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Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases

Published online by Cambridge University Press:  06 April 2009

W. R. McMaster ,
C. J. Morrison ,
M. H. Macdonald  and
P. B. Joshi
W. R. McMaster
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
C. J. Morrison
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
M. H. Macdonald
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
P. B. Joshi
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
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Summary

The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63000 Mr present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and α2–macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS–7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2–terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity. The similarity of GP63 to the family of matrix-degrading proteinases suggests that the proteinase activity of GP63 maybe involved with the pathology of lesion formation in the mammalian host and may also be involved with the promastigote life stage in the sandfly vector. To study the functional role of GP63 proteinase, mutant strains of L. major, deficient in the expression of GP63, are currently being derived by targeted gene deletion. Using this strategy results have demonstrated the deletion of an entire L. major GP63 locus, containing in total six GP63 genes. Strategies to delete the second GP63 gene locus are developed and will determine whether deletion of both loci results in viable promastigotes. L. major strains deficient in the expression of GP63 may then be used to address the function of GP63 glycoprotein in the life cycle of Leishmania.

Keywords

active site mutationLeishmaniasurface metalloproteinaseGP63
Type
Research Article
Information
Parasitology , Volume 108 , supplement S1: Symposia of the British Society for Parasitology Volume 31:Functional molecules on the surface of protozoan parasites , March 1994 , pp. S29 - S36
Copyright
Copyright © Cambridge University Press 1994

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