O-GlcNAcylation Is a Specific Glycosylation Modification

O-GlcNAcylation is an atypical, highly dynamic, and reversible glycosylation modification. O-GlcNAcylation is formed when a single N-acetylglucosamine (GlcNAc) is attached to the hydroxyl group of Ser/Thr of a target protein via an O-glycosidic bond in the β-configuration. Its synthesis involves two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). OGT and OGA coordinate with each other to precisely regulate the O-GlcNAcylation cycle. O-GlcNAcylation differs significantly from classical glycosylation modifications. O-GlcNAcylation involves the addition of only a single GlcNAc without further addition or extension. It can occur in all cell compartments. Most O-GlcNAcylation proteins are found in the nucleus, cytoplasm, and mitochondria. This modification is uncharged has a lower molecular weight and is more abundant than the classical glycosylation modification. The O-GlcNAc glycosylation modification is found to be extremely conserved in worms, fungi, plants, insects, and humans.

Fig.1 Overview of the hexosamine biosynthetic pathway and O-GlcNAcylation. (Chang, et al., 2020)

O-GlcNAcylation Glycoengineering Service at CD BioGlyco

CD BioGlyco has developed a range of O-GlcNAcylation glycoengineering services including targeted introduction and modification, identification, purification, antibody, Inhibitor studies, etc. Our O-GlcNAcylation glycoengineering services are mainly for mice, rats, drosophila, Caenorhabditis elegans, plants, etc.

• Targeted introduction and modification of O-GlcNAcylation

O-GlcNAcylation synthesis is related to OGT and OGA. We altered cell surface O-GlcNAcylation by Gene Editing means such as knock-in or knock-out, etc. By this method, we can study the biological functions of O-GlcNAcylation in plant bodies, fungi, insects, etc.

• O-GlcNAcylation analysis service

The O-GlcNAcylation analysis process includes protein extraction, enzymatic digestion, enrichment, mass spectrometry analysis, etc. We use Chemoenzymatic glycolabeling to study O-GlcNAc. The specific substrate specificity of glycosyltransferases is utilized for glycan chain derivatization of GlcNAc in O-GlcNAc structures. This is followed by the next step of click chemistry via the introduced monosaccharide derivatives with chemical reaction handles. It can add on a detection label such as a fluorescent moiety or an affinity label such as biotin.

We extract the total protein of the samples with lysis buffer. The protein solution is then analyzed by high-performance liquid chromatography (HPLC) classification and tandem mass spectrometry (MS/MS). We will perform database searches and bioinformatics analysis of the resulting data. The molecular weight of serine/threonine to which O-GlcNAcylation occurs is increased by covalently attached monosaccharides. We use mass spectrometry to accurately determine whether the molecular weight has shifted accordingly, and realize the analysis of O-GlcNAcylation peptides and sites. In addition, we also research inhibitors and antibodies for OGT and OGA.

Fig.2 Our O-GlcNAcylation glycoengineering service. (CD BioGlyco)

Publication

Paper Title: Swainsonine inhibits autophagic degradation and causes cytotoxicity by reducing CTSD O-GlcNAcylation

Technology: HPLC- MS/MS

Journal: Chemico-Biological Interactions

IF: 5.1

Published: 2023

Results: Rat primary renal tubular epithelial cells (RTECs) were treated with different concentrations of swainsonine (SW) for 12 hours. The results showed that total O-GlcNAcylation increased at 25 μg/mL and peaked at 50 and 100 μg/mL. In this study, proteome sequencing analysis and modification site identification were performed in control and SW groups by HPLC- MS/MS. The proteins related to the autophagy-lysosomal pathway and glycosylation process were finally screened and further investigated.

Fig.3 SW alters total O-GlcNAcylation in RTECs. (Wang, et al., 2023)

Applications

• O-GlcNAcylation is important for human survival. Glycoengineering techniques have been utilized to study the role of O-GlcNAcylation in human life activities and the underlying physiological mechanisms.
• Homeostatic imbalance of O-GlcNAcylation is the underlying cause of the pathogenesis of many diseases. Glycoengineering techniques were utilized to study the correlation between O-GlcNAcylation abnormalities and disease occurrence.
• Glycoengineering techniques are used to study the biological functions of O-GlcNAcylation in plants.

Advantages

• As a performed O-glycan modification, O-GlcNAcylation is a research hotspot in glycobiology with high application potential.
• We provide a one-stop O-GlcNAcylation glycoengineering service, including glycoengineering, O-GlcNAc sites and glycoforms, inhibitor studies, etc.
• Our analytical technology platform has various advantages, such as accurate site localization, higher identification depth, and good stability of the experimental process.

CD BioGlyco is constantly optimizing glycoengineering technologies. We tailor our glycoengineering services to your specific needs. In addition to O-GlcNAcylation, we also offer O-Glycan Core 1 and O-Glycan Core 2 Glycoengineering Service. Please feel free to contact us with your needs. Our researchers will reply to you for the first time.