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J. Biol. Chem., Vol. 279, Issue 17, 17038-17046, April 23, 2004

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Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence

PROPEPTIDE CLEAVAGE SITES AND AUTOACTIVATION OF THE ZYMOGEN SECRETED FROM GASTRODERMAL CELLS^*

Peter R. Collins, Colin M. Stack¶||, Sandra M. O'Neill, Sean Doyle¶, Thecla Ryan, Gerard P. Brennan**, Angela Mousley**, Michael Stewart**, Aaron G. Maule**, John P. Dalton¶¶, and Sheila Donnelly||||

From the School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland, the ^¶Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Republic of Ireland, the ^**Parasitology Research Group, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom, and the Institute for the Biotechnology of Infectious Diseases (IBID), University of Technology, Sydney (UTS), Westbourne Street, Gore Hill, Sydney, New South Wales 2065, Australia

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys²⁶ with Gly²⁶, failed to autoprocess. However, [Gly²⁶]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.

Received for publication, August 11, 2003 , and in revised form, January 29, 2004.

^* 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.

Supported by a grant received from Enterprise Ireland and Ildana Biotech.

^|| Funded by the Health Research Board (HRB), Ireland.

Funded by a joint North-South Cooperation grant from the HRB (Ireland) and the Research and Development Office (Northern Ireland).

^|||| Funded by The Wellcome Trust.

^¶¶ To whom correspondence should be addressed: Institute for the Biotechnology of Infectious Diseases (IBID), University of Technology, Sydney (UTS), Westbourne Street, Gore Hill, Sydney, NSW 2065, Australia. Tel.: 61-2-9514-4142; Fax: 61-2-9514-4201; E-mail: john.dalton{at}uts.edu.au.

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