Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

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.

Major Surface Glycoproteins of Insect Forms of Trypanosoma brucei Are Not Essential for Cyclical Transmission by Tsetse

“…We have found that a mutant that constitutively expresses GPEET is transmitted very efficiently (unpublished data). The GPEET expressed by late procyclic forms and epimastigote forms was not phosphorylated, however, which is consistent with reports that the activity of the kinase is restricted to early procyclic forms [29].…”

Social Motility of African Trypanosomes Is a Property of a Distinct Life-Cycle Stage That Occurs Early in Tsetse Fly Transmission

“…Moreover, there might also be post-translational modifications or non-peptide moieties that are stage-specific. For example, the activity of the kinase that phosphorylates GPEET is restricted to early procyclic forms [33]. It is also known that early procyclic forms of T. congolense preferentially express PRS, a protease-resistant surface glycoconjugate [34], [35], although an equivalent molecule has not been reported for T. brucei .…”

Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei

“…By 24 h, the cells show the characteristic profile of early procyclic forms, with high levels of GPEET and traces of EP. GPEET is phosphorylated by a membrane-associated kinase [13] that also becomes active within a few hours of triggering differentiation [3,11,14]. If glycerol is present in the medium [3] and the mitochondrial alternative oxidase is active [15], the cells continue to proliferate as early forms and express GPEET.…”

“…If glycerol is present in the medium [3] and the mitochondrial alternative oxidase is active [15], the cells continue to proliferate as early forms and express GPEET. In medium lacking glycerol, the trypanosomes differentiate to late procyclic forms within 7–9 d; this is marked by the replacement of GPEET by the glycosylated isoforms of EP (EP1 and EP3) [11] and loss of GPEET kinase activity [13]. The same sequence of events, expression of GPEET by early procyclic forms and its replacement by EP1 and/or EP3 in established procyclic forms also occurs during tsetse fly infections [12].…”