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, 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.
Tsukuba
Takayuki
T
Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan.
Ikeda
Shinobu
S
Okamoto
Kuniaki
K
Yasuda
Yoshiyuki
Y
Sakai
Eiko
E
Kadowaki
Tomoko
T
Sakai
Hideaki
H
Yamamoto
Kenji
K
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020059:Cathepsin E / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D009154:Mutation; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D014162:Transfection
D001224:Aspartic Acid; D020059:Cathepsin E; D000409:Alanine