Processing of the Papain Precursor
THE IONIZATION STATE OF A CONSERVED AMINO ACID MOTIF WITHIN THE Pro REGION PARTICIPATES IN THE REGULATION OF INTRAMOLECULAR PROCESSING (*)
- Thierry Vernet(§),
- Paul J. Berti(¶),
- Chantal de Montigny,
- Roy Musil(**),
- Daniel C. Tessier,
- Robert Ménard,
- Marie-Claude Magny,
- Andrew C. Storer and
- David Y. Thomas
- From the (1) Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montréal, Québec H4P 2R2, Canada
- § To whom correspondence should be addressed: Institut de Biologie Structurale, Avenue des Martyrs, 38027 Grenoble Cedex 1 France. E mail: Vernet{at}IBS.FR.
Abstract
The cysteine protease papain is synthesized as a 40-kDa inactive precursor with a 107-amino-acid N-terminal pro region. Although
sequence conservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp
, papain precursor numbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate
the function to this conserved motif, we have mutagenized at random each of the 4 residues individually within the pro region
of the papain precursor. Precursor mutants were expressed in yeast, screened according to their ability to be processed through
either a cis or trans reaction, into mature active papain. Three classes of mutants were found. Non-functional propapain mutants of the first class
are completely degraded by subtilisin indicating that they are not folded into a native state. Mutants of the second class
were neutral with respect to cis and trans processing. The third class included mutants that mostly accumulated as mature papain in the yeast vacuole. They had mutations
that had lost the negatively charged Asp
residues and a mutation that probably introduces a positive charge, Phe
His. The precursor of the Phe
His mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe
His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. These results
indicate that the electrostatic status of the conserved motif participates in the control of intramolecular processing of
the papain precursor.
Footnotes
-
↵* This work was supported as part of the Protein Engineering Network of Centres of Excellence sponsored by the government of Canada. This paper is issued as National Research Council of Canada publication No. 38531. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- E-64
-
1-[[(L- trans-epoxysuccinyl)-L-Leucyl]amino]-4-guanidino)butane
- HPLC
-
high performance liquid chromatography.
-
↵2 T. Vernet, D. C. Tessier, J. Chatellier, C. Plouffe, T. S. Lee, D. Y. Thomas, A. C. Storer, and R. Ménard, submitted for publication.
-
↵3 P. J. Berti and A. C. Storer, personal communication.
-
↵4 T. Vernet, D. C. Tessier, and D. Y. Thomas, unpublished results.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
