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J. Biol. Chem., Vol. 278, Issue 38, 36183-36190, September 19, 2003

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Cloning and Biochemical Characterization of Blisterase, a Subtilisin-like Convertase from the Filarial Parasite, Onchocerca volvulus^*,

Catherine B. Poole, Jingmin Jin and Larry A. McReynolds 

From the Molecular Parasitology Division, New England Biolabs, Beverly, Massachusetts 01915

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, ₁-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 k_cat 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.

Received for publication, March 13, 2003 , and in revised form, June 18, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY157026.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary data.

To whom correspondence should be addressed. Tel.: 978-927-5054 (ext. 259); Fax: 978-921-1350; E-mail: mcreynolds{at}neb.com.

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