Identification of the Major Cysteine Protease of Giardia and Its Role in Encystation*
Kelly N. DuBois
1,
Marla Abodeely,
Judy Sakanari,
Charles S. Craik¶,
Malinda Lee,
James H. McKerrow, and
Mohammed Sajid2
From the
Department of Pathology, the Sandler Center for Basic Research in Parasitic Diseases, the Biomedical Sciences Graduate Program, and the ¶Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158
Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.
Received for publication, March 18, 2008
, and in revised form, April 18, 2008.
* This work was supported, in whole or in part, by National Institutes of Health Grant AI35707 from the Tropical Disease Research Unit. This work was also supported by the Sandler Family Supporting Foundation. 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 supplemental Figs. S1–S3 and Table S1.
1 Supported by a NIAID, National Institutes of Health Training Fellowship in Microbial Pathogenesis.
2 To whom correspondence should be addressed: 1700 4th St., QB3/UCSF 509, San Francisco, CA 94158-2550. Fax: 415-502-8193; E-mail: sajid{at}ucsf.edu.
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Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
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