Analysis of the active site and activation mechanism of the Leishmania surface metalloproteinase GP63.
The major surface glycoprotein of Leishmania promastigotes, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) containing a glycosylphosphatidylinositol (GPI) membrane anchor. Recent studies demonstrated that recombinant GP63 (rGP63) expressed by the baculovirus insect cell system was secreted as a glycosylated latent proteinase that required activation for full proteinase activity (Button et al. (1993) Gene 134, 75-81). To extend these studies, the active site of L. major GP63 was characterized by site-directed mutagenesis and the activation mechanism of latent rGP63 was studied using both secreted and cell surface expression systems. To determine whether the proposed active site of L. major GP63 conforms to other well characterized zinc metalloproteinases, the proposed GP63 catalytic Glu-265, corresponding to catalytic Glu-147 of thermolysin, was changed to Asp-265. Using a transient expression system in COS-7 cells, expression of the Asp-265 mutant GP63 gene resulted in rGP63 with no detectable proteinase activity, whereas expression of the wild-type GP63 gene resulted in rGP63 with a level of proteinase activity similar to native GP63. Thus, the mechanism of GP63 proteinase activity is predicted to be homologous to that of other well characterized zinc metalloproteinases. NH2-Terminal sequence analysis revealed that activation with HgCl2 resulted in removal of the pro region, ultimately generating the mature NH2-terminus. This processing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism, since the addition of previously activated rGP63 did not lead to an enhancement of latent rGP63 proteinase activation. The mechanism of activation of GP63 is consistent with the cysteine switch mechanism proposed for matrix metalloproteinases and thus has been conserved from protozoa to mammals.
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
M H
MH
Macdonald
C J
CJ
Morrison
W R
WR
McMaster
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D007891:Leishmania / Q000201:enzymology*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008627:Mercuric Chloride / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D008627:Mercuric Chloride; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania