Production of Glycopeptide Derivatives for Exploring Substrate Specificity of Human OGA Toward Sugar Moiety

Abstract

O-GlcNAcase (OGA) is the only enzyme responsible for removing N-acetyl glucosamine (GlcNAc) attached to serine and threonine residues on proteins. This enzyme plays a key role in O-GlcNAc metabolism. However, the structural features of the sugar moiety recognized by human OGA (hOGA) remain unclear. In this study, a set of glycopeptides with modifications on the GlcNAc residue, were prepared in a recombinant full-length human OGT-catalyzed reaction, using chemoenzymatically synthesized UDP-GlcNAc derivatives. The resulting glycopeptides were used to evaluate the substrate specificity of hOGA toward the sugar moiety. This study will provide insights into the exploration of probes for O-GlcNAc modification, as well as a better understanding of the roles of O-GlcNAc in cellular physiology.

The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

“…Therefore, we first detected if the UDP-6AzGal donor is a substrate of OGT. A one-pot reaction containing UDP-6AzGal, the YAVVPVSK peptide, and OGT was performed as the method we previously reported ( Li et al, 2018 ). Unfortunately, no 6AzGal modified peptide was detected compared to the UDP-GlcNAc group, which is a natural substrate for OGT and almost complete transformation to the GlcNAc-peptide ( Supplementary Figure S3 ).…”

“…Finally, biotin or the fluorescent tag is labeled on the glycan-modified protein through bio-orthogonal click chemical reaction to realize visualization and enrichment of glycoproteins. Currently, an increasing number of peracetylated O-GlcNAc analogues for O-GlcNAc metabolic markers have been developed and characterized, such as modification at the 2-position of HexNAc ( Vocadlo et al, 2003 ; Boyce et al, 2011 ; Tan et al, 2018 ) or at the 4-/6-sites ( Chuh et al, 2014 ; Li et al, 2016 ; Chuh et al, 2017 ; Darabedian et al, 2018 ; Guo et al, 2019 ), and all the reported functionalized GlcNAc analogues demonstrated satisfactory selectivity and efficiency, which exhibited a wide range of substrate tolerance of OGT ( Li et al, 2018 ). Meanwhile, any nonanalogues of GlcNAc, 6-azido-6-deoxy-glucose (6AzGlc) ( Darabedian et al, 2018 ), and 2-azido-2-deoxy-glucose (2AzGlc) ( Zaro et al, 2017 ) are also reported as the substrates of OGT to modify O-GlcNAc proteins.…”

Machine‐Driven Chemoenzymatic Synthesis of Glycopeptide

“…Protein glycosylation mediates a diversity of physiological and pathological processes; this includes but is not limited to immune responses, angiogenesis and tumor cell metastasis, protein folding and degradation, cell‐cell communications, and cell‐pathogen interactions. Structurally defined glycopeptides have significant potential for application in enzymatic activity tests, [4] clinical diagnostics, [5] and in the development of glycopeptide antibiotics and carbohydrate‐based vaccines [6] . There are two main approaches for glycopeptide preparation.…”

The Glucose Metabolic Pathway as A Potential Target for Therapeutics: Crucial Role of Glycosylation in Alzheimer’s Disease

“…For a detailed description of OGT and OGA substrate recognition see Joiner et al, 2019 [ 27 ]. OGT catalyzes the transfer of GlcNAc onto Ser and Thr residues employing Uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) as a substrate, whereas OGA is the enzyme that removes O-GlcNAc from proteins [ 28 , 29 ]. OGT is mainly expressed in three predominant isoforms namely short OGT (sOGT), mitochondrial OGT (mOGT) and nucleocytoplasmic OGT (ncOGT).…”