Isolation and sequencing of two cDNA clones encoding rat spleen cathepsin E and analysis of the activation of purified procathepsin E.
Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase consisting of two identical subunits with a molecular mass of approximately 42 kDa and has a unique subcellular distribution in various rat tissues. In this study, we determined the complete amino acid sequence of rat spleen CE and examined the activation mechanism of the proenzyme purified from this tissue. Two cDNA clones encoding rat CE, termed pTN05 and pTN1, were isolated. Comparison of the amino acid sequences predicted from the respective cDNA sequences revealed that they were essentially identical, except that pTN1 lacked a sequence corresponding to residues Tyr293-Pro325 of the longer cDNA clone (pTN05). Based on the structural analysis of purified enzyme forms, the CE precursor was found to comprise a signal peptide, a prosequence, and a mature protein region of 19, 39, and 337 (pTN05) or 304 (pTN1) residues, respectively. Despite a high degree of similarity in the overall structure of CE between rat and other mammalian species, the first 11 residues in the NH2-terminal sequence of rat mature enzyme were significantly different from those of other species. The purified pro-CE was analyzed for its conversion to the mature form. The results indicated that the maximal conversion occurred at pH 3.0-4.0 in a temperature- and time-dependent manner by autocatalytic cleavage at the site between Phe39-Ser40. This conversion was highly dependent on the protein concentrations of pro-CE and delayed by the presence of exogenous substrates, suggesting the predominance of intermolecular reaction for its conversion to the mature form.
Okamoto
K
K
Department of Pharmacology, Kyushu University Faculty of Dentistry, Fukuoka, Japan.
Yu
H
H
Misumi
Y
Y
Ikehara
Y
Y
Yamamoto
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020059:Cathepsin E; D002403:Cathepsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013154:Spleen / Q000201:enzymology*
D018076:DNA, Complementary; D004792:Enzyme Precursors; D002403:Cathepsins; C067581:procathepsin E; D020059:Cathepsin E