The Importance of N- and O-Glycosylation of Brain Cell Surface Glycoproteins

Abstract

The mammalian brain is unique in its cell types, mainly neurons and glial cells, and the glycoproteins expressed by these cells. Two of the most abundant types of modifications of cell surface glycoproteins are N-glycans linked to Asn residues and O-glycans linked via GalNAc to Ser/Thr residues. Recent studies focused on glycoproteomics, glycomics and glycan localization in the brain reveal major differences in these protein modifications compared to other organs. Deficiencies in glycosylation are associated with the development of multiple brain disorders such as congenital disorders of glycosylation (CDG) that include brain structural abnormalities, epilepsy and seizures to more common disorders including schizophrenia and Alzheimer’s disease. Here we summarize recent advances in the growing field of neuro-glycobiology and highlight key points that could be used as primer for future studies.

Introduction

Glycosylation is the modification of proteins and lipids by macromolecules called glycans, made of several building monosaccharide blocks. All living cells express glycans, with each expressing their own monosaccharides and structures (Gagneux et al. 2022; Cummings 2024). In the secretory pathway, glycans are synthesized by a complex machinery throughout 2 essentials compartments: the endoplasmic reticulum (ER) and the Golgi apparatus, after which they may reside in intracellular organelles, e.g., lysosomes, or travel to the plasma membrane for either secretion or surface membrane residence. There is an increasing interest in studying the brain glycome as it is involved in brain development, functions and neuro-disorders. This review will focus on relatively recent developments regarding the N- and O-glycans (mucin-type R-GalNAc-Ser/Thr modifications) synthesized in the brain, their spatial distribution and their involvement in brain functions.

1. Glycomic profile of N- and O-glycans in the brain

   • N-Glycans

The analysis of brain N- and O-glycans on proteins accelerated in the early 1970s and interest in this field has continued to grow (Margolis et al. 1972). Early studies identified several types of N- and O-glycans and glycan antigens that appeared to be uniquely expressed in the brain, such as the polysialic acid chain on NCAM (Rutishauser and Landmesser 1996; Hildebrandt et al. 2007), the 3-O-sulfated GlcA or L2/HNK-1 epitope present on N- and O-mannose glycans (Morise et al. 2017) and the 4-O-sulfated GalNAc modification being present in N-glycans of pituitary hormones (Baenziger and Green 1988) (Fig. 1). More comprehensive N-glycomic analyses of the mammalian brain in recent years have reported that a majority of all N-glycans are of the high-mannose types with Man5 (Man5GlcNAc2-Asn) representing almost half of the N-glycan pool (Lee et al. 2020; Williams et al. 2022; Helm et al. 2024). High-mannose glycans are universally present during N-glycoprotein biosynthesis at the beginning of the secretory pathway in the ER and within the Golgi apparatus where they are converted to hybrid and complex N-glycans (Stanley et al. 2022). Of the ~30% of N-glycans in the brain which are not high-mannose structures, a majority (80–90%) of those are bisected and fucosylated. Sialylation of N-glycans, considered as one of the hallmarks of fully mature glycans, is relatively low in the brain. Although the level of sialylation may vary between studies -from 2% (Williams et al. 2022) to 50% (Zamze et al. 1998)- there are several lines of evidence confirming that sialylation is relatively low among brain N-glycans. Among them is a low binding of SNA to glycoproteins in brain lysates, a lectin that binds to α2,6-linked sialic acid, and a low expression of sialyltransferases specific to N-glycans (Williams et al. 2022). In terms of abundance, brain N-glycans have a relatively different distribution than other organs that typically have low levels of Man5.

Fig. 1.

