title: Recombinant fungal lectin as a new tool to investigate O-GlcNAcylation processes.
abstract: 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.
authors: CERMAV, UPR5301, CNRS  and Université Grenoble Alpes, 38041 Grenoble, France.
authors: Oriane
authors: O
authors: Machon
authors: University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France.
authors: Steffi F
authors: SF
authors: Baldini
authors: CERMAV, UPR5301, CNRS  and Université Grenoble Alpes, 38041 Grenoble, France.|DCM, UMR5250, CNRS and Université Grenoble Alpes , 38041 Grenoble, France.
authors: João P
authors: JP
authors: Ribeiro
authors: University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France.
authors: Agata
authors: A
authors: Steenackers
authors: CERMAV, UPR5301, CNRS  and Université Grenoble Alpes, 38041 Grenoble, France.
authors: Annabelle
authors: A
authors: Varrot
authors: University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France imberty@cermav.cnrs.fr tony.lefebvre@univ-lille1.fr.
authors: Tony
authors: T
authors: Lefebvre
authors: CERMAV, UPR5301, CNRS  and Université Grenoble Alpes, 38041 Grenoble, France imberty@cermav.cnrs.fr tony.lefebvre@univ-lille1.fr.
authors: Anne
authors: A
authors: Imberty
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000235:genetics; D000363:Agaricales / Q000737:chemistry / Q000235:genetics; D055785:Gene Knockdown Techniques; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000235:genetics*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics*
chemical_list: D037102:Lectins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine
keywords: Fungal lectin; O-GlcNAcylation; rPVL