载体pET 30 Xa/LIC构建AH表达质粒。以pBluescriptII SK+中克隆的全长AH编码基因为模板，通过PCR扩增截短的AH。使用适当的引物对pBluescript II SK+：：2K 3A进行定点诱变。突变体构建体在E.用表达env的HeLa细胞和表达CD 4/CXCR 4的HeLa细胞进行合胞体形成试验，AH由高度保守的三重串联片段组成。构建了六种由一个或两个AH片段组成的截短变异体，并测试了它们对合胞体形成的抑制活性。认为AH的三个片段是合胞体形成抑制活性所必需的。由于片段2中的两个半胱氨酸残基被丝氨酸取代的突变体AH也具有与AH相似的抑制活性，这表明半胱氨酸残基不影响AH的活性。然后，我们鉴定了片段1的LD和QXW共有序列之间的六个氨基酸残基^<15>D、^<23>Y、^<25>L、^<28>N、^<32>Y和^<33>Q是与糖链结合所必需的。此外，其中三个片段的LD-QXW区域被片段1的LD-QXW区域（Seg 1三聚体）取代的突变体AH具有增加的活性。这些结果表明，LD和QXW之间的区域是重要的结合糖链和其他区域需要适当的安排每个片段。

项目成果

H.Chiba et al.: "Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120."Biochem.Biophys.Res.Comm.. 316・1. 203-210 (2004)

H.Chiba等人：“Actinohivin是一种来自放线菌的新型抗人类免疫缺陷病毒蛋白，通过结合gp120的高甘露糖型糖链来抑制病毒进入细胞。”Biochem.Biophys.Res.Comm.. 316・1. 203-210 (2004)

H.Chiba et al.: "Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp 120"Biochem.Biophys.Res.Comm.. 316(1). 203-210 (2004)

H.Chiba 等人：“Actinohivin，一种来自放线菌的新型抗人类免疫缺陷病毒蛋白，通过结合 gp 120 的高甘露糖型糖链来抑制病毒进入细胞”Biochem.Biophys.Res.Comm.. 316（