Abstract
Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH 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 approximately 20-fold less than wild-type cells. The selective conditions chosen fully restored this deficiency in transfectants with the wild-type gene. Under these conditions, all transfectants were found comparable in both the plasmid copy number per cell and elevation of gp63 transcripts. Mutant and wild-type products in the transfectants were then compared quantitatively and qualitatively by specific immunologic and protease assays. 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-intramolecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Substitution of Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp63 as a functional protease is regulated by these posttranslational modification pathways.
Highlights
Leishmanolysin (EC 3.4.24.36) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa
We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition
2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63
Summary
Cloning and Site-directed Mutagenesis of gp Genes—Fig. 1 shows the amino acid residues of gp substituted by oligonucleotide-directed mutagenesis [35] of the genes using a phagemid mutagenesis kit (BioRad). Western Blot and Immunoprecipitation Analyses—SDS-PAGE-resolved proteins were transferred to nitrocellulose (Schleicher & Schuell) for immunoblot analysis [10, 12] using rabbit antiserum raised against denatured and deglycosylated gp63 [5] that was purified from L. amazonensis [8]. Immune complexes were collected by Protein A-Sepharose (Sigma), followed by washing and autoradiography of these samples were performed as described [10, 12]. GPI-PLC Digestion—gp was immunoprecipitated from the culture supernatants of transfectants with mutant N577L using anti-gp polyclonal antibodies cross-linked to Protein A-Sepharose with dimethylpimelimidate. All samples were incubated with or without GPI-PLC for 3 h at 37 °C and subjected to SDS-PAGE/Western blot analysis with the ECL reagents using a 1/10,000 dilution of either anti-gp or anti-CRD antisera. From a set of 5 representative data in the gel assays for gelatinolytic activity, the faster migrating overexpressed band (Fig. 3, panel C, open arrows) was scanned versus the endogenous slower migrating band (solid arrows) from the photographic negatives (Table I, Gelatinolytic activity)
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