UR1268 Unité de Recherches Biopolymères Interactions Assemblages, INRA, F-44300 Nantes, France.
Plant Physiol. 2010 Jul;153(3):1345-61. doi: 10.1104/pp.110.153882. Epub 2010 May 4.
Phloem Protein2 (PP2) is a component of the phloem protein bodies found in sieve elements. We describe here the lectin properties of the Arabidopsis (Arabidopsis thaliana) PP2-A1. Using a recombinant protein produced in Escherichia coli, we demonstrated binding to N-acetylglucosamine oligomers. Glycan array screening showed that PP2-A1 also bound to high-mannose N-glycans and 9-acyl-N-acetylneuraminic sialic acid. Fluorescence spectroscopy-based titration experiments revealed that PP2-A1 had two classes of binding site for N,N',N''-triacetylchitotriose, a low-affinity site and a high-affinity site, promoting the formation of protein dimers. A search for structural similarities revealed that PP2-A1 aligned with the Cbm4 and Cbm22-2 carbohydrate-binding modules, leading to the prediction of a beta-strand structure for its conserved domain. We investigated whether PP2-A1 interacted with phloem sap glycoproteins by first characterizing abundant Arabidopsis phloem sap proteins by liquid chromatography-tandem mass spectrometry. Then we demonstrated that PP2-A1 bound to several phloem sap proteins and that this binding was not completely abolished by glycosidase treatment. As many plant lectins have insecticidal activity, we also assessed the effect of PP2-A1 on weight gain and survival in aphids. Unlike other mannose-binding lectins, when added to an artificial diet, recombinant PP2-A1 had no insecticidal properties against Acyrthosiphon pisum and Myzus persicae. However, at mid-range concentrations, the protein affected weight gain in insect nymphs. These results indicate the presence in PP2-A1 of several carbohydrate-binding sites, with potentially different functions in the trafficking of endogenous proteins or in interactions with phloem-feeding insects.
韧皮部蛋白 2（PP2）是在筛分子中发现的韧皮部蛋白体的组成部分。我们在这里描述拟南芥（Arabidopsis thaliana）PP2-A1 的凝集素特性。使用在大肠杆菌中产生的重组蛋白，我们证明了它与 N-乙酰葡萄糖胺寡聚物的结合。糖基阵列筛选表明 PP2-A1 还与高甘露糖 N-糖基和 9-酰基-N-乙酰神经氨酸唾液酸结合。基于荧光光谱的滴定实验表明，PP2-A1 对 N,N',N''-三乙酰壳三糖具有两类结合位点，一个低亲和力位点和一个高亲和力位点，促进蛋白二聚体的形成。对结构相似性的搜索表明，PP2-A1 与 Cbm4 和 Cbm22-2 碳水化合物结合模块对齐，导致其保守结构域的预测β-折叠结构。我们通过首先通过液相色谱-串联质谱法对丰富的拟南芥韧皮部汁液蛋白进行表征，来研究 PP2-A1 是否与韧皮部汁液糖蛋白相互作用。然后我们证明 PP2-A1 与几种韧皮部汁液蛋白结合，并且糖苷酶处理不能完全消除这种结合。由于许多植物凝集素有杀虫活性，我们还评估了 PP2-A1 对蚜虫体重增加和存活的影响。与其他甘露糖结合凝集素不同，当添加到人工饮食中时，重组 PP2-A1 对桃蚜和烟粉虱没有杀虫特性。然而，在中浓度范围内，该蛋白影响昆虫若虫的体重增加。这些结果表明 PP2-A1 中存在几个碳水化合物结合位点，在内源性蛋白的运输或与韧皮部取食昆虫的相互作用中具有潜在的不同功能。
