Evolutionarily conserved temperature dependency leads to loss of protein O-GlcNAc in mammalian hypothermia

O-N-acetylglucosaminylation (O-GlcNAcylation) is a non-canonical glycosylation of intracellular proteins suggested to be involved in diverse cellular processes, yet its regulation and specific effects on protein function remain elusive. The O-GlcNAc modification of protein serine and threonine residues is cycled by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAc sites lack a strict consensus sequence, suggesting that structural context rather than a defined motif governs the O-GlcNAc site selection. O-GlcNAcylation has been thought to be primarily regulated by the availability of its substrate, UDP-GlcNAc, produced by the hexosamine biosynthetic pathway (HBP). Here, we report a systemic loss of protein O-GlcNAc in Bcs1l mutant mice, a model of mitochondrial complex III deficiency. Although HBP precursor metabolites linked to mitochondrial function were decreased in the Bcs1l mutant liver, UDP-GlcNAc levels in the liver and other tissues were largely unaffected and did not correlate with the O-GlcNAc levels. Consistently, neither boosting UDP-GlcNAc with N-acetylglucosamine supplementation nor overexpression of the HBP rate-limiting enzyme GFPT1 restored O-GlcNAc levels, indicating that the loss was independent of the HBP. Instead, the body temperature of the hypothermic mutant mice showed a strong positive correlation with their O-GlcNAc level. Correcting body temperature via liver-targeted gene therapy, expression of alternative oxidase (AOX), or housing at 35oC prevented the loss of O-GlcNAc. Pharmacological inhibition of OGA restored O-GlcNAc levels in the hypothermic mice but did not affect disease progression, indicating that decreased O-GlcNAc is a physiologically normal consequence of hypothermia. We show that temperature dictates the O-GlcNAc status also across Drosophila species, zebrafish, and cultured mammalian cells. In cultured cells, hypothermia decreased OGT and increased OGA expression transcriptionally, indicating their ratio as a simple regulatory mechanism. Proteome solubility profiling revealed that O-GlcNAc alters the proteome structural and/or interactome landscape. Together, our findings identify temperature as an evolutionarily conserved master regulator of O-GlcNAcylation, which we propose protects the proteome from temperature-dependent fluctuations in both poikilothermic and homeothermic organisms.