Simplified biosynthesis pathways of N- and O-glycans in the brain. N-glycans are linked to Asparagine (Asn) and are initially high-mannose type. They are then converted to a precursor for all hybrid/complex N-glycans after action of the MGAT1 enzyme. This hybrid N-glycan GlcNAc₁Man₅ is then processed by several distinct enzymes to create the unique N-glycans of the brain, including bisected GlcNAc, HNK-1 epitope, sialylated and fucosylated structures, and sulfated terminal epitopes. A detailed explanation of the pathway can be found in Stanley et al. 2022. O-GalNAc glycan synthesis starts with the addition of a single GalNAc by one of twenty GalNAc-Ts to form the Tn antigen which is then converted to T antigen after the action of T-synthase (C1GALT1) and its chaperone Cosmc (C1GALT1C1). This structure is then further elongated by the action of several enzymes to form an array of O-glycans including sialylated species.

What mechanisms underlie the uniqueness of the brain glycome? One of the simplest explanations for the high abundance of Man5 could be a low expression of MGAT1, the unique enzyme required to convert Man5 to the precursor for all hybrid and complex structures (Fig 1). MGAT1 is expressed in the brain although at slightly reduced levels compared to other organs (Williams et al. 2022). Another possibility is the presence of a specific inhibitor of MGAT1, such as the protein GnT1IP-L, which inhibits the MGAT1 activity (Huang et al. 2015), but there is no evidence for its expression in the brain. Man5 is especially abundant in neurons, as iPSC-derived microglia and endothelial cells have relatively higher levels of complex N-glycans, which suggest a specific mechanism in neurons leading to its high abundance (Kiwimagi et al. 2024; Tang et al. 2025). Another consideration is the unique organization of the secretory pathway in neurons (Kennedy and Hanus 2019). Neurons are specialized cells that possess a very large ER that extends from the dendrites to the axon and a relatively restricted Golgi apparatus in their soma and vesicles related to the Golgi apparatus called Golgi satellites in their dendrites. These vesicles are involved in the glycosylation of the glycoproteins in the dendrites, although details regarding Golgi satellites are still unclear as well as the glycosyltransferases that are present in these vesicles. These vesicular structures have been shown to promote a conversion from high-mannose glycans to mature N-glycans on the glycoproteins that transit through them to reach the cell surface (Mikhaylova et al. 2016; Bowen et al. 2017; Kennedy and Hanus 2019). The use of a non-canonical pathway for glycoprotein phosphorylation in neurons has also been proposed, which may contribute to the unique N-glycan profile in the brain (Sironić et al. 2025).

Finally, the low abundance of complex N-glycans in the brain may relate to the high abundance of bisected N-glycans. It is known that MGAT3, the unique enzyme adding bisecting GlcNAc to the N-glycans, is highly expressed in the brain and may represent one of the main mechanisms preventing the generation of complex N-glycans (Fig. 1) (Williams et al. 2022). The presence of the bisecting GlcNAc limits subsequent modifications of N-glycans, including galactosylation and sialylation, as the additional GlcNAc residue may alter the glycan conformation and reduce interactions with glycosyltransferases (Nagae et al. 2016; Nakano et al. 2019).

• O-Glycans

Core 1 O-GalNAc glycans (Galβ1-3GalNAcα-R) are less studied in the brain compared to O-mannose glycans, despite representing ~80% of total brain O-glycans (Williams et al. 2022) (Fig. 1). In contrast to N-glycans, most O-GalNAc glycans are sialylated, with the most abundant structure being the Core1- O-glycan with two sialic acids. It is interesting to note that Neu5Gc, which can be expressed by many mammals including mice, but not by humans or new worlds monkey (Springer et al. 2014), is not detected by mass spectrometry on N-glycans of the murine brain, and is only present in ~1% of the O-glycans (Williams et al. 2022). Neu5Gc is a sialic acid that differs from Neu5Ac by a single atom of oxygen and commonly known to be low in the vertebrate brain, perhaps due to toxicity of this structure to the brain (Davies and Varki 2015), and that the gene permitting the conversion of CMP-Neu5Ac to CMP-Neu5Gc called CMAH, does not have detectable level of transcripts in the mammalian brain (Davies and Varki 2015).

