Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases.
The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) 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 alpha 2-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.(ABSTRACT TRUNCATED AT 250 WORDS)
McMaster
W R
WR
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Morrison
C J
CJ
MacDonald
M H
MH
Joshi
P B
PB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D017125:Genes, Protozoan / Q000235:genetics; D006801:Humans; D007891:Leishmania / Q000201:enzymology* / Q000235:genetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation
D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania