Treumann A, Zitzmann N, Hülsmeier A, Prescott A R, Almond A, Sheehan J, Ferguson M A
Department of Biochemistry, University of Dundee, UK.
J Mol Biol. 1997 Jun 20;269(4):529-47. doi: 10.1006/jmbi.1997.1066.
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.
通过溶剂萃取和反相色谱法，从布氏锥虫前循环型的长期培养物中纯化出一种前循环酸性重复蛋白（PARP）组分。该PARP组分产生了少量具有Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc（Man5GlcNAc2）结构的单一N-连接寡糖。在伴刀豆球蛋白A-琼脂糖上对PARP进行分级分离显示，大部分（80%至90%）的PARP组分不与伴刀豆球蛋白A结合，由含有-Glu-Pro-Pro-Thr-（GPEET-PARP）重复序列且缺乏N-糖基化位点的parpAα基因产物组成。这种形式的PARP以前在蛋白质水平上未被鉴定出来。较小的伴刀豆球蛋白A结合组分被证明富含先前描述的由parpAbeta和/或parpBα基因编码的PARP形式，其含有-Glu-Pro-重复结构域（EP-PARP）和一个N-糖基化位点。对长期和短期培养物的分析表明，前循环细胞最初主要表达EP-PARP，随后逐渐被GPEET-PARP取代。两种形式的PARP都显示含有难以区分的糖基磷脂酰肌醇（GPI）膜锚，其中保守的GPI核心结构被异质性的唾液酸化分支聚乳糖胺样结构取代，预计这些结构会形成致密的表面糖萼，在其上方展示出多阴离子的-Glu-Pro-Pro-Thr-和-Glu-Pro-重复结构域。GPI锚的磷脂酰肌醇（PI）组分被证明是2-O-酰基-肌醇-1-HPO4-(sn-1-硬脂酰-2-溶血甘油)和2-O-酰基-肌醇-1-HPO4-(sn-1-十八烷基-2-溶血甘油)的混合物，其中取代肌醇环的酰基链表现出相当大的异质性。质谱和光散射实验均表明GPEET-PARP的平均质量约为15 kDa，单个糖型范围约为12 kDa至20 kDa，这与其氨基酸和碳水化合物组成一致。测得的平移扩散系数为3.9×10(7) cm2 s(-1)，表明该分子具有高度细长的形状。讨论了这些异常糖蛋白的可能功能。