It is also worth noting the relative absence of the Tn antigen (GalNAcα-S/T) in the mouse adult brain glycoproteins, which is only detectable via some mass spectrometry techniques (Suttapitugsakul et al. 2023), but not others (Williams et al. 2022) and is not easily detected by lectins or antibodies that bind the Tn antigen (Noel et al. 2025). Interestingly, the Tn antigen is expressed during the embryonic development of the mouse brain and is greatly reduced 3 weeks after birth (Akita et al. 2001). Thus, much remains to be discovered about the function and role of O-GalNAc glycans in brain development.

• Conservation during evolution

From an evolutionary point of view, the overall glycome of all tissues is known to vary between species. These differences are thought to be important in development but also to help the host to evade recognition and subsequent infection by specific pathogens through the expression of unique glycans. This concept, termed “red queen effect” highlights the idea of adaptation of the glycome between the host and its pathogens (Varki 2006). Remarkably, however, brain N-glycans seem to be well conserved between vertebrates (Lee et al. 2020; Williams et al. 2022; Klarić et al. 2023). For example, the brain of zebrafish contains a large amount of Man5 in N-glycans consistent with the mammalian brain N-glycome (Hall et al. 2023). This suggests ancestral mechanisms that are conserved within vertebrates favoring the presence of Man5 in the brain, probably benefiting from the immune privilege that the brain possesses due to the blood-brain barrier (Muldoon et al. 2013).

However, some species have some unique features in their brain glycome that distinguish them from others. For example, the absence of Neu5Gc in the mammalian brain, despite its high abundance in other tissues (Davies and Varki 2015; Lee et al. 2020). In zebrafish, Neu5Gc is found on N-glycans, suggesting a molecular evolution that appeared during the emergence of mammals (Yamakawa et al. 2018). In humans, increased galactosylation and the abundance of α2,6 sialic acid are more characteristic of our brain compared to other primates (Klarić et al. 2023).

In summary, glycosylation in the brain has unique features and many mechanisms are involved, most not yet well understood, to generate this atypical glycosylation profile.

1. Spatial distribution of N- and O- glycans in the brain

   • Current methodologies

The spatial distribution of glycans in an organ represents an intense field of research as it can reveal new roles for specific structures based on their location. An emerging technology is MALDI-mass spectrometry imaging (MALDI-MSI), which uses targeted application of PNGase F to release the N-glycans on a tissue section; the released glycans in the area are then detected by mass spectrometry. This technique was first used in 2013 to highlight a specific distribution of N-glycans on a brain section (Powers et al. 2013; Hasan et al. 2021). For example, in cerebellum which possesses 4 layers (outward to inward: molecular layer rich in synapses, Purkinje cells and granular layer with the soma of neurons which together compose the grey matter, and the Arbor Vitae containing the white matter of axonal tracts and myelin), N-glycans are more abundant in the grey matter. Studies on O-glycans with this technology could be performed as an endo-α-N-acetylgalactosaminidase from Actinomyces sp. (called POGase) was recently discovered to cleave a variety of neutral and sialylated O-glycans (core 1 to core 3). However, it does not cleave disialylated core 1 O-glycans limiting its application to brain O-glycans as it is highly abundant (Williams et al. 2022; Zhou et al. 2025). As the resolution and depth of analysis improve, MALDI-MSI will be important to highlight the expression of glycans at the cellular level inside a complex tissue such as the brain (Cumin et al. 2024; Dressman et al. 2025).

Another approach to map the brain glycome is the use of chemical reagents to tag specific types of glycans. For instance, the use of sialic acid derivatization allows for the recognition of sialic acid linkage and subsequent analysis of the glycans by mass spectrometry (de Haan et al. 2020). With this approach, researchers were also able to study the spatial distribution of α2,3 and α2,6 sialylated species in a tissue by MALDI-MSI (Holst et al. 2016). Also, there are methods using modified monosaccharides that allows for the study and identification of every carrier glycan. A monosaccharide can be linked to a biotin or a fluorophore through click chemistry (Kolb et al. 2001; Prescher et al. 2004). This allows for the complete visualization of sialylated glycoconjugates in cultured cells or in a whole organism (Prescher et al. 2004; Baskin et al. 2007).

