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.
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Natthawan
N
Kongkerd
Pichart
P
Uparanukraw
Nimit
N
Morakote
Mohammed
M
Sajid
James H
JH
McKerrow
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