Fig. 6: R196W decreases PRPS1 O-GlcNAcylation and activity.

a,b, HEK293T cells were transfected by indicated plasmids. Interactions between OGT and WT/mutant FLAG-PRPS1 (L152P, R196W and Q133P) were analyzed by co-immunoprecipitation (IP). PRPS1 O-GlcNAcylation in each group was examined (a) (n = 3). PRPS1 enzymatic activity from a is shown (b) (n = 3). c,d, Myc-OGT was transfected into WT or R196W mutant HEK293T cells, and PRPS1 O-GlcNAcylation level (c) and enzymatic activity (d) were examined (n = 3). e, PRPS1 R196W mutation inheritance and family phenotype and conservation of PRPS1 R196 residue across species. The stillbirth (SB) statuses with the gestational week are indicated in the pedigree chart. f, WT or PRPS1 R196W mutant from HEK293T cells was eluted in fractions on the size-exclusion column in the same settings. Indicated fraction samples were used for immunoblotting (n = 3). g, WT or PRPS1 R196W plasmids were replenished into PRPS1 knockout H1299 cells. EdU incorporation assays were performed as in Fig. 5a. PRPS1 protein levels are shown in the lower-right panel (n = 3). h, The indicated cells were implanted into mice by subcutaneous injection. Xenograft volumes were measured on the indicated days. By the end of the experiment, tumors were harvested and weighed and subjected to immunoblotting after euthanization of mice (n = 5). i, WT or mutated FLAG-PRPS1 was overexpressed and immunoprecipitated from HEK293T cells and subjected to PRPS1 enzymatic activity assays (n = 3). PRPS1 protein level in each group is shown in the bottom panel. j, The indicated H1299 cells were implanted into nude mice by subcutaneous injection. Xenograft volumes were measured on the indicated days. By the end of the experiment, tumors were harvested and weighed and subjected to immunoblotting after euthanization of mice (n = 5). Every error bar signifies mean ± s.e.m. Two-tailed Student’s t-tests were employed for statistical evaluation. wk, weeks.

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