ANALYSIS OF THE ACTIVE-SITE AND ACTIVATION MECHANISM OF THE LEISHMANIA SURFACE METALLOPROTEINASE GP63

The major surface glycoprotein of Leishmania promastigotes, referred t o as GP63, is a zinc metalloproteinase of 63 000 M(r) containing a gly cosylphosphatidylinositol (GPI) membrane anchor. Recent studies demons trated that recombinant GP63 (rGP63) expressed by the baculovirus inse ct cell system was secreted as a glycosylated latent proteinase that r equired 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 activatio n mechanism of latent rGP63 was studied using both secreted and cell s urface expression systems. To determine whether the proposed active si te of L. major GP63 conforms to other well characterized zinc metallop roteinases, the proposed GP63 catalytic Glu-265, corresponding to cata lytic Glu-147 of thermolysin, was changed to Asp-265. Using a transien t expression system in COS-7 cells, expression of the Asp-265 mutant G P63 gene resulted in rGP63 with no detectable proteinase activity, whe reas expression of the wild-type GP63 gene resulted in rGP63 with a le vel 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 sequenc e analysis revealed that activation with HgCl2 resulted in removal of the pro region, ultimately generating the mature NH2-terminus. This pr ocessing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism, since the addition of previously activate d rGP63 did not lead to an enhancement of latent rGP63 proteinase acti vation. The mechanism of activation of GP63 is consistent with the cys teine switch mechanism proposed for matrix metalloproteinases and thus has been conserved from protozoa to mammals.