THE ROLE OF PROTEOLYSIS DURING DIFFERENTIATION OF TRYPANOSOMA-BRUCEI FROM THE BLOOD-STREAM TO THE PROCYCLIC FORM

The in vitro differentiation of Trypanosoma brucei from bloodstream to procyclic (insect) forms is accompanied by diminishing variant surfac e glycoprotein (VSG) and increasing levels of procyclin and phosphoeno lpyruvate carboxykinase (PEPCK). In this study, we examined the fate o f several glycolytic enzymes of T. brucei during differentiation. We o bserved a down-regulation of glycosomal phosphoglycerate kinase (gPGK) during differentiation. In contrast, intracellular levels of glycosom al glyceraldehpde-3-phosphate dehydrogenase (gGAPDH), aldolase (ALD), and phosphoglucoisomerase (PGI) remained unchanged during differentiat ion and apparently continued to be synthesized in the procyclic form. To determine the potential role of proteasomes and other proteases dur ing the differentiation process, we tested the effect of lactacystin, a specific inhibitor of proteasome activity, and morpholinourea-Phe-ho moPhe-benz-alpha-pyrone (P27), a selective inhibitor of cysteine prote ases, on the in vitro differentiation of T. brucei. Cells differentiat ed normally in the presence of 1 mu M lactacystin, which confirmed our previous observation that this differentiation does not require cross ing any phase boundaries in the cell cycle (Mutomba and Wang, Mol Bioc hem Parasitol 1996;80:89-102). But the cells thus differentiated did n ot increase in number and retained gPGK. Cells differentiated under 2 mu M P27 also proceeded at a normal rate but failed to multiply and re tained gPGK. However, most of the differentiated cells under 2 mu M P2 7 also retained VSG on the cell membrane surface and expressed higher levels of procyclin suggesting that a cysteine protease(s) may be invo lved in releasing VSG and partially reducing procyclin during differen tiation. This cysteine protease(s) has been tentatively identified in the procyclic cells as a 48 kDa protein through labeling of cysteine p rotease(s) with a biotinylated P27 homolog K02 (morpholinourea-Phe-hom oPhe-vinylsulfone). (C) 1998 Elsevier Science B.V. All rights reserved .