One of the most common approaches to target specific glycans is the use of glycan-binding reagents and general methods of immunohistochemistry. Such an approach increases the resolution, and identifies spatial presentation of glycan structures but has its limitations (Noel et al. 2023). Lectins are not typically specific to a single glycan structure but rather to a feature carried by several glycans, such as bisected GlcNAc, fucosylation and galactosylation (Bojar et al. 2022). Recombinant lectins with modified glycan specificities have been used to identify unique glycan structures (Cummings et al. 2022). For example, hemagglutinin proteins from the Clostridium botulinum complex were engineered to bind either terminal galactose or sialic acid structures, and using these reagents, it was reported that Gal-GalNAc glycoproteins are enriched and spatiotemporally regulated in the nodes of Ranvier (Tulin et al. 2022, 2021). Antibodies to glycan epitopes, such as HNK-1 (Abo and Balch 1981), are very useful as they are highly specific, but they may not readily allow identification of other glycan features within those glycans carrying the epitope and cannot distinguish between glycoproteins or glycolipids carrying this epitope (Dennis et al. 1988). Variable lymphocyte receptor B (VLRB) proteins engineered as VLRB-IgG chimeric proteins are attractive sources of monoclonal antibodies against specific glycan structure in the brain, highlighting specificity in their spatial expression (McKitrick et al. 2021; Lajoie et al. 2022; Tulin et al. 2025a). They have allowed for the visualization of the O blood group in healthy and tumoral tissues, sialylated species in endothelial cells of the brain and also 3-O-sulfated galactose that is not easily detectable with common techniques (McKitrick et al. 2021; Lajoie et al. 2022; Tulin et al. 2025). Another emerging approach to target specific glycans is the use of a recombinant glycosyltransferase; when combined with a biotinylated monosaccharide within a nucleotide sugar, which allows the introduction of biotin to a specific glycan structure recognized by the recombinant enzyme, readily permitting their subsequent visualization on the tissue using a tagged streptavidin (Lopez Aguilar et al. 2017). This approach allowed the labeling of N- and O-glycans on different tissues (Lopez Aguilar et al. 2018) and has been used on brain tissues with identification of the carrier proteins (Noel et al. 2024).

• Spatial differences in abundance for N- and O-glycan expression

The different techniques described above highlight an interesting contrast between glycan classes, where N-glycans are most abundant in grey matter and O-glycans in white matter tracts (Lee et al. 2025; Noel et al. 2025, 2024, 2023). Using lectins, our group showed that high-mannose, complex N-glycans and O-glycans are enriched in different areas of the brain (Fig. 2). For example, within the 3 layers of the cerebellum, complex N-glycans are enriched in the synapse-rich molecular layer while high mannose N-glycans are present in both the molecular and the granular layer. On the other hand, O-glycans are enriched in white matter tracts, although they can also be detected to a lower degree in the other layers (Noel et al. 2025, 2023).

Fig. 2.

Spatial segregation between N- and O-GalNAc glycans. A) Brain regions where the sections were made and schematic. B) Drawing of the brain sections illustrating the different brain regions including the corpus callosum and arbor vitae, which are white matter tracts. C) Brain sections stained with PNA for O-glycans, GNL for high-mannose N-glycans and PHA-E for bisected N-glycans.

High mannose N-glycans are considered intermediates in N-glycan biosynthesis and often are present in intracellular compartments, especially on lysosomal hydrolases (Makrypidi et al. 2012), but many high-mannose-type-N-glycans are also found on cell surface glycoproteins (An et al. 2012; Stavenhagen et al. 2021). Studies using cell surface biotinylation showed that they are present at the cell surface of neurons and modulates synaptic signaling (Hanus et al. 2016). Glycoproteomics analysis have showed that high-mannose glycans are also highly abundant in purified synaptic vesicles, again with Man5 being the most abundant N-glycan of this class (Bradberry et al. 2023).

