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, 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.
McGwire
B S
BS
Department of Microbiology/Immunology, University of the Health Sciences/Chicago Medical School, North Chicago, Illinois 60064, USA.
Chang
K P
KP
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D015870:Gene Expression; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D006639:Histidine / Q000737:chemistry; D007891:Leishmania / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D006639:Histidine; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D015032:Zinc