Abstract
GPEET procyclin is a major glycosylphosphatidylinositol-anchored protein of procyclic (insect stage) trypanosomes in culture and is heavily phosphorylated in the GPEET pentapeptide repeat. The phosphorylation reaction is a late event and occurs during maturation and transport of GPEET or on the parasite surface by an ecto-protein kinase. Initial biochemical characterization of the GPEET kinase activity now shows that it depends on bivalent cations for maximal activity, is stimulated by sulfhydryl group reagents, and is specific for ATP as phosphoryl donor. No kinase activity is detected in bloodstream form trypanosomes in culture, whereas strong phosphorylation is observed in early procyclic forms. In addition, the GPEET kinase activity is absent from procyclic trypanosomes that have repressed GPEET synthesis but can be induced in these same stocks by conditions, which also induce GPEET expression. However, the presence of an active kinase does not depend on the presence of (functional) GPEET because it can be detected in parasites expressing a non-phosphorylatable GPEET mutant protein and in procyclin null mutant trypanosomes. Interestingly, the presence of the glycosylphosphatidylinositol lipid moiety seems necessary for GPEET to become phosphorylated. Together, the results demonstrate that GPEET and its kinase are expressed during the same life cycle stages and that factors that induce the expression of GPEET in vitro also induce the expression of the GPEET kinase.
Highlights
This change of surface protein coat must be under tight control; because the closely packed variant surface glycoprotein (VSG) protects the parasite in the mammalian bloodstream against the immune system of the host, a premature loss of VSG would be lethal for the parasite
Control cells expressing endogenous GPEET showed increased phosphorylation in the presence of exogenous GPEET (Fig. 7B, lane 2). These results show that procyclic form parasites, in which GPEET expression is down-regulated by the removal of glycerol from the culture medium, have no GPEET kinase activity and suggest that the expression of GPEET and its kinase may be linked
Initial biochemical characterization of the GPEET kinase activity using crude membranes from procyclic parasites shows that the reaction is dependent on divalent cations and can be stimulated by reagents that bind to free sulfhydryl groups
Summary
This change of surface protein coat must be under tight control; because the closely packed VSG protects the parasite in the mammalian bloodstream against the immune system of the host, a premature loss of VSG would be lethal for the parasite. These results demonstrate that GPEET is phosphorylated on the surface of procyclic form trypanosomes by an ecto-protein kinase using exogenously added ATP as phosphoryl donor.
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