Processing and trafficking of cysteine proteases in Leishmania mexicana.
Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.
Brooks
D R
DR
Wellcome Centre for Molecular Parasitology, University of Glasgow, The Anderson College, Glasgow G11 6NU, UK.
Tetley
L
L
Coombs
G H
GH
Mottram
J C
JC
D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation; D006031:Glycosylation; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics*; D008247:Lysosomes / Q000201:enzymology / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D003545:Cysteine