Characterization of β cell-specific gene obtained by large cDNA sequencing.

为了研究胰岛β细胞表达序列标签的特征，对小鼠胰岛β细胞系MIN6细胞的表达序列标签进行了大规模的序列分析。其中，3个单独的EST编码一种新的蛋白质，我们将其命名为Isetasin。Isletasin基因编码552个氨基酸。Isletasin含有对S1家族丝氨酸蛋白酶的重复催化三联体，其表达仅限于MIN6细胞或小鼠胰岛。从INS-1细胞系中克隆了大鼠同源基因。小鼠与大鼠氨基酸同源性为94.8%。实时荧光定量逆转录聚合酶链式反应显示胰岛抑素在胰腺β细胞系MIN6、βHC9、INS-1以及分离的小鼠和大鼠胰岛中高表达。免疫印迹分析表明，表达表位标记的异莱菌素的COS-7细胞提取物不是一个完整的蛋白质，而是一种外周蛋白。Isletasin与微体膜的管腔表面密切相关，在那里它经历信号肽的裂解和N-糖基化。在MIN6细胞中对各细胞器标记蛋白和绿色荧光蛋白标记的斯莱他辛或His，myc标记的斯莱他辛的荧光检测表明，斯莱他辛是一种早期的内体蛋白。

To characterize expressed sequence tag (EST) profiling in pancreatic islet β cells, a large-scale cDNA sequencing analysis was carried out for ESTs derived from MIN6 cells, a cell line of murine pancreatic islet β cells. Among these, 3 individual ESTs were found to code one novel protein which we termed isletasin. Isletasin gene encoded 552 amino acids. Isletasin contained repeated catalytic triads for S1 family of serine proteases, and its expression was limited to MIN6 cells or mouse pancreatic islets. Rat homologue was cloned from INS-1 cell line. Identity of amino acids between mouse and rat was 94.8%. Real-time RT-PCR showed high expression of isletasin in pancreatic β cell lines of MIN6, βHC9, INS-1 and pancreatic islets isolated from mice and rats. Immunoblotting of extract from COS-7 cells expressing epitope-tagged isletasin showed that isletasin is not an integral but a peripheral protein. Isletasin is strongly associated with the luminal surface of the microsomal membrane, where it undergoes signal peptide cleavage and N-glycosylation. The fluorescence detection of each organelle marker protein and green fluorescence protein-tagged isletasin or His, myc-tagged isletasin in MIN6 cells revealed that isletasin is an early endosomal protein.