title: A function for a specific zinc metalloprotease of African trypanosomes.
abstract: The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.
authors.affiliation: Interdepartmental Genetics Program, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Grandgenett
authors.forename: Keiko
authors.initials: K
authors.lastname: Otsu
authors.forename: Helen R
authors.initials: HR
authors.lastname: Wilson
authors.forename: Mary E
authors.initials: ME
authors.lastname: Wilson
authors.forename: John E
authors.initials: JE
authors.lastname: Donelson
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan; D002460:Cell Line; D017353:Gene Deletion; D018628:Gene Dosage; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D008018:Life Cycle Stages / Q000502:physiology; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D045726:Metalloproteases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase