Multiple procyclin isoforms are expressed differentially during the development of insect forms of Trypanosoma brucei

Transmission of Trypanosoma brucei by the tsetse fly entails several rounds of differentiation as the parasite migrates through the digestive tract to the salivary glands of its vector. Differentiation of the bloodstream to the procyclic form in the fly midgut is accompanied by the synthesis of a new coat consisting of EP and GPEET procyclins. There are three closely related EP isoforms, two of which (EP1 and EP3) contain N-glycans. To identify the individual EP isoforms that are expressed early during synchronous differentiation in vitro, we exploited the selective extraction of GPI-anchored proteins and mass spectrometry. Unexpectedly, we found that GPEET and all isoforms of EP were coexpressed for a few hours at the onset of differentiation. At this time, the majority of EP1 and EP3 molecules were already glycosylated. Within 24 hours, GPEET became the major surface component, to be replaced in turn by glycosylated forms of EP, principally EP1, at a later phase of development. Transient transfection experiments using reporter genes revealed that each procyclin 3′ untranslated region contributes to differential expression as the procyclic form develops. We postulate that programmed expression of other procyclin species will accompany further rounds of differentiation, enabling the parasite to progress through the fly.