Wednesday, December 31, 2008

The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin

Mol Biochem Parasitol. 2008 Dec 6. [Epub ahead of print]

The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin

Huang R, Que X, Hirata K, Brinen LS, Lee JH, Hansell E, Engel J, Sajid M, Reed S.

Department of Medicine, University of California, San Diego, CA 92103, United States.

Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.

PMID: 19111576 [PubMed - as supplied by publisher]

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