2006年1月
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
FEBS JOURNAL
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- 巻
- 273
- 号
- 1
- 開始ページ
- 219
- 終了ページ
- 229
- 記述言語
- 英語
- 掲載種別
- DOI
- 10.1111/j.1742-4658.2005.05062.x
- 出版者・発行元
- WILEY-BLACKWELL
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-1-positive 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 ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.
- リンク情報
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- DOI
- https://doi.org/10.1111/j.1742-4658.2005.05062.x
- CiNii Articles
- http://ci.nii.ac.jp/naid/80017686839
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/16367762
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000234027900020&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1111/j.1742-4658.2005.05062.x
- ISSN : 1742-464X
- CiNii Articles ID : 80017686839
- PubMed ID : 16367762
- Web of Science ID : WOS:000234027900020