Autocatalytic Processing of Recombinant Human Procathepsin L
CONTRIBUTION OF BOTH INTERMOLECULAR AND UNIMOLECULAR EVENTS IN THE PROCESSING OF PROCATHEPSIN L IN VITRO*
- Robert Ménard‡§,
- Euridice Carmona‡¶,
- Sachiko Takebe‡,
- Éric Dufour‡‖,
- Céline Plouffe‡,
- Patrizia Mason** and
- John S. Mort**
- From the ‡Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada and the**Joint Diseases Laboratory, Shriners Hospital for Children, and Department of Surgery, McGill University, Montreal, Quebec H3G 1A6, Canada
Abstract
The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.
Footnotes
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↵* This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil (to E. C.), NATO, and the Ministère de la recherche et de l’espace, France (to E. D.). This is NRCC Publication 41400. The Biotechnology Research Institute and McGill University are members of the Protein Engineering Network of Centres of Excellence, sponsored by the government of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Fax: 514-496-5143; E-mail: robert.menard{at}nrc.ca.
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↵¶ Visiting scientist from Instituto Butantan, C.P. 65, 05504, São Paulo, Brazil.
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↵‖ Visiting scientist from Institut National de la Recherche Agronomique, BP 1627, 44316 Nantes Cedex 03, France.
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↵1 The abbreviations used are: Cbz-Phe-Arg-MCA, benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-methylcoumarinyl-7-amide hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
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↵2 Residues in the propeptide are identified with the suffix p. Numbering is based on the sequence of human procathepsin L starting at the N-terminal amino acid of the proregion (residue 1p). The C-terminal amino acid of the proregion corresponds to position 96p and is followed by the N-terminal residue (position 1) of the mature enzyme.
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↵3 R. Ménard, E. Carmona, S. Takebe, and R. Coulombe, unpublished data.
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- Received August 27, 1997.
- Revision received November 13, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
