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Molecular & Cellular Proteomics 7:1111-1123, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica

Expansion of a Repertoire of Virulence-associated Factors^*

Mark W. Robinson^,,¶, Jose F. Tort^,||, Jonathan Lowther, Sheila M. Donnelly, Emily Wong, Weibo Xu, Colin M. Stack^,**, Matthew Padula, Ben Herbert and John P. Dalton^,

From the Institute for the Biotechnology of Infectious Diseases and Proteomics Technology Centre of Expertise, University of Technology Sydney, Level 6, Building 4, corner of Thomas and Harris Streets, Ultimo, Sydney, New South Wales 2007, Australia, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, United Kingdom, and ^|| Departamento de Genetica, Facultad de Medicina, Universidad de la República, General Flores 2125, CP 11800 Montevideo, Uruguay

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-SerHis motif for autocatalytic cleavage by cathepsin Ls were preserved.

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				M. W. Robinson and J. P. Dalton Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases Phil Trans R Soc B, September 27, 2009; 364(1530): 2763 - 2776. [Abstract] [Full Text] [PDF]

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