title: Extracellular release of the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metalloprotease, gp63, is independent of GPI phospholipolysis: implications for parasite virulence.
abstract: The major zinc metalloprotease of Leishmania (gp63), an important determinant of parasite virulence, is attached to the parasite surface via a glycosylphosphatidylinositol anchor. Here we report the spontaneous release of proteolytically active gp63 from a number of Leishmania isolates, causing cutaneous and visceral disease. To investigate the mechanism(s) of gp63 release, we transfected a gp63-deficient variant of Leishmania amazonensis with constructs expressing gp63 and various mutants thereof. Surprisingly, approximately half of wild type gp63 was found in the culture supernatant 12 h post-synthesis. Biochemical analysis of the extracellular gp63 revealed two forms of the protein, one that is released from the cell surface, and another, that apparently is directly secreted. Release of cell surface gp63 was significantly reduced when the proteolytic activity of the protein was inactivated by site-specific mutagenesis or inhibited by zinc chelation, suggesting that release involves autoproteolysis. The extracellular gp63 does not contain a glycosylphosphatidylinositol moiety or ethanolamine, indicating that phospholipolysis is not involved in the release process. Release of gp63 is also independent of glycosylation. The finding of proteolytically active, extracellular gp63 produced by multiple Leishmania isolates suggests a potential role of the extracellular enzyme in substrate degradation relevant to their survival in both the mammalian host and the insect vector.
authors.affiliation: Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, USA. b-mcgwire@northwestern.edu
authors.forename: Bradford S
authors.initials: BS
authors.lastname: McGwire
authors.forename: William A
authors.initials: WA
authors.lastname: O'Connell
authors.forename: Kwang-Poo
authors.initials: KP
authors.lastname: Chang
authors.forename: David M
authors.initials: DM
authors.lastname: Engman
mesh_terms: D000818:Animals; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007891:Leishmania / Q000201:enzymology / Q000472:pathogenicity*; D008666:Metalloendopeptidases / Q000378:metabolism*; D014774:Virulence
chemical_list: D017261:Glycosylphosphatidylinositols; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania