Molecular basic research for obtaining genes specifying characteristics of pancreatic α and β cells.

为了表征胰岛β细胞中的表达序列标签（EST）谱，对来自小鼠胰岛β细胞的细胞系MIN 6细胞的3，429个EST进行大规模cDNA测序分析。与GenBank数据库进行同源性比较，共发现369条未知EST，与EST数据库（dbEST）中的EST序列一致。其中，3个独立的EST被发现编码一种新的蛋白质，我们称之为isletasin。Isletasin基因编码短型和长型两种变体mRNA，分别产生482和552个氨基酸的Isletasin。Isletasin含有丝氨酸蛋白酶S1家族的重复催化三联体，其表达仅限于MIN 6细胞或小鼠胰岛。来自表达表位标记的isletasin的COS-7细胞的提取物的免疫印迹显示isletasin不是一个整体而是一个外周蛋白。Isletasin与微粒体膜的腔表面强烈结合，在该处其经历信号肽切割和N-糖基化。在MIN 6细胞中的每个细胞器标记蛋白和绿色荧光蛋白（GFP）标记的isletasin的荧光检测揭示isletasin是早期内体蛋白。总之，通过筛选来自MIN 6细胞的3，429个EST有效获得了其cDNA的isletasin的新蛋白作为早期内体蛋白在胰岛β细胞中特异性表达。

To characterize expressed sequence tag (EST) profiling in pancreatic islet β cells, a large-scale cDNA sequencing analysis was carried out for 3,429 ESTs derived from MIN6 cells, a cell line of murine pancreatic islet β cells. Homology comparison with the GenBank database revealed 369 unknown ESTs, which corresponded to those in the EST database (dbEST). Among these, 3 individual ESTs were found to code one novel protein which we termed isletasin. Isletasin gene encoded two variant mRNAs of a short and long form, from which two forms of isletasin with 482 and 552 amino acids were generated, respectively. Isletasin contained repeated catalytic triads for S1 family of serine proteases, and its expression was limited to MIN6 cells or mouse pancreatic islets. 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 (GFP)-tagged isletasin in MIN6 cells revealed that isletasin is an early endosomal protein. In conclusion, novel protein of isletasin of which cDNA was efficiently obtained by screening 3,429 ESTs derived from MIN6 cells, is specifically expressed in pancreatic islet β cells, as an early endosomal protein.