Mutational and functional analysis of theLeishmaniasurface metalloproteinase GP63: similarities to matrix metalloproteinases

Abstract

SUMMARY: The major surface glycoprotein ofLeishmania, referred to as GP63, is a zinc metalloproteinase of 63000M_rpresent on promastigotes and amastigotes from diverse species ofLeishmania. 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 clonedL. major GP63genes was carried out to determine whether the proposed active site ofLeishmaniaGP63 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. majorrecombinant GP63 (rGP63) has been produced in the baculovirus expression system whererGP63 was secreted as a latent proteinase. To study the activation of baculovirusrGP63, purifiedrGP63 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 ofL. 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 entireL. major GP63locus, containing in total sixGP63genes. Strategies to delete the secondGP63gene locus are developed and will determine whether deletion of both loci results in viable promastigotes.L. majorstrains deficient in the expression of GP63 may then be used to address the function of GP63 glycoprotein in the life cycle ofLeishmania.