Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei.
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 Man alpha1-6(Man alpha1-3)Man alpha1-6(Man alpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc (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 parpA alpha 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 parpAbeta and/or parpB alpha 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 cells 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 10(7) cm2 s(-1) indicates that this molecule has a highly elongated shape. The possible functions of these unusual glycoproteins are discussed.
Treumann
A
A
Department of Biochemistry, University of Dundee, UK.
Zitzmann
N
N
Hülsmeier
A
A
Prescott
A R
AR
Almond
A
A
Sheehan
J
J
Ferguson
M A
MA
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D015800:Protozoan Proteins / Q000737:chemistry*; D014346:Trypanosoma brucei brucei / Q000737:chemistry*
D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D015800:Protozoan Proteins; C053277:procyclic acidic repetitive protein, Trypanosoma