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J. Biol. Chem., Vol. 282, Issue 22, 16532-16543, June 1, 2007

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The Major Secreted Cathepsin L1 Protease of the Liver Fluke, Fasciola hepatica

A LEU^-12 TO PRO^-12 REPLACEMENT IN THE NONCONSERVED C-TERMINAL REGION OF THE PROSEGMENT PREVENTS COMPLETE ENZYME AUTOACTIVATION AND ALLOWS DEFINITION OF THE MOLECULAR EVENTS IN PROSEGMENT REMOVAL^*

Colin M. Stack¹, Sheila Donnelly², Jonathan Lowther, Weibo Xu, Peter R. Collins, Linda S. Brinen^¶3, and John P. Dalton⁴

From the Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Building 4, Harris Street, Ultimo, Sydney, New South Wales 2007, Australia, School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland, and the ^¶Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2140

A protease secreted by the parasitic helminth Fasciola hepatica, a 37-kDa procathepsin L1 (FheproCL1), autocatalytically processes and activates to its mature enzyme (FheCL1) over a wide pH range of 7.3 to 4.0, although activation is more rapid at low pH. Maturation initiates with cleavages of a small proportion of molecules within the central region of the prosegment, possibly by intramolecular events. However, activation to fully mature enzymes is achieved by a precise intermolecular cleavage at a Leu^-12-Ser^-11His^-10 sequence within the nonconserved C-terminal region of the prosegment. The importance of this cleavage site in enzyme activation was demonstrated using an active site variant FheproCL1Gly²⁶ (Cys²⁶ to Gly²⁶) and a double variant FheproCL1Pro^-12/Gly²⁶ (Leu^-12 to Pro^-12), and although both of these variants cannot autocatalytically process, the former is susceptible to trans-processing at a Leu^-12-Ser^-11His^-10 sequence by pre-activated FheCL1, but the latter is not. Another F. hepatica secreted protease FheCL2, which, unlike FheCL1, can readily accept proline in the S2 subsite of its active site, can trans-process the double variant FheproCL1Pro^-12/Gly²⁶ by cleavage at the Pro^-12-Ser^-11His^-10 sequence. Furthermore, the autoactivation of a variant enzyme with a single replacement, FheproCL1Pro^-12, was very slow but was increased 40-fold in the presence of FheCL2. These studies provide a molecular insight into the regulation of FheproCL1 autocatalysis.

Received for publication, December 15, 2006 , and in revised form, March 2, 2007.

^* 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.

¹ Supported by grants obtained from Enterprise Ireland, Republic of Ireland, and Grant DP0557819 from the Australian Research Council, Australia.

² Supported by grants from The Wellcome Trust and National Health and Medical Research Council Grant ID352912, Australia.

³ Supported by the Sandler Family Supporting Foundation and the Sandler Center for Basic Research in Parasitic Diseases.

⁴ Recipient of a New South Wales Government BioFirst Award in Biotechnology. To whom correspondence should be addressed: Institute for the Biotechnology of Infectious Diseases, Bldg. 4, Thomas and Harris St., Faculty of Science, University of Technology Sydney, Ultimo, Sydney, New South Wales 2007, Australia. Tel.: 61-2-95144142; Fax: 61-2-95144201; E-mail: john.dalton{at}uts.edu.au.

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