title: Molecular cloning, expression, and enzymatic analysis of cathepsin X from starfish (Asterina pectinifera).
abstract: Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Bak
authors.forename: Moo-Sang
authors.initials: MS
authors.lastname: Kim
authors.forename: Na Young
authors.initials: NY
authors.lastname: Kim
authors.forename: Hye-Jin
authors.initials: HJ
authors.lastname: Go
authors.forename: Jin Woo
authors.initials: JW
authors.lastname: Han
authors.forename: Hyae
authors.initials: H
authors.lastname: In Jo
authors.forename: Sang Jung
authors.initials: SJ
authors.lastname: Ahn
authors.forename: Nam Gyu
authors.initials: NG
authors.lastname: Park
authors.forename: Joon Ki
authors.initials: JK
authors.lastname: Chung
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: D000818:Animals; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D013215:Starfish / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D002403:Cathepsins