Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis - PubMed (original) (raw)
Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis
Mariana Barboza et al. Mol Cell Proteomics. 2021.
Abstract
N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease.
Keywords: N-glycans; N-glycome; N-glycosylation; glycocalyx; glycomics; mouse brain.
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Figures
Graphical abstract
Fig. 1
Scheme of the cell membrane N-glycomic workflow used for the characterization of region-specific cell surface mouse brain N-glycomes. After extraction, brains were surgically dissected either into forebrain and hindbrain (A) or the functional substructures derived from both regions, including the cortex and hippocampus (from forebrain) and cerebellum (from hindbrain) (B). Tissues dissected were homogenized, cell debris, nucleus, and mitochondria were removed by centrifugation from region-specific brain lysates. Cell membrane fractions enriched in cell surface glycoproteins were obtained by ultracentrifugation as previously described (38). Cell membrane glycans were enzymatically released, purified, and analyzed by nano-LC-Chip-Q-TOF mass spectrometry.
Fig. 2
Comparison of cell membrane N-glycome obtained from the forebrain and hindbrain. Heatmap of N-glycans differentially expressed in the Forebrain (FB) and Hindbrain (HB), ordered from high mannose to complex glycans approximately according to N-glycan biosynthesis (p < 0.05%, as analyzed by two-way ANOVA and the TUKEY post-hoc test) (A). Glycan mass of hybrid and complex glycans increases from top to bottom. Glycan composition is abbreviated as HexHexNAcFucNeuAc where Hex = Hexose, HexNAc = N-Acetylhexosamine, Fuc = Fucose, NeuAc = N-Acetylneuraminic Acid; and numbers indicate the number of each monosaccharide residue. The abundance for each glycan composition was normalized to the abundance of total glycans identified. Data from biological replicates (n = 3) were used (FBM1–3 and HBM1–M3). Partial least squares–discriminant analysis (PLS-DA) score plot of cell surface N-glycan profiles from FB and HB (B). Each symbol represents a mouse (n = 3). Loadings plots from the PLS-DA model showing the N-glycan species (compositions) contributing to the separation in the first dimension (x-axis of the scores plot) for the FB and HB N-glycomes (C).
Fig. 3
Fucosylation and sialylation in the forebrain and hindbrain N-glycome. Comparative distribution of mono (F1), di (F2), tri (F3), tetra (F4), and penta (F5) fucosylated glycans in all fucosylated glycans (A); neutral fucosylated only glycans (B); and sialofucosylated glycans (C). Comparative distribution of mono (S1), di (S2), tri (S3), and tetra-sialylated (S4) species in: all sialylated glycans (D); sialylated only glycans (E), and sialofucosylated species (F). Combined distribution of fucose and sialic acid residues (FxSy) sorted by the number of fucose residues (x) in (G) or sialic acid residues (y) in (H). Asterisks indicate the statistical significance between groups compared (∗p < 0.05%; ∗∗p < 0.01%; ∗∗∗p < 0.001%).
Fig. 4
Region-specific expression of bisecting N-glycans in forebrain and hindbrain. Comparison of most abundant and differentially expressed glycans containing bisecting GlcNAc in forebrain and hindbrain N-glycomes (A). Glycan composition is abbreviated as HexHexNAcFucNeuAc where Hex = Hexose, HexNAc = N-Acetylhexosamine, Fuc = Fucose, NeuAc = N-Acetylneuraminic Acid; and numbers indicate the number of each monosaccharide residue. The abundance for each glycan composition was normalized to the abundance of total glycans identified. Glycan structures are presented with symbols recommended by the CFG (
http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml
). Asterisks indicate the statistical significance between groups compared (∗p < 0.05%; ∗∗p < 0.01). Tandem MS/MS spectra of select N-glycans containing bisecting GlcNAc in the Forebrain and Hindbrain (B). The fragment ion with m/z 790.3 (C fragment of Hex1HexNAc3 + H20) was used to identify bisecting GlcNAc. Putative structures are shown with the corresponding accurate masses.
Fig. 5
Comparison of cell membrane N-glycome obtained from the cortex, hippocampus, and cerebellum. Relative abundances of N-glycan groups in the cerebral cortex, hippocampus, and cerebellum (A), and total levels of fucosylation and sialylation (B). Asterisks indicate the statistical significance between groups compared (∗p < 0.05%; ∗∗p < 0.01%; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). Heatmap of N-glycans differentially expressed in the cortex, hippocampus and cerebellum, ordered from high mannose to complex glycans approximately according to N-glycan biosynthesis (p < 0.05%, as analyzed by two-way ANOVA and the TUKEY post-hoc test) (C). Glycan mass of hybrid and complex glycans increases from top to bottom. Glycan composition is abbreviated as Hex_HexNAc_Fuc_NeuAc where Hex = Hexose, HexNAc = N-Acetylhexosamine, Fuc = Fucose, NeuAc = N-Acetylneuraminic Acid; and numbers indicate the number of each monosaccharide residue. Abundance for each glycan composition was normalized to the abundance of total glycans identified. Data from six biological replicates were used. Partial least squares–discriminant analysis (PLS-DA) scores plots for cell membrane N-glycan profiles of the cortex, hippocampus, and cerebellum (D), and cortex and hippocampus (F). The 95% confidence interval is indicated as an ellipse, and each symbol represents a mouse (n = 6). The loadings plots (E and G) from the PLS-DA model show glycans contributing to the separation in the first dimension (x-axis of the scores plot) for cortex, hippocampus, and cerebellum (E), and cortex and hippocampus (G). C, complex; H, hybrid.
Fig. 6
Region-specific distribution of most abundant glycans in the cortex, hippocampus, and cerebellum N-Glycomes. Comparative distribution of high mannose glycans (A), neutral fucosylated glycans (B), and sialofucosylated glycans (C) in the cerebral cortex, hippocampus, and cerebellum N-Glycomes. Glycan composition is abbreviated as follow HexHexNAcFucNeuAc where Hex = Hexose, HexNAc = N-Acetylhexosamine, Fuc = Fucose, NeuAc = N-Acetylneuraminic Acid; and numbers indicate the number of each monosaccharide residue. Values are reported as the average relative abundance of six biological replicates (n = 6). Asterisks indicate the statistical significance between groups compared (∗p < 0.05%; ∗∗p < 0.01%; ∗∗∗p < 0.001).
References
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