POSTTRANSLATIONAL REGULATION OF A LEISHMANIA HEXXH METALLOPROTEASE (GP63) - THE EFFECTS OF SITE-SPECIFIC MUTAGENESIS OF CATALYTIC, ZINC-BINDING, N-GLYCOSYLATION, AND GLYCOSYL PHOSPHATIDYLINOSITOL ADDITION SITES ON N-TERMINAL END CLEAVAGE, INTRACELLULAR STABILITY, AND EXTRACELLULAR EXIT

Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encod ed by multicopied genes in Leishmania and implicated in the infectivit y of these parasitic protozoa. We examined posttranslational regulatio n of gp63 expression by site-specific mutagenesis of the predicted cat alytic/zinc-binding sites in the H(264)EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition. Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient variant, which produced gp63 similar to 20-f old less than wild-type cells. The selective conditions chosen fully r estored this deficiency in transfectants with the wild-type gene. Unde r these conditions, all transfectants were found comparable in both th e plasmid copy number per cell and elevation of gp63 transcripts. Muta nt and wild-type products in the transfectants were then compared quan titatively and qualitatively by specific immunologic and protease assa ys. The results indicate the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. The propeptide of the inactive mutant products was cleaved, suggestive of a non-intramo lecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing t o a role for zinc binding in in vivo stability of gp63. 3) The three p otential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Su bstitution of Asn-577 causes release of all mutant products, indicativ e of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp6 3 as a functional protease is regulated by these posttranslational mod ification pathways.