Biochemical analysis of the novel tumor suppressor gene product RECK

新型抑癌基因产物RECK的生化分析

基本信息

批准号：
08660101
负责人：
MAKI Masatoshi
金额：
$ 1.41万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660101/
关键词：
tumor suppressor gene protease baculovirus monoclonal antibody

项目摘要

RECK is a novel tumor suppressor gene which reverses normally the activated-ras-transformed mouse fibroblast cells. In malignant transformed cells, its expression is suppressed. Forced exogenous expression of the gene in the cancer cells suppresses colony formation in soft agar and inhibits tumor metastasis. We characterized the gene product as follows :(1) Partial cDNA was expressed in E.coli, and the purified recombinant protein was subjected to immunize mice for preparation of monoclonal antibodies. Hybridomas were screened with ELISA method. (2) RECK cDNA was expressed in insect cells using baculovirus system. Western analysis using the monoclonal antibodies revealed that the molecular mass of the expressed protein was about 100 kDa. It agreed well with the value estimated from the amino acid sequence and suggested low glycosylation. (3) RECK contains signal sequence at N-terminus and hydrophobic sequence at C-terminus and is transported onto the membrane outside of the cell. Deletion of the C-terminal hydrophobic region caused secretion of RECK into the culture medium. We established a method of affinity purification of the secreted type of RECK by substituting the C-terminal sequence with a tag of histidine hexamer. (4) Potential reactive site residue (P1) of the Kazal domain of RECK is Asp and it suggests caspase-like proteases as target enzymes. RECK did not inhibit caspase-1. It did not interact with granzyme B,either.

RECK是一种新型的肿瘤抑制基因，通常逆转激活的Ras转换的小鼠成纤维细胞。在恶性转化细胞中，其表达被抑制。该基因在癌细胞中的强迫外源表达抑制了软琼脂中的菌落形成，并抑制肿瘤转移。我们表征了基因产物如下：（1）在大肠杆菌中表达部分cDNA，并对纯化的重组蛋白进行免疫小鼠进行免疫以制备单克隆抗体。用ELISA方法筛选杂交瘤。（2）使用杆状病毒系统在昆虫细胞中表达RECK cDNA。使用单克隆抗体的西方分析表明，表达蛋白的分子质量约为100 kDa。它与氨基酸序列估计的值很好地一致，并建议低糖基化。（3）RECK在N末端包含信号序列和C末端的疏水序列，并将其转运到细胞外的膜上。 C末端疏水区的缺失导致RECK分泌到培养基中。我们通过用组氨酸六聚体标签代替C末端序列来建立了分泌类型的RECK类型的亲和力纯化方法。（4）RECK KAZAL结构域的潜在反应性位点残基（P1）是ASP，它表明胱天冬酶样蛋白酶作为靶酶。 RECK没有抑制caspase-1。它也没有与粒状B相互作用。