Machon Oriane, Baldini Steffi F, Ribeiro João P, Steenackers Agata, Varrot Annabelle, Lefebvre Tony, Imberty Anne
CERMAV, UPR5301, CNRS and Université Grenoble Alpes, 38041 Grenoble, France.
University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France.
Glycobiology. 2017 Jan;27(2):123-128. doi: 10.1093/glycob/cww105. Epub 2016 Oct 18.
Glycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here, we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. Recombinant PVL (Psathyrella velutina lectin) (rPVL) displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA (wheat germ agglutinin). Labeling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by western blot. The staining of whole cell lysates with labeled-rPVL was dramatically decreased in response to O-GlcNAc transferase knockdown and seen to increase after pharmacological blockade of O-GlcNAcase. Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins.
糖基化是一组翻译后修饰，具有多种结构，并参与众多生物学过程。因此，研究糖基化需要不同的技术方法和可靠的工具，凝集素就是其中一部分。在此，我们描述了重组蘑菇凝集素PVL用于区分O-GlcNAc糖基化的用途，O-GlcNAc糖基化是一种修饰，即单个N-乙酰葡糖胺残基连接到存在于胞质、细胞核和线粒体区室中的蛋白质上。通过热位移分析和表面等离子体共振实验验证，重组PVL（绒毛小皮伞凝集素）（rPVL）对GlcNAc的亲和力明显强于Neu5Ac残基，因此是小麦胚芽凝集素（WGA）的极佳替代品。用生物素或辣根过氧化物酶（HRP）标记rPVL，可通过蛋白质印迹法有效利用它。用标记的rPVL对全细胞裂解物进行染色，在O-GlcNAc转移酶敲低后显著减少，而在O-GlcNAcase的药理学阻断后增加。此外，HRP-rPVL似乎比抗O-GlcNAc抗体RL2更敏感。因此，rPVL是一种选择性检测O-GlcNAc化蛋白质的有效新工具。
