Overview of two similar modification: O-GlcNAc and O-GalNAc modifications.

Protein glycosylation represents one of the most ubiquitous and functionally diverse forms of post-translational modification and regulates many cellular and physiological processes. O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) modifications are the two simplest forms of O-linked glycosylation, each involving the attachment of a single monosaccharide (GlcNAc or GalNAc) to the hydroxyl group of amino acid residues, primarily serine or threonine. Structurally, these two-type glycosylation differ only by the stereochemistry at a single hydroxyl group, yet they exhibit markedly different biological functions. In recent years, numerous studies have reported the functions of O-GlcNAc and O-GalNAc modifications, but no comparative analysis of these two types of modifications has been reported. Moreover, accumulating evidence indicates that O-GlcNAc and O-GalNAc modifications can interfere with each other, primarily at the level of experimental detection, owing to their structural similarity and their potential coexistence within the same cellular compartments or even on the same protein, which collectively complicate their accurate identification and discrimination. This review provides a comparative overview of O-GlcNAc and O-GalNAc modifications, focusing on their structural and biosynthetic characteristics, functional divergence, potential cross-interference and methodological approaches including biological tools and chemical methods for their discrimination. A comprehensive elucidation of these modifications will not only refine our current understanding but also drive the development of more precise and mechanistically informed approaches for their investigation.