School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
Institut für Physiologische Chemie und Pathobiochemie, University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):622-7. doi: 10.1016/j.bbrc.2014.06.024. Epub 2014 Jun 17.
The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier.
南瓜韧皮部凝集素（PP2）是一种 RNA 结合、防御相关的、几丁寡糖特异性的、Mr 为 48 kDa 的同源二聚体凝集素，在南瓜（Cucurbita maxima）的筛管和伴胞中高浓度表达。在本研究中，PP2 在甲醇营养酵母毕赤酵母中表达，带有酿酒酵母α-因子序列，以将重组蛋白导向分泌途径，作为重组 PP2 正确折叠和翻译后糖基化的前提。先前的计算建模和配体对接研究预测了 PP2 表面上的一个假定的几丁寡糖结合位点，该位点分为三个亚位点，每个亚位点中的两个氨基酸残基被确定为与几丁寡糖（CHO）相互作用的可能候选物。在这项工作中，突变分析和血凝试验被用来验证预测残基在蛋白质糖结合活性中的作用。结果表明，将第 2 亚位点的丝氨酸 104 突变为丙氨酸（S104A）导致蛋白的凝集活性降低约 90%，表明丝氨酸 104 对于 CHO 与 PP2 的结合至关重要。此外，L100A（在第 1 亚位点）和 K200A（在第 3 亚位点）分别使凝集素活性降低约 40%，表明这两个残基也显著有助于 PP2 与糖的结合。总之，这些发现证实了所有三个亚位点都在不同程度上参与了 PP2-碳水化合物相互作用，并证实了早期提出的计算模型的有效性。
