Expression and function of the Trypanosoma brucei major surface protease (GP63) genes.
The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.
LaCount
Douglas J
DJ
Department of Biochemistry, University of Iowa, Iowa City 52242, USA.
Gruszynski
Amy E
AE
Grandgenett
Paul M
PM
Bangs
James D
JD
Donelson
John E
JE
D000595:Amino Acid Sequence; D000818:Animals; D015971:Gene Expression Regulation, Enzymologic; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*
D015800:Protozoan Proteins; D008666:Metalloendopeptidases