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. 2024 Oct 14;15(21):4033–4045. doi: 10.1021/acschemneuro.4c00399

Unique Glycans in Synaptic Glycoproteins in Mouse Brain

Maxence Noel †, Suttipong Suttapitugsakul †, Mohui Wei †, Catherine Tilton †, Akul Y Mehta †, Yasuyuki Matsumoto †, Jamie Heimburg-Molinaro †, Robert G Mealer †,‡, Richard D Cummings †,*
PMCID: PMC11587512  PMID: 39401784

Abstract

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The synapse is an essential connection between neuronal cells in which the membrane and secreted glycoproteins regulate neurotransmission. The post-translational modifications of glycoproteins with carbohydrates, although essential for their functions as well as their specific localization, are not well understood. Oddly, whereas galactose addition to glycoproteins is required for neuronal functions, galactosylation is severely restricted for Asn-linked on N-glycans in the brain, and genetic evidence highlights the important roles of galactose in brain functions and development. To explore this novel glycosylation, we exploited an orthogonal technology in which a biotinylated sialic acid derivative (CMP-biotin-Sia) is transferred to terminally galactosylated proteins by a recombinant sialyltransferase (rST6Gal1). This approach allowed us to identify the carrier proteins as well as their localization on brain sections. Immunohistochemical analysis of the biotinylated glycoproteins in brain sections demonstrates that they are largely positioned in the pre- and postsynaptic membranes. Consistent with this positioning, glycoproteomic analyses of the labeled glycoproteins identified a number of them that are involved in synaptic function, cell adhesion, and extracellular matrix interactions. The discovery of these galactosylated N-glycoproteins and their relative confinement to synapses provide novel insights into the unusual and specific nature of protein glycosylation in the brain.

Keywords: brain, galactosylated glycoproteins, glycoproteomics, biotinylated CMP-sialic acid, ST6Gal1, click-chemistry

Introduction

Synapses in the brain display unique types of membrane and secreted glycoproteins whose glycan modifications are essential for their normal functions in neurotransmission.1−3 Aberrant alterations in glycosylation of Asn residues (N-glycosylation) and of Ser/Thr residues (O-glycosylation) in brain glycoproteins are associated with many developmental and cognitive disorders, including schizophrenia.4−6 Interestingly, the mammalian brain glycan repertoire is unique in many respects. Overall, brain N-glycans include a relatively high level of oligomannose-types and low level of complex types containing sialic acid and galactose; in non-neuronal cells and tissues, the complex type N-glycans are much more abundant.7−14 Nevertheless, galactose plays a central role in brain function as highlighted by dysfunctions in its synthesis, transport, or transfer onto glycoproteins that are associated with several neurological disorders.15−17 For instance, loss-of-function mutations in the β-1,4-galactosyltransferase 1 (B4GALT1), which prevents the generation of galactose-based sequences (Galβ1-4GlcNAc), causes a congenital disorder of glycosylation (B4GALT1-CDG) associated with severe neurologic dysfunctions.16 In addition, deficiency in manganese, an essential cofactor of galactosyltransferases, also leads to neurodisorders.18,19 Galactosylated glycoproteins are involved in many neurological processes ranging from synaptic pruning and activity to recognition by glycan-binding proteins and complement factors;20−23 thus, modifications in galactose metabolism are most likely to impact these pathways. However, the galactose-carrying proteins are unknown, and it is critical to define these glycoproteins to understand their functional pathways in the brain.

Although the identification of glycans and glycoproteins present in the brain is an active area of research, spatial brain expression patterns of specific glycans remain poorly understood. Recent approaches using immunohistochemistry, mass spectrometry (MS), and MS imaging have begun to reveal some of the unique features of brain glycans and their expression,7,11−13,24−30 although many structures, in particular those of low abundance, remain relatively underexplored. Among the various approaches to identify specific glycoproteins are bioorthogonal labeling and enzymatic tagging. The former has made a tremendous impact on the understanding of the roles of glycoconjugates, such as proteins and lipids, in human health and disease.16,31−34 This approach, however, requires the labeling of living cells with a chemically modified metabolic precursor. For example, to label N-glycoproteins carrying sialylated galactose, precursors of sialic acid are taken up by cells, incorporated into different glycoconjugates, and labeled with a tag through click chemistry.35 By contrast, the enzymatic tagging approach can be done on any sample, living or fixed, and can also be directed to specific glycan classes.36−41

We have exploited the enzymatic tagging approach to identify galactosylated N-glycoproteins in murine brain using a biotinylated CMP-sialic acid derivative that can be efficiently and specifically transferred to galactosylated N-glycans by a recombinant α2,6 sialyltransferase 1 (rST6Gal1). This tagging approach leverages the specificity of ST6Gal1, a sialyltransferase that preferentially transfers sialic acids to β-1,4-linked galactose residues of N-glycans, to allow the addition of a biotinylated sialic acid derivative to the glycoproteins at the cell surface, which can be further used for glycoproteomic studies and direct visualization within tissue.39,42−44

Here we use for the first time this approach to identify both the specific distributions of galactosylated N-glycoproteins in the brain and many of their protein carriers. We have observed that galactosylated N-glycoproteins are associated with multiple biological pathways, including synaptic function and cell–cell adhesion. These findings expand our knowledge of the relationship between brain function and galactosylation and will promote future studies of this critical modification.

Results

Synthesis of Biotinylated CMP-Sialic Acid and Enzymatic Characterization of Recombinant ST6GAL1

In our studies, we exploited the unique activity of the enzyme ST6Gal1, which specifically modifies terminal galactose in N-glycans and exhibits preference for α3-branched mannose antennae.45−47 For this, we prepared a recombinant form of the human ST6Gal1 (rST6Gal1) and chemically synthesized a CMP-sialic acid derivative covalently linked to biotin (CMP-biotin-Sia). The addition of such a biotin tag easily enables direct visualization of the spatial distribution of galactosylated glycoproteins in tissues and permits the purification, isolation, and identification of the labeled N-glycoproteins (Figure 1).48

Figure 1.

Figure 1

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Reaction schematic to label rare galactosylated glycoproteins using recombinant ST6Gal1 and a biotinylated sialic acid. General reaction to label galactosylated glycoproteins with a biotinylated sialic acid derivative and recombinant ST6Gal1, which specifically transfers the biotinylated sialic acid from the donor substrate to the acceptor glycans in a tissue sample of interest. Labeled glycoproteins can then be easily analyzed using multiple methodologies based on biotin/streptavidin interactions including protein blotting, fluorescence imaging/localization, and affinity enrichment and glycoproteomics.

To validate this approach, we first synthesized and characterized CMP-biotin-Sia and its utilization by rST6Gal1. To synthesize CMP-biotin-Sia, we slightly modified the basic strategy of Sun et al. in which CMP-sialic acid synthetase converts a sialic acid monosaccharide carrying an azido group on C-9 (SiaNAz) to CMP-SiaNAz using CTP.40 We then proceeded directly with a Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click-chemistry reaction to link an alkyne-modified biotin to CMP-SiaNAz in the presence of CuSO4, ascorbate sodium, and BTTAA to generate CMP-biotin-Sia (Figure 2A). After purification by size-exclusion chromatography, the structure of the final CMP-biotin-Sia compound was confirmed by 1H and 13C NMR and quantified using a standard curve of CMP (Figures S1 and S2A–D).40

Figure 2.

Figure 2

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Biotinylated sialic acid can be effectively utilized as substrate by rST6Gal1. (A) A modified sialic acid carrying an azido group on the ninth carbon (SiaNAz) is converted to CMP-SiaNAz by the CMP-sialic acid synthetase (CSS). A biotin group is then chemically inserted on the CMP-SiaNAz by a copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction, generating biotinylated CMP-sialic acid (CMP-Biotin-Sia). (B) Enzymatic properties of recombinant ST6Gal1 toward its natural donor substrate (CMP-Neu5Ac) and biotinylated CMP-SiaNAz. One hundred micrograms of asialofetuin; 0.5 μg of rST6Gal; and 0, 20, 100, 200, 500, and 1000 μM of donor substrate were used for the enzymatic reaction. rST6Gal1 activity was measured indirectly through the liberation of phosphate using the malachite phosphate detection kit (R&D systems) according to the manufacturer’s protocol.

To determine the ability of rST6Gal1 to utilize CMP-biotin-Sia as a substrate, we compared the activity of rST6Gal1 using CMP-biotin-Sia with CMP-Neu5Ac (Figure 2B). We observed that rST6Gal1 has a higher Km (332 vs 63 μM) and higher Vmax (435 vs 70 pmol/min) using CMP-biotin-Sia as the donor compared to CMP-Neu5Ac; however, the similar ratio of Vmax/Km (∼1.2) for each donor indicates a similar catalytic efficiency (Figure 2B).

rST6Gal1 Transfers Biotin-Sia to N-Glycans

To assess the activity of rST6Gal1 toward galactosylated N-glycoprotein acceptor substrates, we prepared total brain lysate or brain lysate, which can be pretreated with enzymes to effect specific glycan removal or modification as an aid to identifying the endogenous glycan acceptors for rST6Gal1. The concentrations of rST6Gal1 and CMP-biotin-Sia were optimized across a dose range to reach a maximum intensity on Western blots using brain lysates as acceptors and streptavidin-800 for detection (Figure S3). The deglycosylating enzymes used were PNGase F, which removes all N-glycans;49 Endo H, which removes oligomannose/hybrid N-glycans;50 β-galactosidase, which removes terminal β-linked galactose;51 and neuraminidase (NeuA), which removes all terminal sialic acids52 (Figure 3A). Although we did not assess the total efficiency of the glycosidase digestion, we observed that rST6Gal1 transferred biotin-Sia from CMP-biotin-Sia specifically to galactosylated N-glycans based on the sensitivity of the products to treatment with PNGase F and enhancement of staining following sialidase treatment, which exposed additional β-linked galactose and decreased staining after β-galactosidase treatment. Interestingly, Endo H treatment had little effect on rST6Gal1 products, indicating that the labeled N-glycans are primarily of the complex type rather than the hybrid type that is sensitive to Endo H. We also performed these glycosidase treatments after rST6Gal1 sialylation, and only PNGase F treatment was able to reduce the signal (Figure S4). Interestingly, the NeuA enzyme is unable to cleave the biotinylated sialic acid once it is added to glycoproteins (Figure S4). These results confirm that rST6Gal1 modifies galactosylated and complex N-glycans on a number of glycoproteins in brain lysates.

Figure 3.

Figure 3

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rST6Gal1 preferentially sialylates complex galactosylated brain N-glycans containing LacNAc. (A) Brain lysate was pretreated with neuraminidase A, β-galactosidase, Endo H, or PNGase F and incubated with rST6Gal1 and the biotinylated CMP-biotin-Sia where indicated. Tagged proteins were revealed using fluorescent streptavidin-800, with total protein stain shown for the loading control. The protein band of rST6Gal1 that self-biotinylates is indicated in the lane of “no lysate”. (B, C) The top 10 glycans sialylated by 90 ng/μL of rST6Gal1 and 600 μM of biotinylated sialic acid from the CFG glycan array with (B) or without (C) pretreatment with NeuA. Data presented as the normalized mean ± 1 SD, with the highest value set to 100%. Glycan ID and raw data can be found in the Supporting Information.

To explore the types of glycans sialylated by rST6Gal1 and its specificity, we performed the same types of sialylation reactions toward glycans on a glycan microarray using rST6Gal1 and CMP-biotin-Sia (Figure 3B, Figure S5A–C). As expected, most glycans that contain N-acetyllactosamine (LacNAc) units Galβ1-4GlcNAcβ-R on the α1-3Mannose branch were modified by rST6Gal1-transferred biotinylated sialic acid.53 In addition, we observed that pretreatment of the microarray with NeuA to remove sialic acids enhanced the labeling of additional glycans (Figure 3C, Figure S5B,C). The microarray results also indicate that rST6Gal1 has some preference for poly-N-acetyllactosamine chains. Notably, although rST6Gal1 exhibited some activity toward β1-4-linked galactose in core-2 and -4 O-GalNAc glycans, it did not modify glycans having substitutions to the LacNAc structures, such as those with the Lewis x structure Galβ1-4(Fucα1-3)GlcNAcβ-R (Figure 3B,C, Figure S5A–C). Taken together, these results indicate that rST6Gal1 can successfully transfer the biotinylated sialic acid to the terminal galactose.

Galactosylated N-Glycoproteins Localize to the Synapse

Using these validated tools, we sought to spatially localize the acceptor glycoproteins in brain tissue that could be modifiable by rST6Gal1. For this, we used paraformaldehyde-fixed and lipid-cleared brain sections and enzymatically modified them using CMP-biotin-Sia and rST6Gal1 followed by standard fluorescent imaging techniques of biotinylated products.39 Pretreatment of sections with PNGase F prior to incubation with CMP-biotin-Sia and rST6Gal1 dramatically reduced the labeling; this result demonstrates that the enzyme primarily tags N-glycans in the brain (Figure 4A).

Figure 4.

Figure 4

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rST6Gal1-modified glycoproteins enrich and colocalize at the synapse. (A) The coronal section of the mouse cerebellum and medulla was incubated with rST6Gal1 and the biotinylated CMP-sialic acid with or without pretreatment of PNGase F and assayed using streptavidin phycoerythrin-conjugated (Strep-PE). The synapse-rich molecular layer (ML) was highly labeled by rST6Gal1 as well as the fourth ventricle (4th V), with an additional signal detected in the arbor vitae (AV) and much less labeling in the cell-dense granular layer (GL). Scale bar = 1 mm. (B) High magnification of Purkinje cells costained with VGLUT1 or PSD-95, markers of pre- and postsynaptic structures, respectively. Punctae in the Purkinje cells were isolated by Imaris to highlight the colocalization between PSD-95 or VGLUT1 (green) with rST6Gal1 (red) and Hoechst 33342 (blue). Scale bar: 5 μm.

In the cerebellum, the brightest staining was observed in the molecular layer, which contains a high density of synapses. We also observed labeling of the fourth ventricle, but less staining was observed for the cell-dense granular layer (Figure 4A). These results correlate with our recent study in which we used the lectin RCA-I, which is a terminal galactose-specific lectin,54 to localize expression of galactosylated glycoproteins.26 This staining was higher than that with staining with CMP-Biotin-Sia or streptavidin (Strep-PE alone) (Figure S6A). Similar patterns of labeling specific layers were observed within other brain regions, including the cortex and hippocampus (Figure S6B).

Because we observed intense labeling in regions rich in Purkinje cells of the cerebellum, we further assessed the presence of labeled glycans in the synapses. Individual synapses can be directly observed on the soma of these large cell bodies and colabeled with pre- and postsynaptic markers such as VGLUT155,56 and PSD-95,57 respectively. Many punctae were labeled by rST6Gal1 and CMP-biotin-Sia and colocalized with those for VGLUT1 and PSD-95. These results indicate a spatial enrichment of galactosylated glycoproteins at specialized synaptic structures (Figure 4B).

Glycoproteomic Identification of Galactosylated Glycoproteins in Murine Brain

To identify the acceptor glycoproteins tagged by rST6Gal1 and CMP-biotin-Sia, we prepared total brain lysates from six wild-type mouse brains and labeled the glycoproteins in the lysate by enzymatic tagging. Our reasoning was to explore total brain lysates rather than synaptic preparations or some other restricted population. The total tagged glycoproteins were enriched by absorption to streptavidin beads followed by elution and analyzed using ultrahigh-performance liquid chromatography mass spectrometry (UHPLC-MS). As a control, these results were compared to those in which the treatment omitted the CMP-Biotin-Sia.

These analyses revealed more than 1000 individual glycopeptides that mapped to 49 different glycoproteins (Figure 5A). As a secondary analysis, we compared this list of rST6Gal1-modified N-glycoproteins with those identified in other glycoproteomics analysis, although each utilized different techniques with varying depths of coverage (Figure S7A).58,59 Large data sets from our group and others showed that over 75% of the N-glycoproteins modified by rST6Gal1 were detected in the total N-glycoproteome pool, whereas there is a much smaller overlap (∼13%) with our recent study investigating the brain O-GalNAc glycoproteome (Figure S7A, Supporting Information).59

Figure 5.

Figure 5

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Gene Ontology analysis of rST6Gal1-modified glycoproteins shows an enrichment for receptor activities, cell–cell adhesion, and synapses. (A) Glycoproteins were labeled by rST6Gal1 and biotin-Sia, enriched with streptavidin beads, and identified by mass spectrometry. Label-free-quantification of glycopeptides was performed and compared with a negative control (without Biotin-CMP-Sia) to determine the relative abundance of the glycoproteins in the fraction with Biotin-CMP-Sia. Only glycoproteins found in both replicates and expressed in the brain are shown. (B) Gene Ontology analysis showing the molecular functions associated with the glycoproteins labeled by rST6Gal1. (geneontology.org; Fisher exact test). (C) GO analysis using the database SynGO (syngoportal.org) highlighting the number of genes associated with the pre- and postsynaptic compartments (p value: 9.2 × 10–09 and 3.9 × 10–05, respectively). See the Supporting Information for details.

Almost half (24/49) of the rST6Gal1-modified glycoproteins found in the present study overlapped with glycoproteins that had altered levels in a mouse model expressing the schizophrenia risk allele in SLC39A8, which was shown by MALDI-MS to have significant changes in the large, branched N-glycans (tri- and tetra-antennary) and those containing galactose, sialic acid, and fucose (Figure S7B).19 In contrast, the rST6Gal1-modified glycoproteins in this study had minimal overlap (2/49, 4%) with glycoproteins reported to be altered in symptomatic Alzheimer’s disease samples.12 Many (18/49, 37%) of the rST6Gal1 modified glycoproteins were present in synaptic vesicles identified by Bradberry et al., consistent with a role of these structures at the synapse (Figure S7C).2

Gene ontology analysis showed that many of the identified glycoproteins are involved with cell–cell adhesion, axon guidance and regeneration, and synapses (Figure 5B, Figure S8A–C). Finally, analysis using the synaptic gene ontology database SynGO indicated that 11 and 8 genes are significantly associated with the pre- and postsynaptic compartment (p value= 9.2 × 10–09 and 3.9 × 10–05), respectively (Figure 5C, Figure S7C, Supporting Information). Taken together with immunohistochemical analyses, these results demonstrate that galactosylated N-glycans are significantly enriched in the synapse.

Discussion

Our studies demonstrate that galactosylated N-glycans are uniquely distributed in the mouse brain and occur on multiple brain glycoproteins, many of which are confined to the pre- and postsynaptic compartment and have functions in cell–cell adhesion, axon guidance, and regeneration. These studies were prompted by our earlier results highlighting the relatively minor expression of terminally galactosylated N-glycans in the total glycome of the brain; this low abundance has complicated the identification of glycoproteins containing this specific modification within total brain samples through conventional techniques.13

Among animal tissues, the brain is often considered to be the most complex in terms of structure and physiology. However, its vast glycome, which includes both the glycoproteome and the glycolipidome, is poorly understood compared to other tissues in terms of its structure and spatiotemporal presentations. Although studies are progressing in this area, much remains to be learned about the total glycan and glycoprotein repertoires and the spatial location of such glycoconjugates in the brain.12,19,26 In terms of organization and spatial presentation, the brain is unique in many ways compared to other tissues, and its study has the potential to unravel new functions of glycosylation in the brain; this importance underscores the need to develop new tools to understand it.26

The use of glycomic technologies incorporating modified monosaccharides through various bioorthogonal and tagging technologies, which generally require living samples, is providing considerable knowledge of glycan functions in human health and diseases.44,60−63 Previous studies using tissues other than brain have focused on the use of enzymatic labeling to target specific galactosylated glycoconjugates, allowing more in-depth studies compared to a global labeling of the glycoconjugates. This strategy allowed for the visualization of labeled glycoproteins on tissue sections as well as their identification in cell culture.39,40,61 However, the study of the galactosylated glycoproteins in an organ is a relatively unexplored area and has thus far not been readily approachable by bioorthogonal technologies.

We chose to examine brain sections and total brain glycoproteins using biotinylated sialic acid (CMP-Biotin-Sia) that can be enzymatically transferred to galactosylated N-glycans by rST6Gal1 for further visualization of glycoproteins on brain slides and for their identification in a whole brain lysate. ST6Gal1 is the key enzyme in mammals for addition of α2,6-sialic acid to galactosylated N-glycans and is widely expressed, but the highest levels are found in liver and hematopoietic tissues.64 The recombinant enzyme has robust kinetics and activity.65 Although the presence of biotin on the sialic acid of CMP-biotin-Sia might limit the transfer efficiency of the molecule by rST6Gal1, our enzymatic analyses indicate that the enzyme transfers Biotin-Sia to acceptors with a slightly better catalytic efficiency (Vmax/Km) than its counterpart lacking biotin. In relation to this, a prior study created an even larger CMP-sialic acid donor molecule containing sialic modified by biotin and a heparin sulfate oligosaccharide; this large derivative was also successfully transferred by rST6Gal1, which further highlights the ability of rST6Gal1 to efficiently recognize large, modified donor substrates.66

Our results also demonstrate that galactosylated N-glycans are accessible to rST6Gal1, yet relatively few N-glycans are sialylated,13 and often, α2,3-sialylation appears to be more abundant than α2-6-sialylation.27 Glycans with modified LacNAc residues, however, would not be identifiable by our strategy. For example, the Lewis X-type sequence Galβ1-4(Fucα1-3)GlcNAcβ-R is found in murine brain glycoproteins,13,67 but it is not an acceptor for rST6Gal1. Glycans containing α2-fucosylated galactose have been identified in murine brain,68 but the rST6Gal1 does not modify galactose residues in such glycans. Thus, glycoproteins that we have identified are relatively specific in carrying the terminal galactose, but other glycoforms may also occur. Whether such modified galactosylated glycans exist on the same glycoproteins as those tagged in our study by rST6Gal1 remains to be defined.

There are a few limitations to our approach that need to be considered. First, the galactosylated glycoproteins we have identified and associated with synapses represent only a subset of the total known synaptic glycoproteins.2,69 Consistent with our studies, however, few glycans in such glycoproteins contain terminal, unmodified galactose residues such as those we identified. Thus, our results suggest a strong differential glycosylation of protein species within synapses that is deserving of future studies with regard to their glycan functions. Second, although we observed the transfer of biotin-Sia from rST6Gal1 onto N-glycans, consistent with outcomes of prior studies on this enzyme,43,46 we also observed that rST6Gal1 has weak activity toward galactose residues in O-glycans containing the core 2 and core 4 backbones. However, in our results, the N-glycans labeled by this strategy were releasable by PNGase F, indicating that the endogenous glycans tagged by this strategy are not O-linked. The results raise the possibility, however, that endogenous O-glycans at a lower level and resistant to PNGase F might contain such modifications. Third, we also observed that rST6Gal1 can modify GlcNAc residues in some types of N-glycans. The presence of α2–6-linked sialic acid on terminal GlcNAc has been observed previously, and there is a prediction that ST6Gal1 can catalyze this reaction.70,71 Such terminal GlcNAc structures are commonly found in hybrid type-N-glycans in the mouse brain.13 However, the glycans that we labeled in our technology were not sensitive to Endo H, which cleaves such hybrid N-glycans, thus indicating that our tagging is restricted to galactose within complex N-glycans. Fourth, we did not analyze all the glycans on our identified glycoproteins and all their sites of glycosylation due to the issues of abundance and restrictions of the technology. Thus, the galactosylated glycoproteins we identified may have other types of non-N-glycans, e.g., O-glycans and other modifications.

Our results on the cerebellum slides indicate that terminal galactosylated N-glycans are enriched in the molecular layer, which is rich in synapses, but we observed staining in the arbor vitae, which contains axons of the Purkinje cells and the oligodendrocytes producing the myelin sheath. Our observations relate to the other issue of the natural presence of α2,6-sialylated galactose in the brain. It is reported that the loss of ST6Gal1 in mice leads to a general loss of all α2,6-sialylated glycans, suggesting that the major type of sialylation is α2,6.72,73 Histological studies indicate that α2,6-linked sialylated glycans generated by the ST6Gal1 in mice are widely distributed in the brain parenchyma and not concentrated in synapses; interestingly, the complete lack of ST6Gal1 in mice leads to a loss of all such sialylated glycans in the brain.73 It is important to note that whereas deletion of galactosylation of N-glycans has a profound neural phenotype, the deletion of the ST6Gal1 that could modify such galactosylated glycans has only a modest neural phenotype.73 Such studies combined with ours suggest a relatively differential expression and function of α2,6-sialylated N-glycans in the brain compared with those that are terminally galactosylated.

Many of the glycoproteins identified in this study overlap with those from previous studies that focused on modified glycoproteins in several neurodegenerative disorders. For instance, the schizophrenia-associated variant A391T in the manganese transporter SLC39A8 alters the N-glycosylation of some glycoproteins that were also found in this study.19 This observation indicates that some of the identified galactosylated glycoproteins are altered when the concentration of manganese is modified when this schizophrenia-associated variant is expressed.19 The glycoproteins MOG and NPTN, identified in our approach, have also been associated in a recent glycoproteomics studies on Alzheimer’s disease.12 A mutation in the ST6GAL1 gene is associated with the development of this brain disorder and might indicate that the differential sialylation of MOG and NPTN might be important for the development of Alzheimer’s disease.74 Such results further suggest a direct biological role of galactosyl residues in brain functions. Indeed, the restricted localization of galactosylated glycoproteins in the brain suggests that they may have relatively unique functions, as highlighted in diseases where galactosylation is altered, such as CDGs. Individuals suffering from diseases impacting the synthesis of galactosylated glycans, such as galactosemia or B4GALT1-CDG (CDG type IId) caused by a deficiency in the β-1,4-galactosyltransferase 1, both diseases are associated with severe neurological disorders.16,75 These disorders can help to identify the impacted glycoproteins, as well as many others associated with altered galactosylation through either defects in glycosyltransferases or cofactors or other pathways that could be addressed by this approach.16,19,75−77

In summary, we identified a unique set of galactosylated glycoproteins enriched in synapses. Such galactosylated N-glycoproteins are involved in axon guidance, synapse transmission, and cell–cell adhesion. The contribution of glycosylation in brain functions is still underappreciated, but our study provides a glimpse into the differential localization of glycosylation types in the brain. More generally, this approach can be utilized on any organ to identify the glycoproteins where galactosylation is altered. These and related approaches will enhance our discovery of the expression and roles of galactosylated glycoproteins in organ function and their involvement in the development of diseases. Our results further highlight the potential use of biotinylated monosaccharides that can be specifically transferred by glycosyltransferases,78 allowing the modified glycans to be visualized on slides and identified at the organ level for their spatial presentation.

Material and Methods

Mouse Information

Twelve week old male mice on a C57BL/6J background were maintained in standard housing conditions, provided normal chow and water ad lib, and maintained on a 12 h day/light cycle. All mice were housed and maintained in accordance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) at the Beth Israel Deaconess Medical Center under approved protocol #02-2022.

Reagents

SiaNAz was purchased from Carbosynth (MA30919). Pyrophosphatase from S. cerevisiae (I1643), CMP-sialic acid synthetase from N. meningitidis (C1999), biotin-PEG-alkyne (764213), β-galactosidase (G5160), and cOmplete Mini EDTA-free Protease inhibitor (11836170001) were purchased from Sigma-Aldrich. Endo H (P0702S), PNGase F (P0704S), and NeuA (P0722L) were purchased from NEB. IRDye 800CW streptavidin (926-32230) and total protein staining (926-11010) solutions were purchased from Li-Cor. The antibody against PSD-95 (MABN68) was purchased from Millipore, and that against VGLUT1 (Ab272913) was purchased from Abcam. Secondary anti-mouse-488 (A11059) and anti-rat-488 (A21212) were purchased from Molecular Probes. The CFG arrays were obtained from The Scripps Research Institute.79 Streptavidin agarose beads (20347) were purchased from ThermoScientific. All other reagents were purchased from SIGMA unless otherwise noted.

Synthesis of Biotinylated CMP-SiaNAz

Five milligrams of SiaNAz (0.015 mmol), 15.76 mg of CTP (0.033 mmol), 1.5 U of CSS, and 3 U of pyrophosphatase were mixed with 500 μL of Tris-HCl buffer (100 mM Tris-HCl, 20 mM MgCl2, pH 8.8) and incubated for 4 h at 37 °C. The synthesis of the CMP-SiaNAz was assessed by 31P NMR after the solution was lyophilized. The lyophilizate was solubilized in 200 μL of 250 mM Tris-HCl buffer (pH 7.4), and then 600 μL of biotin-PEG4-alkyne (9 mg) was added to 72 μL of CuSO4 (Cf: 6.7 mM), 90 μL of ascorbate sodium (Cf: 8.5 mM), and 100 μL of BTTAA (0.0056 mmol). The mixture was incubated for 3 h at room temperature (RT) and lyophilized again. The lyophilizate was resuspended in 200 μL of water, placed in a P2 column overnight, and eluted with 0.1 M NH4HCO3 at 4 °C in the dark. Positive fractions were identified with orcinol, mixed and lyophilized again for NMR analysis, and quantified with a standard curve of CMP.

Expression of Recombinant ST6Gal1

Recombinant ST6Gal1 (rST6Gal1) lacking the first 74 amino acids but containing a His-tag was purified according to the protocol described in Moremen et al.48 In brief, plasmids were purchased from DNASU (HsCD00413052). HEK-293 Freestyle cells were cultured in suspension to reach a density of 1 × 106 cells in 50 mL of fresh media, transfected with 150 μg of plasmid and 450 μg of PEI (polyethylenimine), and incubated at 37 °C on a rotatory incubator/shaker. After 24 h, the cell culture was diluted with 50 mL of media containing valproic acid to reach a final concentration of 2.2 mM valproic acid. The medium was harvested 3 days later, and recombinant ST6Gal1 enzyme was enriched by using Ni2+ column, dialyzed against 100 mM NaCl pH 7.5, and concentrated to reach 2 mg/mL.

Kinetic Analysis

rST6Gal1 activity was measured by using the malachite green phosphate detection kit (R&D Systems #DY996), where 0.5 μg of ST6Gal1 was incubated with 100 μg of asialofetuin, the indicated concentration of donor substrate, and 50 ng of phosphatase with MES buffer (100 mM, pH 6.5) in 25 μL final volume.48 Samples were incubated at 37 °C for 30 min followed by the addition of 30 μL of reagent A, 100 μL of water, and 30 μL of reagent B according to manufacturer’s protocol. Optical density was determined using a microplate reader (SpectraMax i3x, Molecular Devices) at 620 nm.

Western Blotting

Four hundred micrograms of brain lysates was precleared by using 800 μL of streptavidin beads (4 mg/mL) for 1 h at RT to remove the nonspecific background likely caused by the biotin-dependent carboxylases in the brain as previously described.13 Fifty micrograms was digested before or after the sialylation reaction with 50 U of NeuA, 5.4 mU of β-galactosidase, 500 U of Endo H or PNGase F, and the appropriate buffers in a final volume of 12 μL and incubated for 2 h at 37 °C. Sialylation was performed with 1.5 μg of rST6Gal1, 1× MES buffer (100 mM MES, pH 6.5), and 100 μM of biotinylated CMP-sialic acid in a final volume of 30 μL for 2 h at 37 °C. After sialylation, 8 μL of Ni2+ beads was incubated with the lysates to remove rST6Gal1 for 1 h at RT. The remaining sample and supernatant were then removed and directly assessed via Western blot. Samples were loaded and separated by SDS-PAGE gels (4–12%) and transferred onto a nitrocellulose membrane by using iBlot transfer stacks (Invitrogen, #IB301031). After total protein staining and imaging, the membrane was blocked with 5% BSA (ThermoFisher, #BP1600-1) in TBS with 0.1% Tween-20 (TBS-T) for 30 min followed by incubation with streptavidin-800 diluted 1:10,000 in TBS-T-BSA 5% for 1 h at RT in the dark, washed three times for 15 min in TBS-T, and imaged using a Li-COR Odyssey CLx scanner.

Glycan Microarray

Glycan microarray slides were dried in a desiccator for 20 min and rehydrated in the TSM Wash (TSMW) buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.05% Tween-20).79 Slides were then incubated with 835 mU of NeuA (where indicated) in 100 μL of an aqueous glycobuffer 1 (5 mM CaCl2, 50 mM sodium acetate (pH 5.5), NEB) overnight in a humidifying chamber at 37 °C. After four sequential washes with TSMW, TSM (TSMW without Tween-20), and water, 90 ng/μL of rST6Gal1 was incubated with 600 μM of CMP-biotin-Sia in 80 μL of 100 mM of the MES buffer (pH 6.5) for 2 h at 37 °C in a humidifying chamber. Slides were then washed four times each with TSMW and TSM. Streptavidin Cy5 (Jackson ImmunoResearch) was incubated at 0.5 μg/mL in the TSM binding buffer (TSMW with 1% BSA) on the slides for 1 h at RT in a humidifying chamber. After four sequential washes with TSMW, TSM, and water, slides were dried by centrifugation and scanned at 635 nm using a GenePix 4400A microarray scanner (Molecular Devices). The image was analyzed using the Genepix Pro software followed by averaging of signal intensities using Microsoft Excel and final data analysis visualization using GLAD.80

Brain Tissue Staining

Mice at 12 weeks of age were euthanized with CO2 gas in accordance with AVMA guidelines and transcardially perfused with ice-cold PBS containing heparin at 10,000 U/L (Sigma #3149) for 2 min at 6 mL/min followed by ice-cold 4% PFA in 0.1 M PBS (pH 7.4) for 5 min at 5 mL/min. Whole brains were then removed from the skull, postfixed in 4% PFA overnight, and transferred to PBS until processing. Fixed brains were paraffin embedded and cut in 3 μm coronal sections with a microtome at the BIDMC Pathology Core Facility. Slides were then deparaffinized using a standard xylene/ethanol gradient protocol and placed in an antigen retrieval solution (0.1 M citric acid and 0.1 M sodium citrate, pH = 6), incubated in a pressure cooker at boiling temperature for 3 min, and stored at 4 °C in TBS. The staining protocol was adapted from Lopez Aguilar et al.39 In brief, slides were pretreated with denaturing buffer for 5 min at 95 °C and washed with TBST-0.1% three times for 3 min. If indicated, slides were incubated overnight with 5 μL of PNGase F with glycobuffer 2 (50 mM sodium phosphate, pH 7.5, NEB) and NP-40 in 80 μL final volume in a humidified chamber on a small rotator at 37 °C. After three washes with PBS, the tissue was blocked with PBS-5% BSA for 1 h at RT and incubated with 90 ng/μL of ST6Gal1 and 600 μM of CMP-biotin-Sia in a final volume of 80 μL of the MES buffer (100 mM, pH 6.5) for 2 h at 37 °C. After three washes with PBS, if indicated, slides were incubated with anti-VGLUT1 or anti-PSD95 antibodies at 1:100 dilution in BSA 5% for 2 h at 37 °C. Slides were then washed with PBS three times and incubated with streptavidin phycoerythrin-conjugated (Invitrogen, #12-4317-87) and anti-mouse-488 or anti-rabbit-488 at 1:200 for 1 h at 37 °C. After three washes with PBS, the tissue was incubated with Hoechst 33342 (Invitrogen) at 1:1000 for 10 min and washed again three times in PBS. A cover glass was added with the mounting medium (Invitrogen, P36930). The sample was dried overnight and analyzed by confocal microscopy using ImageJ and Imaris software (Oxford Instruments).

Enrichment of Biotinylated Glycoproteins

Six whole brains from male wild-type C57BL/6J mice (Jackson Laboratories, strain #000664) were individually lysed in 500 μL of the lysis buffer (50 mM Tris, 150 mM NaCl, 1.0% v/v Triton-X-100, pH 7.6) with the protease inhibitor (Roche, #46931320019) using mortar and pestle and briefly sonicated for 10 s two times on ice. An additional 500 μL was added to reach 1 mL final volume, and the protein concentration was measured by a BCA assay. Three lysates were incubated with 800 μL of MES 2.5× (250 mM, pH 6.5), 85 μg of ST6Gal1, and 200 mM of CMP-biotin-Sia in 2 mL total volume overnight at 37 °C, and three lysates were incubated without the CMP-biotin-Sia as a negative control. Lysates were then filtered through Amicon Ultra-15 centrifugal filters (Millipore) with 30,000 kDa MWCO with five washes of 2 mL of PBS and centrifuged at 4000 rpm for 1 h at 4 °C to remove the free CMP-biotin-Sia from the media. Lysates were pooled, and 40 mg of brain lysates was incubated with 150 μL of streptavidin beads in a final volume of 1 mL of the lysis buffer overnight at 4 °C. Beads were recovered and washed 10 times with TBS-tween 0.1% for 5 min.

Identification of Glycopeptides by Mass Spectrometry

The biotin-Sia-containing proteins were eluted from the streptavidin beads three times with 200 μL of 8 M guanidine hydrochloride solution (pH 1.5) at 56 °C. The eluates were combined, transferred to a Microcon 30 kDa centrifugal unit (Millipore), and centrifuged at 14,000g (4 °C) until dried. The proteins were washed with 400 μL 25 mM ammonium bicarbonate pH = 8.5 three times. The purified proteins were suspended in a buffer containing 25 mM ammonium bicarbonate and digested with 5 μL of sequencing grade modified trypsin (Promega, V511C) overnight with end-over-end rotation at 37 °C. The next day, the filter was centrifuged at 14,000g to collect the peptides. Centrifugation was repeated twice with the 25 mM ammonium bicarbonate solution, and the eluates were pooled and dried with lyophilization. Glycopeptides were separated from nonglycopeptides by HILIC as previously described.12 Briefly, the peptides were dissolved in a solution containing 80% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). The HILIC SPE column was packed in house using microcrystalline cellulose (Sigma, #435236). The column was equilibrated with 80% ACN and 0.1% TFA, the peptides were loaded, the column was washed with 80% ACN and 0.1% TFA, and the enriched glycopeptides were eluted with 0.1% TFA. The glycopeptides were then dried with lyophilization.

Peptide sequencing was performed on a Dionex UltiMate 3000 UHPLC system coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoScientific). The glycopeptides were dissolved in a solution containing 0.1% formic acid (FA) in water, loaded onto a C18 precolumn (C18 PepMap 100, 300 μm × 5 mm, 5 μm, 100 Å, ThermoScientific) with 15 μL/min solvent A (0.1% FA in water) for 3 min, and separated on a C18 analytical column (PicoFrit 75 μm ID × 150 mm, 3 μm, New Objective) using a gradient of 2–40% solvent B (80% ACN and 0.1% FA in water) over 97 min followed by a gradient of 40–90% over 3 min. The mass spectrometer was operated in a data-dependent acquisition mode, where the 15 most intense ions from MS1 are further sequenced in MS2. The ion source voltage was 2100 V. The ion transfer tube temperature was 275 °C. MS1 was performed in the Orbitrap at the resolution of 120,000 with the mass range of 800–2000 m/z. Normalized automatic grain control (AGC) target was set to 100% with a maximum ion injection time of 50 ms. The quadrupole was used to isolate precursor ions with an isolation window of 1.5 m/z. Dynamic exclusion was employed for 10 s with a mass tolerance of 10 ppm. Stepped HCD (higher-energy collisional dissociation) with collision energies of 20, 30, and 40% was used to fragment the peptides. MS2 detection was performed in the Orbitrap with the resolution of 30,000. The normalized AGC target was set to 100% with a maximum injection time of 250 ms.

Raw files were searched using pGlyco312 against the mouse proteome database downloaded from UniProt (June 21, 2021, reviewed, containing both canonical and noncanonical sequences, 25,368 entries). Trypsin was set as an enzyme with the specificity set to specific. The maximum number of missed cleavages was 2. Oxidation of methionine (+15.9949 Da) and acetylation of the protein N-terminus (+42.0106 Da) were set as variable protein modifications. The maximum variable modification was two. Peptide length was from 6 to 40. The glycan database used was the default “pGlyco-N-Mouse”. Variable glycan modification of Neu5Ac was selected for the biotin tag with a mass shift of +773.3266 Da. The maximum variable modification on glycans was four. Precursor ion mass tolerance was 10 ppm, and fragment ion mass tolerance was 20 ppm. Identified glycopeptides were inspected manually. Glycan structures were assigned from the glycan composition following the study by Williams et al.13 Label-free quantification was performed with pGlycoQuant to determine the abundance of glycopeptides from the condition with CMP-sialic acid compared with the controlled condition (without CMP-sialic acid).81 All default parameters were used for the quantification with DDA LabelFree selected as the quantification type. For glycopeptide quantification, those with 0 intensity were removed. Raw files can be accessed through Glycopost with the identifier GPST000447.

Acknowledgments

We acknowledge the Confocal Imaging Core at the Beth Israel Deaconess Medical Center and the National Center for Functional Glycomics (NIH grant R24GM137763 to R.D.C.) for the resources, technology, and expertise as well as the grants NS072030 and RR005351 related to the HMS/BCH Center for Neuroscience Research and the generation of materials related to human and bacterial glycosylation enzymes. R.G.M. is supported by NIH grant 5K08MH128712.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.4c00399.

  • Supplementary Figures S1–S8, including NMR profiles and spectra, Western blots, glycan microarray data, immunofluorescent staining, and glycoproteomics analyses (PDF)

  • Raw data files (XLSX)

Author Present Address

§ Current address: Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA

Author Present Address

∥ Current address: Office of Pharmaceutical Quality Research, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, USA

Author Contributions

M.N. performed chemical synthesis, enzyme production, kinetic analysis, Western blotting, slide staining, and data analysis; helped conceptualize the project; and wrote the manuscript. S.S. performed enrichment with Y.M. and proteomics acquisition. M.W. helped in chemical synthesis and performed NMR acquisition and interpretation. C.T., A.Y.M., and J.H.M. performed glycan array analysis and data interpretation. R.G.M. helped conceptualize the project. R.D.C. supervised M.N., S.S., M.W., and Y.M.; helped conceptualize the project; wrote the manuscript with M.N. and J.H.M.; and provided funding. All authors contributed feedback and edits to the manuscript.

The authors declare no competing financial interest.

Supplementary Material

cn4c00399_si_001.pdf (11.1MB, pdf)
cn4c00399_si_002.xlsx (761.5KB, xlsx)

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