Increased expression and secretion of r-Gsp protein, rat counterpart of complement C1s precursor during differentiation in rat C6 glioma cells

大鼠 C6 神经胶质瘤细胞分化过程中补体 C1s 前体的大鼠对应物 r-Gsp 蛋白的表达和分泌增加

基本信息

批准号：
13671429
负责人：
SHINODA Jun
金额：
$ 1.86万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The gene, termed r-gsp, was originally isolated during identification of differentiation-associated molecules in rat C6 glial cells. Its mRNA expression was markedly increased during cAMP-induced glial cell differentiation. The deduced amino acid sequence of r-gsp was homologous to those of complement C1s precursors of hamsters and humans. In the present study, we raised anti-peptide antibody against r-Gsp protein and analyzed its change during cAMP-induced differentiation. The 90 kDa r-Gsp protein increased time-dependently and reached the maximal level (〜7.6-fold increase ) at 24 h in response to dibutyryl cyclic AMP (dbcAMP) and theophylline. Moreover, it was secreted into the medium and then was cleaved to form disulfide-linked fragments, one of which was 30 kDa, similar to C1s, suggesting its processing in the extracellular space. In fact, the partially purified r-Gsp from culture medium was cleaved by active human C1r to form a 30 kDa polypeptide. Moreover, secreted r-Gsp protein cleaved human C4a to yield C4a' and associated with human serum C1-esterase inhibitor, strongly suggesting that r-Gsp protein is rat C1s. However, in C6 cells overexpressing r-Gsp, their morphology and proliferation rate were similar to those in parent C6 cells. These results suggest that r-Gsp protein could not induce glial differentiation alone, and suggest that r-Gsp protein was secreted as a proenzyme and processed in culture medium.

该基因被称为r-gsp，最初是在鉴定大鼠C6神经胶质细胞中的分化相关分子时分离出来的，其mRNA表达在cAMP诱导的神经胶质细胞分化过程中显着增加。推测的r-gsp的氨基酸序列与该基因同源。在本研究中，我们制备了针对 r-Gsp 蛋白的抗肽抗体，并分析了其在 cAMP 诱导分化过程中的变化。 r-Gsp 蛋白随时间依赖性增加，并在二丁酰环 AMP (dbcAMP) 和茶碱的作用下在 24 小时达到最大水平（约 7.6 倍增加），此外，它被分泌到培养基中，然后被裂解形成二硫键。 - 连接的片段，其中一个为 30 kDa，与 C1 类似，表明其在细胞外空间中加工。事实上，是从培养基中部分纯化的 r-Gsp。被活性人 C1r 裂解形成 30 kDa 的强多肽，此外，分泌的 r-Gsp 蛋白裂解人 C4a 产生 C4a' 并与人血清 C1-酯酶抑制剂相关，表明 r-Gsp 蛋白是大鼠 C1s。在过表达r-Gsp的C6细胞中，其形态和增殖率与亲代C6细胞相似，这些结果表明r-Gsp蛋白不能单独诱导神经胶质细胞分化。并表明 r-Gsp 蛋白作为酶原分泌并在培养基中加工。