STRUCTURAL CHARACTERIZATION OF 2 FORMS OF PROCYCLIC ACIDIC REPETITIVEPROTEIN EXPRESSED BY PROCYCLIC FORMS OF TRYPANOSOMA-BRUCEI

A procyclic acidic repetitive protein (PARP) fraction was purified fro m long-term cultures of Trypanosoma brucei procyclic forms by a solven t-extraction and reverse phase chromatography procedure. The PARP frac tion yielded small quantities of a single N-linked oligosaccharide wit h the structure Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1- 3)Man beta 1 4GlcNAc (Man(5)GlcNAc(2)). Fractionation of PARP on Con A -Sepharose revealed that the majority (80 to 90%) of the PARP fraction did not bind to Con A and was composed of the parpA alpha gene produc t that contains repeats of -Glu-Pro-Pro-Thr- (GPEET-PARP) and that lac ks an N-glycosylation site. This form of PARP has not been previously identified at the protein-level. The minor Con-A-binding fraction was shown to be rich in the previously described form of PARP, encoded by the parpA beta and/or parpB alpha. genes, that contains a -Glu-Pro- re peat domain (EP-PARP) and an N-glycosylation site. Analysis of longer and shorter-term cultures suggested that procyclic tells initially exp ress predominantly EP-PARP that is gradually replaced by GPEET-PARP. b oth forms of PARP were shown to contain indistinguishable glycosylphos phatidylinositol (GPI) membrane anchors, where the conserved GPI core structure is substituted by heterogeneous sialylated branched polylact osamine-like structures that are predicted to form a dense surface gly cocalyx above which the polyanionic -Glu-Pro-Pro-Thr- and -Glu-Pro- re peat domains are displayed. The phosphatidylinositol (PI) component of the GPI anchor was shown to be a mixture of yo-inositol-1-HPO4-(sn-1- stearoyl-2-lyso-glycerol) and -inositol-1-HPO4-(sn-1-octadecyl-2-lyso- glycerol), where the acyl chain substituting the inositol ring showed considerable heterogeneity. Mass spectrometric and light scattering ex periments both suggested an average mass of approximately 15 kDa for G PEET-PARP, with individual glycoforms ranging from about 12 kDa to 20 kDa, that is consistent with its amino acid and carbohydrate compositi on. A measured translational diffusion coefficient of 3.9 x 10(7) cm(2 ) s(-1) indicates that this molecule has a highly elongated shape. The possible functions of these unusual glycoproteins are discussed. (C) 1997 Academic Press Limited.