Identification and characterization of a cathepsin L-like cysteine protease from Gnathostoma spinigerum.
Gnathostoma spinigerum is a causative agent of human gnathostomiasis, a common parasitic disease involving skin and visceral organs, especially the central nervous system. In this study, we identified a cDNA encoding a cathepsin L-like cysteine protease (GsCL1) from the lambdaZAP cDNA library of G. spinigerum advanced third-stage larva (aL3) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1484bp encoded 398 amino acids which contained a typical signal peptide sequence (23 amino acids), a pro-domain (156 amino acids), and a mature domain (219 amino acids) with an approximate molecular weight of 24kDa. The deduced amino acid sequence of GsCL1 gene showed 53-64% identity to cathepsin L proteases of various organisms including a cathepsin L family member (cpl-1) of Caenorhabditis elegans. Recombinant proGsCL1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. The expressed enzyme displayed optimal protease activity toward Z-Phe-Arg-AMC substrate at pH 6.0 but not toward Z-Arg-Arg-AMC. The activity was sensitive to cysteine protease inhibitors E-64 and K11777. The preference for large hydrophilic and aromatic residues in the P2 position (I, L, F, W, U, V) was typical of cathepsin L proteases. Mouse anti-GST-proGsCL1 serum showed reactivity with 35-, 38- and 45-kDa proteins in the aL3 extracts. These proteins were shown to localize inside the intestinal cells of aL3.
Kongkerd
Natthawan
N
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Uparanukraw
Pichart
P
Morakote
Nimit
N
Sajid
Mohammed
M
McKerrow
James H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006039:Gnathostoma / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D021382:Protein Sorting Signals; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse