Mammalian brain glycoproteins exhibit diminished glycan complexity compared to other tissues

Abstract

Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

Fig. 1. The brain N-glycome was distinct in its restricted glycan repertoire.

A Schematic of workflow analyzing the brain glycome in human and mouse samples. B Representative MALDI MS data of permethylated N-glycans from mouse and human cortex showed a predominance of low-molecular-weight structures, both with a major peak of Man-5 at m/z: 1579. In contrast, N-glycans from mouse and human plasma have a primary peak of A2G2S2 (m/z: mouse 2852, human 2792) and more complex and high-molecular-weight structures.

Fig. 2. Protein N-glycomics revealed an abundance of high-mannose and fucosylated/bisected structures across the mouse brain.

A Representative MALDI spectra of protein N-glycans isolated from four brain regions show a similar overall pattern. B The 10 most abundant N-glycan masses differ between brain regions (n = 6 per region, male mice). Data presented as mean percent abundance +/− SEM for the 10 most abundant N-glycan masses averaged across regions. Corresponding glycan structures are presented above each peak, with distinct isomers corresponding to the same mass shown in brackets. For single-factor ANOVA calculations, df = (3,20), F_crit = 3.098, p values *<0.05, **<0.01, and ***<0.001. C) Categorical analysis of N-glycans demonstrates greater abundance of complex structures in the cerebellum, with a heat map showing percent change from the average of four regions. CTX cortex, HIP hippocampus, STR striatum, CBLM cerebellum. Source data are provided as a Source Data file.

Fig. 3. Endo H treatment distinguished high-mannose and hybrid structures from complex and paucimannose N-glycans.

The intensity of the 15 most abundant N-glycan masses from a representative male mouse cortex sample is shown after treatment with different glycosidases, grouped by high-mannose (green), hybrid (blue), mixed (pink), or complex and paucimannose structures (gray), with glycan names shown below (F - fucose; G - galactose; S - sialic acid; A - antenna; B - bisected; and H - hybrid). The y-axis (arbitrary units; au) represents the signal intensity (arbitrary units; au) for each mass measured directly from the MALDI without normalization and scaled such that Man-5 (A and B) and FA1B (A and C) were equal for visual comparison. Distinct isomers corresponding to the same mass are shown in brackets. A Treatment with PNGase F removed all N-glycans. B Treatment with Endo H removed only high-mannose and hybrid N-glycans. A placeholder (#) is included because the Endo H fragment corresponding to F2A1G1BH4 is indistinguishable from that of the same glycan without a core fucose (F1A1G1BH4), due to Endo H cleavage between the two core GlcNAc residues and proximal to the core fucose. This results in only one structure present in the +Endo H spectra corresponding to two unique parent glycans. C) PNGase F treatment after Endo H removed complex and paucimannose N-glycans, which were insensitive to Endo H treatment.

Fig. 4. Protein O-glycomics revealed a higher proportion of O-GalNAc glycans compared to O-Man in mouse brain.

A Representative MALDI spectra of protein O-glycans isolated from four regions show a consistent pattern across the brain. B The 10 most abundant O-glycan masses in the brain include both O-GalNAc and O-mannose (O-Man) type structures (CTX = 3, HIP = 2, STR = 4, CBLM = 4, male mice). Data presented as mean percent abundance +/− SEM. Corresponding glycan structures are presented above each peak, with distinct isomers corresponding to the same mass shown in brackets. For single-factor ANOVA calculations, df = (3,9), F_crit = 3.862. p values *<0.05, **<0.01, and ***<0.001. C Categorical analysis of O-glycans revealed differences in the abundance of O-GalNAc, O-Man, and NeuGc-containing glycans between regions. Analyzed independently, O-GalNAc and O-Man glycans varied in their proportion of core 1, core 2, and sialylated structures. Data presented as a heat map showing percent change from the average of four brain regions. Source data are provided as a Source Data file.

Fig. 5. Protein glycosylation showed minimal sex differences in the brain compared to plasma in mice.

Comparison of the top 10 most abundant protein N-glycans in plasma (A), cortex (B), and cerebellum (C) reveals differences between sexes, with much greater divergence between male and female mice in the plasma than in either brain region. D Comparison of the 10 most abundant protein O-glycans in the cortex revealed minor variation between genders, though limited sample size prevented statistical analysis. For plasma samples, male = 8, female = 6. For brain N-glycans, male = 6, female = 4. For brain O-glycans, male = 3, female = 2. Corresponding glycan structures are shown above each assigned peak, with distinct isomers corresponding to the same mass shown in brackets. Data presented as mean +/− SEM for percent abundance of each peak, with unpaired two-tailed t-tests assuming unequal variance performed for sex comparisons of individual glycans. p values *<0.05, **<0.01, and ***<0.001. Source data are provided as a Source Data file.

Fig. 6. Lectin blotting supports a predominance of high-mannose, fucose, and bisected N-glycans in the brain.

Protein lysate from a representative male mouse cortex and cerebellum with human plasma as a positive control was treated with or without PNGase F and visualized using biotinylated lectins (ConA, GNL, PHA-E, AAL, RCA, and SNA) in addition to immunoblotting for actin and staining for total protein. Non-specific binding of lectins to PNGase F is noted by an asterisk (*) near 35 kDa, as shown in the Total Protein stain. Protein blotting of brain lysate with each lectin has been repeated at least three times each with similar results. A schematic with common lectin-binding sites is shown for reference. Source data are provided as a Source Data file.

Fig. 7. Differential RNA expression between brain regions in mice spans multiple glycosylation pathways and correlates with glycomics results.

RNA seq data from cortex and cerebellum (n = 4 each, male mice) revealed differential expression of enzymes involved in several glycosylation pathways, including the synthesis of N-glycans (A), O-GalNAc glycans (B), and O-Man glycans (C). Transcripts in red have significantly increased expression in the cortex relative to the cerebellum, and those in the blue are decreased, as determined using the EdgeR method with gene cutoffs of 2-fold change in expression value and false discovery rates (FDR) below 0.05. *Tmtc1-4 add O-linked Man but these residues are not extended further. Mouse brain RNA seq results for N-acetylglucosaminyltransferases (D), fucosyltransferases (E), and O-Man specific enzymes (F) demonstrated high RNA levels of Mgat3, Fut8, Fut9, and Mgat5b, which correlate with results from glycomics. Data for TPM presented as mean +/− SEM. For D-F, n = 4 independent samples for each brain region from different mice. Human RNA seq data from GTEx portal showed a similar expression profile for N-acetylglucosaminyltransferases (G), fucosyltransferases (H), and O-Man pathway enzymes (I) in the brain between humans and mice, but this pattern is distinct from human liver and lung. Source data are provided as a Source Data file.

Fig. 8. FUMA GENE2FUNC analysis of 354 glycosylation enzymes and related genes in humans revealed a specific downregulation in the brain.

A Heat map demonstrating expression pattern of all glycosylation genes in humans, with a black bar above the 13 columns representing brain regions. B Tissue specific analysis showing down-regulation in the brain compared to all other 29 tissue types, with significant differentially expressed gene (DEG) sets using a two-sided t-test (P_bon < 0.05 and absolute fold change ≥ 0.58) highlighted in red.