Lucas John J, Hayes Gary R, Kalsi Hardip K, Gilbert Robert O, Choe Yongchool, Craik Charles S, Singh Bibhuti N
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 4257 Weiskotten Hall, 766 Irving Avenue, Syracuse, NY 13210, USA.
Arch Biochem Biophys. 2008 Sep 15;477(2):239-43. doi: 10.1016/j.abb.2008.05.018. Epub 2008 Jun 11.
Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis and affecting cattle herds world-wide, resulting in inflammation of the genital tract, infertility and huge economic losses. The parasite secretes a cysteine protease (CP8), which induces cytotoxicity and apoptosis in bovine vaginal and uterine epithelial cells. Mallinson et al. [D.J. Mallinson, J. Livingstone, K.M. Appleton, S.J. Lees, G.H. Coombs, M.J. North, Microbiology 1995, 141 (12) 3077-3085.] originally reported a partial DNA sequence of T. foetus CP8 based on PCR cloning of T. foetus genomic DNA. Here we report the biochemical properties of the CP8 enzyme. Kinetic properties and the substrate specificity profile of T. foetus CP8 were studied using positional scanning synthetic combinatorial libraries and Michaelis-Menten kinetic analysis of three synthetic fluorogenic substrates. The preferred substrate Z-Leu-Arg-MCA prevented host-cell death/apoptosis induced by CP8. In addition, the DNA sequence was completed by 3' and 5' rapid amplification of cDNA ends (RACE) and the full-length amino acid sequence was obtained.
