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

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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.