FUNCTIONAL AND DNA ALALYSIS OF THE NOVEL PROTEIN (R21 PROTEIN) INVOLVED IN MEMBRANE FUSION

参与膜融合的新型蛋白质（R21 蛋白质）的功能和 DNA 分析

• 批准号: 10670416
• 负责人: NAKAMURA Minoru
• 金额: $ 2.3万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670416/
• 关键词: MEMBRANE FUSION; HTLV-I; CXC CHEMOKINE; HTLV-I RECEPTOR; VSV-PSEUDOTYPE; REPORTER GENE ACTIVATION ASSAY; 膜 融合; HTLV-1 レセプター

By immunizing rats with cocultured HTLV-I-positive ILT8M2 and HTLV-I-negative MOLT-4 cells, we isolated one monoclonal antibody (mAb), designated as mAb R21, which enhances the syncytium formation induced by coculturing ILT8M2 cells with MOLT-4 cells. Since the enhancing activity by mAb R21 of syncytium formation was observed only in the presence of a factor contained in fetal calf serum(FCS) which seems to bind to mAb R21, we purified this serum factor from FCS using a mAb R21- coupled Sepharose 4B column. The partial amino acid sequence of the purified protein indicates that R21 protein is a novel bovine serum protein which has approximately 90% amino acid homology with bovine platelet factor 4, a member of CXC chemokine family.Next, to study the mechanism of viral entry and cell fusion mediated by HTLV-I env. glycoproteins, and to identify the HTLV-I cellular receptor(s), we constructed a cell fusion-dependent reporter gene activation assay and a vesicular stomatitis virus (VSV) pse … More udotype assay. The cell fusion-dependent reporter gene activation assay, in which T7 RNA polymerase in donor cells co-expressing env glycoproteins activates a luciferase gene in recipient cells upon cell fusion, revealed that a broad range of cells are susceptible to HTLV-I env-mediated cell fusion. This indicated that cell-to-cell transmission of HTLV-I can occur in a wider range of cells than previously reported. The VSV pseudotype assay, which consists of recombinant VSV containing the green fluorescent protein gene instead of the receptor-binding G protein gene(VSV△GィイD1*ィエD1) and complemented with HTLV-I env glycoproteins (VSV△GィイD1*ィエD1-Env), revealed that VSV△GィイD1*ィエD1-Env infects efficiently only HepG2 and 293T cells, indicating that cell-free HTLV-I transmission is less efficient than cell-to-cell transmission. The characterization of the HTLV-I receptor(s) by various chemical modifications of HepG2 and 293T cells indicated that some glycosaminoglycans and phospholipids but not proteins on the cell surface may play a major role in HTLV-I env-mediated viral entry. The expression cloning of HTLV-I receptor is now underway using cDNA library from HepG2 cells. Less

用HTLV-Ⅰ阳性的ILT 8 M2细胞和HTLV-Ⅰ阴性的MOLT-4细胞共培养免疫大鼠，获得了一株能增强ILT 8 M2细胞与MOLT-4细胞共培养诱导合胞体形成的单克隆抗体（mAb），命名为mAb R21。由于只有在胎牛血清（FCS）中含有似乎与mAb R21结合的因子的存在下才观察到mAb R21对合胞体形成的增强活性，因此我们使用mAb R21偶联的Sepharose 4 B柱从FCS中纯化了该血清因子。纯化蛋白的部分氨基酸序列分析表明，R21蛋白是一种新的牛血清蛋白，与CXC趋化因子家族成员牛血小板因子4（Bovine platelet factor 4）的氨基酸同源性约为90%。为了鉴定HTLV-I细胞受体，我们构建了细胞融合依赖的报告基因激活试验和水泡性口炎病毒（VSV）的特异性表达试验。 ...更多信息 Udotype分析细胞融合依赖性报告基因激活测定，其中共表达env糖蛋白的供体细胞中的T7 RNA聚合酶在细胞融合时激活受体细胞中的荧光素酶基因，揭示了广泛的细胞对HTLV-I env介导的细胞融合敏感。这表明HTLV-I的细胞间传播可以在比以前报道的更广泛的细胞中发生。VSV假型试验，由含有绿色荧光蛋白基因而非受体结合G蛋白基因的重组VSV组成（VSV△G β D1* VSV β D1）并与HTLV-Ⅰ env糖蛋白互补（VSV△G腺病毒D1* 腺病毒D1-Env），表明VSV△G腺病毒D1* 腺病毒D1-Env仅有效地感染HepG 2和293 T细胞，表明无细胞HTLV-1传输比细胞到细胞传输效率低。通过HepG 2和293 T细胞的各种化学修饰对HTLV-I受体的表征表明，细胞表面上的一些糖胺聚糖和磷脂而不是蛋白质可能在HTLV-I env介导的病毒进入中起主要作用。HTLV-1受体的表达克隆正在利用HepG 2细胞的cDNA文库进行。少

