Extended Data Fig. 2: OGT interacts with PRPS1 and OGT O-GlcNAcylates PRPS1 at S83 and T166.
From: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation
a,b, The plasmids encoding full-length (FL) or truncated forms of OGT (M1 and M2) were introduced into HEK293T cells (a). GST, GST-PRPS1 full-length (FL), and truncated mutants (M1 and M2) proteins were purified from E. coli BL21 cells (b). Immunoprecipitation/ pull-down and immunoblot were performed. Schematics illustrating OGT and PRPS1 truncated mutants are presented. OGT TPR ___domain represents OGT tetratricopeptide repeat (TPR) ___domain; PRPS1 PRT ___domain represents PRPS1 phosphoribosyl transferase (PRT)-type I ___domain. (n = 3). c, The indicated plasmids were introduced into H1299 cells. Cells were treated with or with no OSMI-1 (50 µM) for 24 h. Immunoprecipitation and immunoblot analyses were performed with anti-FLAG beads and indicated antibodies. (n = 3). d-h, The indicated siRNAs were introduced into A549 cells (d); A549 cells were cultured with or without glucose for 24h (e); A549 cells were treated with or without OSMI-1 (50 µM) for 24h (f); A549 cells were transfected with or without HA-OGT plasmids (g); transfected with or without HA-OGA plasmids (h). The cells from all groups were transfected with FLAG-vector or FLAG-PRPS1 plasmids. Immunoprecipitation and immunoblot analyses were conducted with anti-FLAG beads and indicated antibodies. (n = 3). i, HEK293T cells were transfected with or without Myc-OGT plasmids. PRPS1 O-GlcNAcylation was analyzed using the chemoenzymatic labeling method. Immunoblotting was conducted with the indicated antibodies. (n = 3). j, Mass spectrometry analyses of PRPS1 O-GlcNAcylation sites. k, Alignment of protein sequences containing PRPS1 S83 (top) and T166 (bottom) sites from different species. l, Modeling of human PRPS1 hexamer structure (Protein Data Bank code: 2H06). The S83 and T166 residues are indicated.
