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

该基因被命名为r-gsp，最初是在鉴定大鼠C6胶质细胞分化相关分子时分离出来的。在camp诱导的神经胶质细胞分化过程中，其mRNA表达明显增加。推导出的r-gsp的氨基酸序列与仓鼠和人的补体C1s前体同源。在本研究中，我们提出了针对r-Gsp蛋白的抗肽抗体，并分析了其在camp诱导的分化过程中的变化。90 kDa r-Gsp蛋白对二丁基环AMP （dbcAMP）和茶碱的反应具有时间依赖性，在24 h达到最高水平（约7.6倍）。此外，它被分泌到培养基中，然后被裂解形成二硫化物连接的片段，其中一个是30 kDa，类似于C1s，表明它在细胞外空间加工。事实上，从培养基中部分纯化的r-Gsp被活性人C1r裂解形成30kda的多肽。此外，分泌的r-Gsp蛋白可裂解人C4a生成C4a'，并与人血清c1酯酶抑制剂相关，强烈提示r-Gsp蛋白是大鼠C1s。而过表达r-Gsp的C6细胞，其形态和增殖速率与亲本C6细胞相似。上述结果表明，r-Gsp蛋白不能单独诱导胶质细胞分化，可能是作为原酶分泌，在培养基中加工而成。

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.