Extracellular release of the surface metalloprotease, gp63, from Leishmania and insect trypanosomatids.
Protease activity was found in spent culture medium collected from Leishmania donovani, L. mexicana, L. major, as well as the insect trypanosomatids, Crithidia luciliae and Leptomonas seymouri. Released protease activity increased linearly over time and was correlated to promastigote density. In SDS-PAGE, zymogram gels showed that the protease's molecular weight ranged from 43-100 kDa. Spent culture medium proteases were blocked by the metallo-protease inhibitors, 1,10-phenanthroline and Z-Tyr-Leu-NHOH, but not by bestatin, leupeptin, ABESF, pepstatin A, E-64 or aprotinin. Monoclonal and/or polyclonal antibodies to the leishmanial gp63 reacted with the released Crithidia, Leptomonas, L. major and L. donovani proteases. Cell surface biotinylation and immune precipitation using gp63-specific antibodies showed that >34% of the released protease originated from the surface. Antibodies against the Trypanosoma brucei variable surface glycoprotein cross-reactive determinant (CRD) did not recognize this activity, suggesting that the gp63 is not cleaved from the cell surface by a parasite phospholipase, but is released by an alternative mechanism.
Jaffe
Charles L
CL
Cell Biology Section, Div. of Intramural Research, NIAID, NIH, Bethesda, MD 20892, USA. cjaffe@cc.huji.ac.il
Dwyer
Dennis M
DM
D000818:Animals; D003421:Crithidia / Q000201:enzymology* / Q000254:growth & development; D017077:Culture Media, Conditioned / Q000737:chemistry; D007313:Insecta / Q000469:parasitology; D007891:Leishmania / Q000201:enzymology* / Q000254:growth & development; D008666:Metalloendopeptidases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014351:Trypanosomatina / Q000201:enzymology* / Q000254:growth & development
D017077:Culture Media, Conditioned; D011480:Protease Inhibitors; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania