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Spatial and Temporal Diversity of Glycome Expression in Mammalian Brain

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Spatial and Temporal Diversity of Glycome Expression in Mammalian Brain Spatial and temporal diversity of glycome expression in mammalian brain Jua Leea,b, Seungshin Hac, Minsoo Kimc, Seong-Wook Kimc, Jaekyung Yuna,b, Sureyya Ozcand,e, Heeyoun Hwangf, In Jung Jia,b, Dongtan Yina,b, Maree J. Websterg, Cynthia Shannon Weickerth,i,j, Jae-Han Kimk, Jong Shin Yooa,f, Rudolf Grimma,l, Sabine Bahnd, Hee-Sup Shinc,1,2, and Hyun Joo Ana,b,1,2 aGraduate School of Analytical Science & Technology, Chungnam National University, 34134 Daejeon, South Korea; bAsia-Pacific Glycomics Reference Site, 34134 Daejeon, South Korea; cCenter for Cognition and Sociality, Institute for Basic Science, 34051 Daejeon, South Korea; dDepartment of Chemical Engineering and Biotechnology, University of Cambridge, CB2 1QT Cambridge, United Kingdom; eDepartment of Chemistry, Middle East Technical University, 06800 Ankara, Turkey; fResearch Center for Bioconvergence Analysis, Korea Basic Science Institute, 28119 Cheongju, South Korea; gLaboratory of Brain Research, The Stanley Medical Research Institute, Chevy Chase, MD 20815; hSchizophrenia Research Laboratory, Neuroscience Research Australia, Randwick, NSW 2031, Australia; iSchool of Psychiatry, University of New South Wales, Sydney, NSW 2052, Australia; jDepartment of Neuroscience & Physiology, Upstate Medical University, Syracuse, NY 13210; kDepartment of Food and Nutrition, Chungnam National University, 34134 Daejeon, South Korea; and lAgilent Technologies Inc., Santa Clara, CA 95051 Contributed by Hee-Sup Shin, October 5, 2020 (sent for review July 9, 2020; reviewed by Carlito B. Lebrilla, David M. Lubman, Alcino J. Silva, and Jeongmin Song) Mammalian brain glycome remains a relatively poorly understood and properties of the glycome (16). For the same reason, most area compared to other large-scale “omics” studies, such as genomics brain glycosylation studies have focused on the cellular level and and transcriptomics due to the inherent complexity and heterogene- target proteins in specific cell-types, not in subregions of the brain. ity of glycan structure and properties. Here, we first performed spatial Every eukaryotic cell in nature is surrounded by a rich surface and temporal analysis of glycome expression patterns in the mam- coat composed of glycolipids and glycoproteins, termed the glyco- malian brain using a cutting-edge experimental tool based on liquid calyx, which forms the interface between the cell and its outside chromatography-mass spectrometry, with the ultimate aim to yield world and acts as a mediator and transducer of environmental in- valuable implications on molecular events regarding brain functions formation to the cell (9). In particular, glycans play a pivotal role in and development. We observed an apparent diversity in the glycome various functions depending on neural cell interactions such as expression patterns, which is spatially well-preserved among nine neurite outgrowth and fasciculation, synapse formation and matu- BIOCHEMISTRY different brain regions in mouse. Next, we explored whether the ration, and modulation of synaptic transmission and plasticity (12). glycome expression pattern changes temporally during postnatal Recently, it has been reported that glycosylation presents an in- brain development by examining the prefrontal cortex (PFC) at differ- credible level of structural diversity according to different biosystems, ent time point across six postnatal stages in mouse. We found that ranging from species-specificity (17) to organ-specificity (11), that is glycan expression profiles were dynamically regulated during postna- further extended by micro- and macroheterogeneity. In particular, tal developments. A similar result was obtained in PFC samples from anatomical and cell differentiation studies have shown that glyco- humans ranging in age from 39 d to 49 y. Novel glycans unique to the sylation in brain function may be spatially and temporally relevant in brain were also identified. Interestingly, changes primarily attributed to sialylated and fucosylated glycans were extensively observed dur- Significance ing PFC development. Finally, based on the vast heterogeneity of glycans, we constructed a core glyco-synthesis map to delineate the Deciphering molecular mechanisms at the glycome level in glycosylation pathway responsible for the glycan diversity during the mammalian brain remains a missing piece of the puzzle in mo- PFC development. Our findings reveal high levels of diversity in a lecular neuroscience due to the intrinsic complexity of glycosyl- glycosylation program underlying brain region specificity and age de- ation and the lack of analytical tools. Here, we uncovered the pendency, and may lead to new