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Fishing for Glyco-Proteins

O-linked β-N-acetylglucosamine (O-GlcNAc) modification sites are present in a vast number of proteins, including antibodies, and play important roles in very different biological processes like transcription, translation, protein turnover and regulation in hyperglycemia[1].

Enzymatic modification of O-GlcNAc sites can be achieved using UDP-GalNAz and a modified glycosyl-transferase (e.g. GalT(Y289L)[2]) or a glycosynthase (e.g. EndoS(D233A/Q)[3]) in combination with Copper-free Strain-promoted Azide-Alkyne Click-labeling (SPAAC).

Many such molecules are available which gives significant flexibility and makes it possible to find a labelling molecule for your experiment. Such molecules enable the identification and isolation of proteins harboring these post-translational modifications[1], but very importantly also could be a promising tool for future tumor-screening and radioimmuno-treatment in cancer therapy[4].

Figure 1: Chemo-enzymatic labeling of antibodies.

Figure 1: Chemo-enzymatic labeling of antibodies. Azide labeling of glycosylated serine or threonine residues by modified enzymes like GalT(Y289L) or EndoS(D233A/Q) opens the possibility to specifically mark antibodies using copper-free Strain-promoted Azide-Alkyne Click-labeling (SPAAC). Afterwards antibodies easily can be screened or sorted by fluorescence, for example.

Selected References:

[1] Yang et al. (2017) Protein O-GlcNAcylation: emerging mechanisms and functions.” Nature reviews. Molecular cell biology 18 (7):452.
[2] Ma et al. (2019) O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry. Anal. Chem. 91 (4):2620.
[3] Huang et al. (2012) Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134 (29):12308.
[4] Walsh et al. (2021) Site-selective modification strategies in antibody-drug conjugates. Chem. Soc. Rev. 50 (2):1305.

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