Cloning and biochemical characterization of blisterase, a subtilisin-like convertase from the filarial parasite, Onchocerca volvulus.
Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.
Poole
Catherine B
CB
Molecular Parasitology Division, New England Biolabs, Beverly, Massachusetts 01915, USA.
Jin
Jingmin
J
McReynolds
Larry A
LA
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics*
D000590:Amino Acid Chloromethyl Ketones; D029742:Caenorhabditis elegans Proteins; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D010455:Peptides; D021382:Protein Sorting Signals; D004492:Edetic Acid; D005982:Glutathione Transferase; D013381:Subtilisins; C093968:bli-4 protein, C elegans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase