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Extracellular release of the surface metalloprotease, gp63, from Leishmania and insect trypanosomatids

Charles L Jaffe1, Dennis M Dwyer

  • 1Cell Biology Section, Div. of Intramural Research, NIAID, NIH, Bethesda, MD 20892, USA. cjaffe@cc.huji.ac.il

Parasitology Research
|August 19, 2003
PubMed

Insights

Researchers detected protease activity in Leishmania and related insect trypanosomatids. This metalloprotease, identified as gp63, is released from the cell surface via a non-phospholipase mechanism.

Area of Science:

  • Parasitology
  • Molecular Biology
  • Biochemistry

Background:

  • Leishmania species cause leishmaniasis, a significant global health concern.
  • Surface glycoproteins like gp63 play crucial roles in parasite survival and virulence.
  • Understanding protease secretion mechanisms is vital for developing targeted therapies.

Purpose of the Study:

  • To characterize the protease activity secreted by Leishmania and related trypanosomatids.
  • To identify the nature and origin of the released proteases.
  • To elucidate the mechanism of gp63 release from the parasite surface.

Main Methods:

  • Culturing Leishmania donovani, L. mexicana, L. major, Crithidia luciliae, and Leptomonas seymouri.
  • Measuring protease activity in spent culture medium over time and correlating it with cell density.
  • SDS-PAGE and zymogram analysis to determine protease molecular weight.
  • Inhibitor assays using specific metalloprotease inhibitors.
  • Immunological detection using antibodies against leishmanial gp63 and Trypanosoma brucei VSG CRD.
  • Cell surface biotinylation and immunoprecipitation.

Main Results:

  • Protease activity was detected in the culture medium of all tested species, increasing linearly with time and cell density.
  • Zymography revealed proteases with molecular weights ranging from 43-100 kDa.
  • The activity was specifically inhibited by metalloprotease inhibitors (1,10-phenanthroline, Z-Tyr-Leu-NHOH).
  • Antibodies against leishmanial gp63 recognized the released proteases from all species.
  • Cell surface biotinylation indicated that over 34% of the released protease originated from the parasite surface.
  • The release mechanism was not mediated by a phospholipase, as indicated by the lack of cross-reactivity with antibodies against the VSG CRD.

Conclusions:

  • Leishmania and related trypanosomatids secrete active metalloproteases, including gp63.
  • The released gp63 is significantly derived from the cell surface.
  • gp63 is released through a mechanism distinct from phospholipase-mediated cleavage.

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