Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei

Achim Treumann, Nicole Zitzmann, Andreas Hulsmeier, Alan R. Prescott, Andrew Almond, John Sheehan, Michael A. J. Ferguson

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    Abstract

    A procyclic acidic repetitive protein (PARP) fraction was purified from long-term cultures of Trypanosoma brucei procyclic forms by a solvent-extraction and reverse phase chromatography procedure. The PARP fraction yielded small quantities of a single N-linked oligosaccharide with the structure Mana1-6(Man a1-3)Mana1-6(Mana1-3)Manß1-4GlcNAcß1-4GlcNAc(Man5GlcNAc2). 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 parpAa gene product that contains repeats of -Glu-Pro-Pro-Thr- (GPEET-PARP) and that lacks 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 parpAß and/or parpBa genes, that contains a -Glu-Pro- repeat domain (EP-PARP) and an N-glycosylation site. Analysis of longer and shorter-term cultures suggested that procyclic tells initially express predominantly EP-PARP that is gradually replaced by GPEET-PARP. Both forms of PARP were shown to contain indistinguishable glycosylphosphatidylinositol (GPI) membrane anchors, where the conserved GPI core structure is substituted by heterogeneous sialylated branched polylactosamine-like structures that are predicted to form a dense surface glycocalyx above which the polyanionic -Glu-Pro-Pro-Thr- and -Glu-Pro- repeat domains are displayed. The phosphatidylinositol (PI) component of the GPI anchor was shown to be a mixture of 2-O-acyl-myo-inositol-1-HPO4-(sn-1-stearoyl-2-lyso-glycerol) and 2-O-acyl-myo-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 experiments both suggested an average mass of approximately 15 kDa for GPEET-PARP, with individual glycoforms ranging from about 12 kDa to 20 kDa, that is consistent with its amino acid and carbohydrate composition. A measured translational diffusion coefficient of 3.9 x 107 cm2 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.

    Original languageEnglish
    Pages (from-to)529-547
    Number of pages19
    JournalJournal of Molecular Biology
    Volume269
    Issue number4
    DOIs
    Publication statusPublished - 20 Jun 1997

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