The Role Of O-GlcNAcylation Of G6PD In The Metabolism Of Cancer Cells

To satisfy the requirement of rapid cell profiferation, cancer cells need to consume substantial glucose, increase glycolysis and convert glucose to lactate even under sufficient oxygen, which is called "the Warburg effect". The Warburg effect is a pattern of metabolic reprogramming of cancer cells, which occurs in many metabolic pathways, including glucose transport, glycolysis, pentose phosphate pathway (PPP), glutaminolysis, tricarboxylic acid (TCA) cycle, electron transport chain and so on.The PPP plays an important role in macromolecular biosynthesis and maintaining cellular redox balance in rapid proliferating cells. Up-regulation of the PPP has been shown in several types of cancer. However, how the PPP is regulated to confer a selective growth advantage on cancer cells is not well understood.Glucose-6-phosphate dehydrogenase (G6PD) is the first rate-limiting enzyme of PPP, catabolizing glucose-6-phosphate which originates from glycolysis to 6-phosphate glucose acid -δ-lactone and NADPH. In our study, we identified the O-linked N-acetylglucosamine modification (O-G1cNAcylation) of G6PD on S84 by chemoenzymatic method and mass spectrometry (MS). Mutation of S84 to valine (G6PD S84V) abolish O-G1cNAcylation. We demonstrated that glycosylation significantly promotes G6PD activity. Crosslinking of G6PD with glutaraldehyde showed that highly glycosylated forms G6PD readily form an active dimer and tetramer.Previous study showed that over 1000 nucleic/cytosolic proteins are O-GlcNAcylated and this modification acts as a sensor, responding dynamically to extracellular nutrition and stress. After the treatment of hypoxia/high glucose stimuli/serum stimuli, we found G6PD glycosylation is sensitive to these extracellular stimuli. The glycosylation level of G6PD increased significantly with the treatment of hypoxia and increasing concentration of glucose and serum. Meanwhile, high glycosylation of G6PD increased the uptake of glucose, producing more intermediate metabolites, promoting nucleic acid and lipid biosynthesis and reducing equivalents for antioxidant defense, which provides growth advantages for cell proliferation.Xenograft study showed the glycosylation of G6PD benefits tumor growth, and blocking its glycosylation significantly impaired tumor growth. In addition, we obtained a total of 39 pairs of primary human lung cancer tissue samples with matched adjacent normal lung tissues, and determined the level of glycosylation by the chemoenzymatic tagging approach and normalization to G6PD protein levels. Among these samples, eight pairs showed minimal G6PD expression in either cancer or normal tissues that prevented reliable quantification. Among the remaining 31 pairs of samples,16 pairs showed relatively higher level of glycosylated G6PD in cancer tissues than the matched normal tissues and among 31 pairs of samples,20 pairs showed relatively higher level of O-linked β-N-acetylglucosamine transferase (OGT) expression in cancer tissues than in the matched normal tissues. Thus, the increased G6PD glycosylation correlates with the increased OGT expression in lung cancer.Substantial G6PD mutants exist in many diseases, especially in the hemolytic anemia. There is also comprehensive research on G6PD in tumor, but many studies focus on the mutation, few on its posttranslational modification. Our findings reveal a mechanistic understanding of how O-glycosylation directly regulates the PPP to confer a selective growth advantage to tumors and shed new light on the therapy of cancer by blocking the glycosylation of G6PD.