Fig. 6: HmwC functions as an O-GlcNAc transferase on OsdY and AlgL.
From: A protein O-GlcNAc glycosyltransferase regulates the antioxidative response in Yersinia pestis
a, b HmwC O-GlcNAcylates OsdY at Ser-83 and AlgL at Ser-5. Purified recombinant GST-HmwC was incubated with His-OsdY, His-OsdYSer83A, His-AlgL or His-AlgLSer5A in the mixture containing UDP-GlcNAc, OGT assay buffer, and ddH2O at 37 °C for 2 h, then 2xSDS loading buffer was added to stop the reaction. All the samples were separated in 12% SDS-PAGE and immunoblotted with mouse monoclonal anti-GST, anti-His, or anti-O-GlcNAc antibodies. c, d The O-GlcNAcylation of OsdY and AlgL showed dose-dependent on concentration of HmwC. e D217 and H276 of HmwC were predicted to be the key residues for substrate binding and enzyme activity, respectively. Protein structure prediction was accomplished using Alphafold2 software (Supplementary Data 6). f GST pull-down assay of binding affinity between OsdY and HmwC and its mutants. Purified recombinant His-tagged OsdY was incubated at 4 °C overnight with glutathione-sepharose 4B beads conjugated with GST-HmwC, GST-HmwC-D217A and GST-HmwC-H276A, respectively. Then the beads were intensively washed and analyzed by SDS-PAGE and immunoblotting. g GST pull-down assay of binding affinity between AlgL and HmwC and its mutants, using the method the same as described in f. h, i Analysis of the O-GlcNAcylation of OsdY and AlgL by HmwC, HmwC-D217 or HmwC-H276A was carried out according to the method described in a. All above experiments were performed at least in triplicate with similar results, and a representative result was shown here.
