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J. Biol. Chem., Vol. 280, Issue 49, 40845-40856, December 9, 2005

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Characterization of a Novel Filarial Serine Protease Inhibitor, Ov-SPI-1, from Onchocerca volvulus, with Potential Multifunctional Roles during Development of the Parasite^*

Louise Ford1, David B. Guiliano12, Yelena Oksov¶, Asim K. Debnath||, Jing Liu, Steven A. Williams**, Mark L. Blaxter, and Sara Lustigman3

From the Laboratory of Molecular Parasitology, the ^¶Laboratory of Electron Microscopy, and the ^||Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, the Molecular and Biochemical Parasitology Group, Division of Cell and Molecular Biology, Imperial College of Science and Technology, London SW7 2AY, United Kingdom, the ^**Clark Science Center, Smith College, Northampton, Massachusetts 01063, and the Institute of Evolutionary Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JT, United Kingdom

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Received for publication, April 22, 2005 , and in revised form, September 7, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ013154 [GenBank] , DQ013161 [GenBank] , DQ011671 [GenBank] , and DQ011672 [GenBank] .

^* 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.

¹ Both authors contributed equally to this work.

² Funded by the Wellcome Trust. Work in Edinburgh was funded by the UK Medical Research Council. Initial work at Smith College by D. B. G. was funded by a Hampshire College Division III Howard Hughes Medical Institute Award and the Edna McConnell Clarke Foundation.

³ To whom correspondence should be addressed: Laboratory of Molecular Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th St., New York, NY 10021. Tel.: 212-570-3119; Fax: 212-570-3121; E-mail: slustigman{at}nybloodcenter.org.

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