O-GlcNAcylation Modulates Expression and Abundance of N-Glycosylation Machinery in an Inherited Glycosylation Disorder

Much of the N-glycosylation machinery was identified decades ago, but the regulatory mechanisms influencing normal and disease states are only beginning to be discovered. Recent studies highlight a role for key metabolites and metabolic enzymes in this process, revising the view of the relationship between different metabolic pathways. Using a multi-omic approach in a zebrafish, we discovered a mechanism where O-GlcNAcylation directly influences the expression and abundance of two enzymes essential for the earliest steps of N-linked glycosylation, the nus1-encoded NgBR and Dpagt1. Here we show that phosphomannomutase (pmm2) deficiency is associated with increased levels of UDP-GlcNAc and protein O-GlcNAcylation. Biochemical studies show that O-GlcNAc modification of the NgBR and Dpagt1 increases their abundance in pmm2m/m mutants. Modulating O-GlcNAc levels, NgBR abundance or Dpagt1 activity exacerbated cartilage phenotypes in pmm2 mutants, suggesting that O-GlcNAc mediated increases in the N-glycosylation machinery protects against more severe pathology. These findings highlight nucleotide-sugar donors as metabolic sensors that coordinate two different glycosylation pathways, demonstrating how their interplay is relevant to disease outcome in the most common congenital disorder of glycosylation. Overall design: We did bulk total RNA-seq with ribo-depletion of whole zebrafish larvae at 2 ages- 5dpf and 8dpf for two genotypes- wildtype WT and Mutant where the mutant is a loss of function of Pmm2. Each sample consisted of 15 larvae and we sequenced 5 biological replicates per genotype and age combination.