studies exploring the role of glycans variation and diversity of glycome expression in human and in spatiotemporally diverse brain functions. mouse brain samples according to spatial and temporal differ- ences. We further constructed a comprehensive synthesis map mammalian | brain | spatiotemporal | glycosylation | LC-MS using glycans structurally elucidated by LC-MS/MS and found strong evidence on the conservation and developmental diver- he complexity of molecular mechanisms involved in mam- gence of human and mouse prefrontal cortex N-glycome. Our Tmalian brain region specificity and development has begun to data could be the reference for future mammalian brain glycome emerge thanks to recent advances in “omics” technologies. Si- study. Furthermore, it provides valuable information on human multaneous monitoring of vast numbers of molecules in the brain glycome, which has languished in relative obscurity. distinct subregions (1–4) and developmental stages of brain (5–8) have facilitated the mapping of the genome, transcriptome, Author contributions: M.J.W., C.S.W., R.G., S.B., H.-S.S., and H.J.A. designed research; J.L., proteome, and metabolome in animals as well as human. Gly- S.H., M.K., S.-W.K., J.Y., S.O., H.H., I.J.J., D.Y., M.J.W., C.S.W., J.-H.K., J.S.Y., R.G., and S.B. performed research; J.L. and H.J.A. analyzed data; and J.L., H.-S.S., and H.J.A. wrote cosylation, one of the most common posttranslational modifi- the paper. cations of proteins (9), has long attracted considerable attention Reviewers: C.B.L., University of California, Davis; D.M.L., University of Michigan; A.J.S., because of its tremendous level of complexity, and therefore University of California, Los Angeles; and J.S., Cornell University. potentially important roles in the central nervous system (10), Competing interest statement: H.J.A. and Carlito B. Lebrilla are coauthors on a 2019 especially in region-specific regulation (11), development (12), method article. and differentiation (13, 14). Recent studies indicated that pro- Published under the PNAS license. files of the neuronal genome, transcriptome, and proteome were See online for related content such as Commentaries. tightly linked to the glycome (15). However, to our knowledge, 1H.-S.S. and H.J.A. contributed equally to this work. comprehensive glycome studies for investigating the spatiotem- 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. poral diversities of glycan in the brains of human and animals This article contains supporting information online at https://www.pnas.org/lookup/suppl/ have not been reported due to the limited availability of samples doi:10.1073/pnas.2014207117/-/DCSupplemental. and the lack of appropriate analytical tools for probing structures First published November 2, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2014207117 PNAS | November 17, 2020 | vol. 117 | no. 46 | 28743–28753 Downloaded by guest on September 26, 2021 mammalian, but do not provide sufficient information on the di- containing brain-unique moieties, such as sialylated N,N-diacetyl versity and complexity of glycosylation. The challenge arises from lactosediamines (sialylated GalNAc-GlcNAc, sialylated Lacdi- the structural and quantitative profile of glycosylation from brain NAc) LacdiNAc in extracts of human and mouse brain samples, matrices. Glycans are inherently unpredictable because they are respectively. Glycans containing similar structural traits, including synthesized stepwise in a nontemplate-driven manner by numer- sialylated LacdiNAc and Lewis Y(B) epitope, readily showed ous glycosyltransferases that are in competition with each other, region-specificity. In addition, we observed that several glycan unlike nucleic acids or proteins that are synthesized in a template- groups with mainly sialic acid and fucose residues were signifi- driven manner (18). In addition, only a small fraction of the cantly altered during PFC development in mouse and human. protein glycosylation repertoire can be decoded depending on Finally, we constructed a PFC N-glycome synthetic map to pin- genomic DNA sequences since glycosylation is highly influenced point developmental stage-specific pathways and provide an by the physiological status of cells (19). Particularly, determining overall picture of the diversity of glycan expression. Our study brain glycosylation is analytically challenging because the amount demonstrates spatiotemporal divergence of glycome patterns in of carbohydrates in the brain is very small compared with other the brain across the entire cellular milieu that transcend cellular- major components, such as fat. Highly sensitive analytical tools or target protein-level resolution. spanning the range from glycan extraction to glycan detection are required to comprehensively probe heterogeneous glycans bound to Results proteins in the brain. Liquid
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