项目成果

Shinji Shimoda,Judy V.D.Water,Aftab Ansari,Minoru Nakamura et al: "Identification and precursorfrequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis." J.Clin.Invest.102. 1831-1840 (1998)

Shinji Shimoda、Judy V.D.Water、Aftab Ansari、Minoru Nakamura 等人：“原发性胆汁性肝硬化线粒体自身抗原中常见 T 细胞表位基序的鉴定和前体频率分析。”

Ishibashi, H., Shimoda, S., Shigematsu, H., Ichiki, Y., Nakamura, M., Hayashida, K., and Gershwin, E.M.: "Immune pathophysiology of primary biliary cirrhosis. Progress in Hepatology - Liver and Immunology"Volume5. edited by M. Yamanaka, G. Toda, and T. Ta

Ishibashi, H.、Shimoda, S.、Shigematsu, H.、Ichiki, Y.、Nakamura, M.、Hayashida, K. 和 Gershwin, E.M.：“原发性胆汁性肝硬化的免疫病理生理学。肝病学进展 - 肝脏和免疫学

B.Kitae,K.Usuku,Y.Yamamoto,S.Yashiki,M.Nakamura,et al: "Human CD4^+ T lymphocytes recognize a highly conserved epitope of human T lymphotropic virus type I(HTLD-1)env gp21 restricted by HLADRB^*0101." Clin.Exp.Immunol.111. 278-285 (1998)

B.Kitae、K.Usuku、Y.Yamamoto、S.Yashiki、M.Nakamura 等人：“人类 CD4^ T 淋巴细胞识别人类 T 淋巴细胞病毒 I 型 (HTLD-1)env gp21 的高度保守表位，受

K. Okuma, M. Nakamura, S. Nakano, and Y. Niho.: "Identification of a novel bovine serum protein which is involved in human T-cellleukemiavirus type I(HTLV-I)-induced syncytium formation."Arch. Virol.. 144. 1-18 (1999)

K. Okuma、M. Nakamura、S. Nakano 和 Y. Niho.：“鉴定一种新型牛血清蛋白，该蛋白参与人 T 细胞白血病病毒 I 型 (HTLV-I) 诱导的合胞体形成。”Arch。

Shinji Shimoda, Minoru Nakamura, Hirohisa Shigematsu, Hironori Tanimoto, Toshihumi Gushima, and Hiromi Ishibashi.: "Mimicrypeptides of human PDC-E2 163-176 peptide the immunodominantT cell epitope of primary biliary cirrhosis."Hepatology. (in press). (200

Shinji Shimoda、Minoru Nakamura、Hirohisa Shigematsu、Hironori Tanimoto、Toshihumi Gushima 和 Hiromi Ishibashi。：“人 PDC-E2 163-176 肽的模拟肽，原发性胆汁性肝硬化的免疫显性 T 细胞表位。”肝病学。