Characterisation and cellular localisation of a GPEET procyclin precursor in Trypanosoma brucei insect forms

Abstract

The procyclins represent the major surface molecules of Trypanosoma brucei insect forms and consist of two classes of proteins that are characterised by internal tandem dipeptide (EP) or pentapeptide repeats (GPEET) and are attached to the membrane by a complex glycosylated glycosylphosphatidylinositol (GPI) anchor. Two different forms of GPEET can be distinguished by their differential reactivity with anti-GPEET antibodies. A major component of 22-32 kDa is recognised by a monoclonal antibody which binds to the phosphorylated form of GPEET, and a minor component of 20 kDa is recognised by a polyclonal antiserum which was raised against a synthetic GPEET peptide. The relationship between the two forms was established by (i) enriching for the 20 kDa form and determining its precise mass using MALDI-TOF mass spectrometry; (ii) studying the expression of the two forms during synchronous differentiation of pleomorphic T. brucei bloodstream forms to procyclic forms; (iii) analysing their sub-cellular distribution by immunofluorescence microscopy; and (iv) pulse-chase labelling using tritiated GPI precursors. The results indicate that the 20 kDa form represents a biosynthetic precursor of GPEET, which has just started to receive components of the poly-N-acetyllactosamine repeat of the GPI anchor.