Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis.
We cloned a cDNA encoding cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis (NsCtB). Nucleotide sequence of the isolated clone encoded a preproenzyme of 328 amino acids, comprising a 15-residue putative signal peptide, a 60-residue propeptide and the 253-residue mature enzyme. The mature NsCtB was 53% identical to human cathepsin B and conserved all the structural features characteristic of cysteine protease. The presence of an occluding loop in the mature region, a unique feature of cathepsin B, suggested the shrimp protein to be cathepsin B. Northern blot analysis revealed expression of NsCtB transcripts exclusively in the hepatopancreas tissues, suggesting a possible digestive role of this enzyme. An interesting feature of NsCtB was its remarkably high negative charge in comparison with other cysteine proteases, which was predicted to effectively locate and guide the positively charged residues of a substrate into the binding cleft. We also observed a repertoire of cysteine protease activities in the acidic milieu of shrimp hepatopancreas using synthetic substrates specific to various cathepsins. The activity profile revealed cathepsin B as the single most dominant enzyme with a specific activity comparable to that attributable to combined activities of other cathepsins. This activity could be blocked by E-64, a cysteine protease inhibitor, but not by Z-Phe-Tyr (t-Bu)-CHN(2), a specific inhibitor of cathepsin L.
Aoki
Hitoshi
H
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, 113-8657, Tokyo, Japan.
Ahsan
Md Nazmul
MN
Watabe
Shugo
S
D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004792:Enzyme Precursors; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010179:Pancreas / Q000201:enzymology*; D033542:Pandalidae; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment
D018076:DNA, Complementary; D004792:Enzyme Precursors; D011480:Protease Inhibitors; D002401:Cathepsin B