The procyclin repertoire of Trypanosoma brucei - Identification and structural characterization of the Glu-Pro-rich polypeptides

The surface of the insect stages of the protozoan parasite Trypanosoma bruc ei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glyc oproteins known as procyclins. One type of procyclin, the EP isoform, is pr edicted to have 22-30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequ ences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractiona tion techniques. To facilitate their structural and functional characteriza tion, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoi re expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise informati on about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. brucei clone the glyc osylated products of the EP3 gene and two different products of the EP1 gen e (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated p roducts of the GREET and EP2 genes were detected. Because all procyclin gen es are transcribed polycistronically, the latter finding indicates that the expression of the GREET and ER2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic t rypanosomes has been characterized at the protein level.