Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Probing synaptic N-glycome fluctuations in circadian rhythm (#117)

Edward S.X. Moh 1 2 , Lasse Dissing-Olsen 3 , Nicolle H Packer 1 2 , Beth Stevens 3 , Morten Thaysen-Andersen 1
  1. Biomolecular Discovery and Design Research Centre, Sydney, NSW, Australia
  2. ARC Centre of Nanoscale BioPhotonics, 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 and is the driver of the sleep/wake cycle. Sleep has been shown to be crucial for memory consolidation, which is supported by the fact that structural and functional changes of neuronal synapses occur during sleep.  Thus, we hypothesized that circadian rhythm regulates the synaptic N-glycan signatures and that alterations in the synaptic N-glycome play functional roles in the synapse modulation that take place during sleep.

With this hypothesis, the N-glycome and the underpinning N-glycosylation enzymes of neuronal synaptosomes comprising of both pre- and post-synaptic nerve endings of mice brains isolated during the light phase (10 AM, n=5) and during the dark phase (9 PM, n=4) were investigated using deep quantitative glycomics and proteomics. Synaptosomes were isolated by density centrifugation. Synaptosomal enrichment was verified using specific synaptic protein markers. Released -glycans and tryptic peptides from synaptosome membrane protein fractions were analysed by LC-MS/MS. The N-glycomics provided a detailed synaptosome N-glycan map encompassing 56 biosynthetically-related, mostly asialylated structures displaying prominent core fucosylation and GlcNAc-, mannose-capped and Lewis-type epitopes. Surprisingly few qualitative and quantitative AM-to-PM N-glycome differences were observed. Label-free proteomics confidently identified and quantified 193 glyco-enzymes in the synaptosomes of which 42 glycosyltransferases and glycosidases are involved in N-glycoprotein biosynthesis. Importantly, circadian regulation of known clock proteins (e.g. BMAL1 and PER1) was observed, which verified the experimental design and the proteomics data. Half (22) of the N-glyco-enzymes were significantly regulated during sleep, indicating that these are not rate-limiting in the biosynthetic machinery for N-glycoproteins in order to maintain a relatively constant N-glycosylation over the sleep-wake cycle. Conclusively, the synaptosomal N-glycome does not fluctuate significantly with circadian rhythm, but this study provides a detailed map of synaptic glycan structures for further glycobiological interrogation.