Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc

Abstract

Studies of post-translational modification by β-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc specific antibodies that can be employed for detection, isolation, and site localization. We have obtained a large panel of O-GlcNAcspecific IgG MAbs having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by largescale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc modified proteins, including a large number of novel glycoproteins. A substantial number of the glycoproteins were only enriched by one of the antibodies and this observation combined with results of inhibition ELISAs suggests that the antibodies in addition to their O-GlcNAcdependence also appear to have different, but overlapping, local peptide determinants. The MAbs made it possible to delineate differentially modified proteins of liver in response to traumahemorrhage and resuscitation in a rat model.

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…The two most commonly used pan-specific antibodies for detecting O-GlcNAc-modified proteins are CTD110.6 , a mouse IgM antibody raised against the C-terminus of RNA Pol II, and RL2 (Snow et al 1987), a mouse IgG antibody raised against O-GlcNAc-modified components of the nuclear pore complex. Recently, a number of monoclonal IgG antibodies have been introduced for detecting OGlcNAc-modified proteins including 1F5.D6 (14), 18B10.C7(3), and 9D1.E3(10) (Teo et al 2010). These antibodies were raised against a three-component immunogen-containing epitope regions from the casein kinase α-subunit 15, a mouse major histocompatibility complex class II-restricted helper T-cell epitope, and a Toll-like receptor 2 agonist (Teo et al 2010).…”

“…Recently, a number of monoclonal IgG antibodies have been introduced for detecting OGlcNAc-modified proteins including 1F5.D6 (14), 18B10.C7(3), and 9D1.E3(10) (Teo et al 2010). These antibodies were raised against a three-component immunogen-containing epitope regions from the casein kinase α-subunit 15, a mouse major histocompatibility complex class II-restricted helper T-cell epitope, and a Toll-like receptor 2 agonist (Teo et al 2010). Like RL2, these antibodies are mouse IgGs and appear to only recognize O-GlcNAc in certain three-dimensional environments.…”

“…Examples of O-GlcNAcylated proteins include chromatinassociated proteins and histones, transcription factors, ribosomal proteins, proteasomal proteins, cytoskeletal proteins, and many different types of signaling proteins such as kinases and metabolic enzymes (Carapito et al 2009;Gurcel et al 2008;Rexach et al 2008;Teo et al 2010;Vosseller et al 2006;Wang et al 2010a, b;Wells et al 2002b). O-GlcNAc is thought to regulate these proteins in a manner analogous to protein phosphorylation.…”

“…Introduction O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a dynamic post-translational modification (PTM) of more than 3,000 nuclear, cytoplasmic, and mitochondrial proteins (Carapito et al 2009;Gurcel et al 2008;Rexach et al 2008;Teo et al 2010;Vosseller et al 2006;Wang et al 2010a, b;Wells et al 2002b;Torres and Hart 1984). While O-GlcNAc appears more common in metazoans, there is evidence for this PTM in simple eukaryotes such as Aspergillus niger (Machida and Jigami 1994) and in some prokaryotes (Shen et al 2006).…”

Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

“…This moiety is a dynamic post‐translational modification of proteins that is involved in many cellular processes and has been linked to major diseases such as diabetes and Alzheimer's disease 5. There is much interest in agents that bind O‐GlcNAc for use in detection and separation methods for modified proteins 6. A few years ago, we reported that the tricyclic receptor 4 (Figure 1) binds the simplest O‐GlcNAc model 2 with K a =630  m −1 , and the O‐GlcNAcylated peptide 3 with K a =1000  m −1 4e.…”