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

该基因被称为r-GSP，最初是在鉴定大鼠C6神经胶质细胞的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的表达显著增加。R-GSP的氨基酸序列与金黄地鼠和人补体C1s前体的氨基酸序列具有同源性。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诱导分化过程中的变化。90 kDa r-GSP蛋白呈时间依赖性增加，并在24 h达到最大值(约7.6倍)。此外，它被分泌到培养基中，然后被切割成二硫键连接的片段，其中一个大小为30 kDa，类似于C1s，表明它是在细胞外空间加工的。事实上，从培养液中部分纯化的r-GSP被活性的人C1r切割成30 kDa的多肽。此外，分泌的r-GSP蛋白可裂解人C4a产生C4a‘，并与人血清酯酶抑制物结合，强烈提示r-GSP蛋白为大鼠C1s。然而，在高表达r-GSP的C6细胞中，它们的形态和增殖速度与亲本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，提示r-GSP蛋白是以酶原形式分泌并在培养上清液中加工的。

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.