tailieunhanh - Báo cáo khoa học: Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, process-ing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. | ềFEBS Journal Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation expressed in human embryonic kidney 293T cells Takayuki Tsukuba1 Shinobu Ikeda2 Kuniaki Okamoto2 Yoshiyuki Yasuda1 Eiko Sakai2 Tomoko Kadowaki1 Hideaki Sakai2 and Kenji Yamamoto1 1 Department of Pharmacology Graduate Schoolof DentalScience Kyushu University Fukuoka Japan 2 Department of OralMolecular Pharmacology Graduate Schoolof BiomedicalSciences Nagasaki University Japan Keywords aspartic proteinase cathepsin E mutation processing sorting Correspondence K. Yamamoto Department of Pharmacology Graduate Schoolof Dental Science Kyushu University Higashi-ku Fukuoka 812-8582 Japan Fax 81 92 642 6342 Tel 81 92 642 6337 E-mail kyama@ Received 10 October 2005 revised 7 November 2005 accepted 14 November 2005 doi To study the roles of the catalytic activity propeptide and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis processing and intracellular trafficking we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1positive endosomal organelles as determined by immunofluorescence microscopy. Consistently pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A indicating its transport from the endoplasmic reticulum ER to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide Leu23-Phe58 and the putative ER-retention sequence Ser59-Asp98 were neither processed nor transported to the endosomal compartment. The mutant lacking the .