Recent advances in demystifying O‐glycosylation in health and disease

Abstract

O-Glycosylation is one of the most common protein post-translational modifications (PTM) and plays an essential role in the pathophysiology of diseases. However, the complexity of O-glycosylation and the lack of specific enzymes for the processing of O-glycans and their O-glycopeptides make O-glycosylation analysis challenging. Recently, research on O-glycosylation has received attention owing to technological innovation and emerging O-glycoproteases. Several serine/threonine endoproteases have been found to specifically cleave O-glycosylated serine or threonine, allowing for the systematic analysis of O-glycoproteins. In this review, we first assessed the field of protein O-glycosylation over the past decade and used bibliometric analysis to identify keywords and emerging trends. We then summarized recent advances in O-glycosylation, covering several aspects: O-glycan release, site-specific elucidation of intact O-glycopeptides, identification of O-glycosites, characterization of different O-glycoproteases, mass spectrometry (MS) fragmentation methods for site-specific O-glycosylation assignment, and O-glycosylation data analysis. Finally, the role of O-glycosylation in health and disease was discussed.

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

“…305 O-glycosylation has been found to be associated with genetic disorders, infectious diseases, immune-related diseases, neurodegenerative diseases, and cancers. 306,307 Chemical and enzymatic approaches have been implemented to generate proteins with O-glycosylation mimetics. The site-directed mutated cysteine residue is transformed into Dha by alkylating reagents and reacted with active glycosylation derivatives.…”

“…In addition to the roles in protein localization and cell signaling, emerging evidence has demonstrated the involvement of O -glycosylation in protein aggregation and phase separation , …”

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography–Mass Spectrometry

“…Motivated by the urge to understand O -glycan regulation and function at the cellular level, many analytical strategies targeting these molecules have been developed in recent years. − In these efforts, mass-spectrometry-based glycomics has become one of the most powerful methods for the global analysis of glycans in complex biological samples. To obtain the deepest structural insights in protein O -glycans, the oligosaccharide are usually released from their protein carrier, taking advantages of 96-well plate sample preparation and structural characterization provided by tandem MS. − A proven approach for in-depth O -glycan analysis is based on porous graphitized carbon nanoliquid chromatography coupled to negative mode tandem mass spectrometry (PGC nano-LC-MS/MS) as initially established by Packer and co-workers. − This analytical platform for glycan alditols features extensive glycan isomer separation as well as negative mode collision induced dissociation (CID) resulting in cross-ring fragments to help to elucidate glycan structures.…”

Deciphering Protein O-GalNAcylation: Method Development and Disease Implication

“…Protein glycosylation mainly consists of N-glycosylation on asparagine (Asn or N) and O-glycosylation on serine (Ser or S), threonine (Thr or T), or tyrosine (Tyr or Y). , It is a complex and dynamic non-template-driven biosynthetic process that depends on the presence of glycoenzymes, sugar donors, and precursors, the accessibility of protein substrates, and cell signaling in the cellular microenvironment. − Aberrant glycosylation is often attributed to altered biosynthesis resulting from abnormal changes in these factors; thus, pathophysiological states of the body are associated with unique glycosylation. ,− The characterization of N-glycosylation is more mature than that of O-glycosylation due to advances in analytical methods and the availability of N-glycosidases with high specificity for the conserved N-X-S/T, which is a consistent motif for N-glycosylation. The latter still needs further exploration due to its structural diversity, lack of general O-glycosidases, and difficulty in assigning O-glycosylation sites. , The analysis of O-glycosylation has recently been advanced by the newly discovered O-glycoproteases that can cleave the N- or O-termini of mucin-type O-glycoproteins. − These O-glycoproteases tend to digest only certain types of O-glycosylated peptides, but are challenging for O-GalNAcylation analysis. Here, we focus on truncated mucin-type O-glycosylation (O-GalNAcylation), as it is an important modification of proteins whose abnormal changes are associated with many diseases.…”

“… 12 , 13 The analysis of O-glycosylation has recently been advanced by the newly discovered O-glycoproteases that can cleave the N- or O-termini of mucin-type O-glycoproteins. 14 − 17 These O-glycoproteases tend to digest only certain types of O-glycosylated peptides, but are challenging for O-GalNAcylation analysis. Here, we focus on truncated mucin-type O-glycosylation (O-GalNAcylation), as it is an important modification of proteins whose abnormal changes are associated with many diseases.…”