Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Circadian rhythm impacts the synaptic glycosylation machinery but not the N-glycosylation signatures in mice brains (#457)

The Huong Chau 1 , Edward Moh 1 2 , Lasse Dissing-Olesen 3 , Nima Sayyadi 1 2 , Beth Stevens 3 , Nicolle Packer 1 2 , Morten Thaysen-Andersen 1
  1. Biomolecular Discovery and Design Research Centre, Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. ARC Centre of Nanoscale BioPhotonics, Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  3. Department of Neurology, F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

Circadian rhythm, the “body clock”, plays central roles in diverse facets of physiology. Sleep is crucial for memory consolidation, a notion supported by the fact that structural and functional changes of the neuronal synapses occur during sleep. Thus, we hypothesised that circadian rhythm regulates the synaptic protein N-glycosylation and that alterations of the synaptic N-glycome, in turn, may impact the synapse modulation. As a first step to test this hypothesis, the N-glycome and the underpinning N-glycosylation enzymes of neuronal synaptosomes of mice brains isolated during the light (n = 5, “sleep”) and dark phase (n = 4, “awake”) were investigated using quantitative LC-MS/MS-based glycomics and proteomics. The synaptosomes were density-separated, their purity verified using synaptic protein markers and their membrane protein extracts obtained. In accordance with previous literature, glycomics identified 56 biosynthetically-related, mostly asialylated structures displaying prominent core fucosylation and GlcNAc- or mannose-capped and Lewis-type epitopes. Surprisingly few sleep-wake cycle N-glycome differences were observed within the detailed synaptic N-glycome map. Label-free proteomics confidently identified and quantified 193 glyco-enzymes in the synaptosomes of which 42 glycosyltransferases and glycosidases were found to be involved in N-glycoprotein biosynthesis. Importantly, circadian regulation of known clock proteins (e.g. BMAL1, PER1) was observed, which verified the experimental design and the proteomics data. Interestingly, approximately half of the N-glyco-enzymes including both the catabolic hydrolases and anabolic glycosyltransferases were significantly regulated during sleep, indicating that these are not rate-limiting in the N-glycoprotein biosynthesis. In summary, our data indicate that the neuronal protein N-glycosylation machinery but not the resulting synaptosomal N-glycome fluctuates with circadian rhythm. This study also provides a high-resolution quantitative map of the murine synaptic N-glycome useful for future glycobiological explorations.