Fig. 5: Identification of Potential O-GlcNAcylated Substrates of HmwC through Quantitative O-GlcNAc Proteomics.

a Principal component analysis of O-GlcNAc proteomics data and statistical plot (b) showing differentially O-GlcNAcylated peptides and proteins between ΔhmwC and WT strains grown under Fv or Mh conditions. c Venn diagram analysis of the O-GlcNAcylated proteins in b. Proteins were illustrated in red for up-regulation, blue for down-regulation, and gray for those with opposite direction in alterations under the two conditions. d Statistical plot for the prediction and classification of subcellular localization of differentially O-GlcNAcylated proteins. e Statistical plot showing the GO term enrichment analysis of the O-GlcNAcylated proteins in b. f LC-MS/MS analyses of the corresponding peptide sequences of the substrate proteins OsdY (left) and AlgL (right). The letter “S” with “he” above in the figure represents the O-GlcNAcylated site. g Glycosylation of OsdY by HmwC. To define the specificity of sugar donor utilization by HmwC, glycosylation assays were carried out in the reaction buffer with or without HmwC in the presence of different UDP activated sugars. After the glycosylation reactions, samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue (up) or subjected to detection using the Pierce™ Glycoprotein Staining Kit (down). Glycosylated OsdY protein was indicated by arrow: ‘a’ is glycosylated OsdY reacted with UDP-GlcNAc and ‘b’ was glycosylated positive control. The experiment was independently repeated at least three times, yielding consistent results. h The interactions enable the stable presence of UDP-GlcNAc in the protein HmwC bind-pocket. Pink represents UDP, green represents GlcNAc, and blue represents active amino acid sites. Ser-435, Leu-492, Asn-517, Ser-277, Agr-280, His-276, and Gly-367 of HmwC form 10 hydrogen bonds (blue solid line) with UDP-GlcNAc. Pro-369 exhibits strong hydrophobic interactions (gray dotted line) with UDP-GlcNAc, while the active group amino in Lys-438 forms a salt bridge (yellow dotted line) with UDP in the molecular ligand.