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

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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 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.