Molecular cloning, tissue distribution and enzymatic characterization of cathepsin X from olive flounder (Paralichthys olivaceus).
In this study, we have cloned a cDNA encoding for cathepsin X (PoCtX) from the olive flounder, Paralichthys olivaceus. The presence of an HIP motif, which is conserved in the unique cathepsin X family, PoCtX, clearly shows its relation to the cathepsin X group, apart from the cathepsin L or B subfamily. The results of RT-PCR and real-time PCR analyses revealed ubiquitous PoCtX expression in normal and LPS-stimulated tissues. The cDNA encoding for the proenzyme of PoCtX (proPoCtX) was expressed in Escherichia coli as a 57 kDa fusion protein with glutathione S-transferase. Its activity was quantified via the cleavage of the synthetic fluorogenic peptide substrate Z-Phe-Arg-AMC, and the optimal pH for the protease activity was 5. The recombinant proPoCtX was inhibited by antipain and leupeptin. The PoCtX protein from P. olivaceus muscle extracts was purified 9.48-fold via a one-step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. Western blotting and ELISA were conducted in order to evaluate the reaction ability and detection-specificity of the anti-proPoCtX polyclonal antibody to native PoCtX and recombinant proPoCtX proteins. Our findings indicate that the P. olivaceus cathepsin X is highly conserved within the cathepsin X subfamily in terms of its amino acid sequence, tissue expression, and biochemical activity.
Ahn
Sang Jung
SJ
Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
Kim
Na Young
NY
Jeon
Soo Jin
SJ
Sung
Ji Hea
JH
Je
Ju Eun
JE
Seo
Jung Soo
JS
Kim
Moo-Sang
MS
Kim
Joong Kyun
JK
Chung
Joon Ki
JK
Lee
Hyung Ho
HH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005432:Flounder / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002403:Cathepsins; D056657:Cathepsin K