Complex N-glycans are also present in the synaptic compartment, but their distribution seems more restricted (Bradberry et al. 2023). Staining of the cerebellum with various lectins targeting complex N-glycans consistently revealed strong labeling of the molecular layer, which is densely packed with synapses (Noel et al. 2023). This observation has been confirmed using the recombinant sialyltransferase ST6GAL1, which sialylates terminal galactose residues in complex type N-glycans (Fig. 1). Using biotin-labeled sialic acid in such studies allows for the visualization of the locations of glycoproteins with galactose-terminating N-glycans within fixed tissue sections (Noel et al. 2024). This observation confirmed previous discoveries based on glycoproteomics analysis, which revealed an enrichment of complex N-glycans in synapses (Bradberry et al. 2023, 2022; Handa-Narumi et al. 2018). These N-glycans have several roles such as regulating synaptic plasticity, membrane excitability and synaptic pruning (Cronin et al. 2005; Hildebrandt and Dityatev 2015; Linnartz-Gerlach et al. 2016). Synapse degradation is also regulated by complement factors whose activity is modulated by glycosylation (Mealer et al. 2020b). Desialylated human neurites were opsonized by the complement component C1q in vitro, although this increased degradation might be due to an altered structural conformation of the targeted glycoproteins by C1q (Jefferis 2007; Linnartz-Gerlach et al. 2016). Factor H, a regulator of the alternative complement pathway, can bind directly to sialylated species at the surface of N. gonorrhoeae, blocking the complement pathway activation. However, the precise glycan structures recognized by factor H and its roles on human synapses are not known (Ram et al. 1998). Some glycan structures possess a unique staining profile, such as glycans carrying the HNK-1 epitope, which occurs in a striped pattern within the molecular layer of the cerebellum (Marzban et al. 2004). Such unique stripes have been observed in different populations of Purkinje cells that have distinguishable expressions of protein markers. These different populations have different capabilities in inducting long-term depression, which is essential for neuronal excitability (Voerman et al. 2022). It is therefore conceivable that HNK-1 could be involved in such mechanisms, considering the shared pattern between these markers.

O-GalNAc glycans show the opposite pattern of N-glycans, with an enrichment in the white matter. Both sialylated and non-sialylated O-GalNAc glycans are enriched at the nodes of Ranvier, an essential structure that increase the speed of conduction through myelinated axons (Noel et al. 2025). Enrichment and glycoproteomics analysis identified the extracellular matrix molecules termed lecticans (neurocan, versican and brevican) as main carriers of this class of glycans. Lecticans are important for the formation and maintenance of the nodes of Ranvier and impairing the synthesis of O-GalNAc glycans in neurons reduced the size of the nodes (Noel et al. 2025).

Given their high abundance at the nodes of Ranvier and considering that O-glycans can carry a small amount (1%) of Neu5Gc (Williams et al. 2022), it might be possible for this monosaccharide to be present at the nodes of Ranvier. However, by using an antibody targeting this sialic acid, Naito-Mitsui et al. demonstrated that the small amount of Neu5Gc in the brain is present mostly in endothelial cells and some macrophages/ microglia in rat brain (Naito-Matsui et al. 2017), suggesting some possible restriction or absence of Neu5Gc in neurons, perhaps related to the potential negative effects this monosaccharide can have on neural functions (Naito-Matsui et al. 2017). Glycoproteomics analysis of recombinant lecticans showed different distributions of NeuAc and NeuGc across both N- and O-glycans for each protein, though it is mostly likely these effects are a result of the species and tissue from where they are purified (Downs et al. 2023).

Altogether, N- and O-glycosylation features appear uniquely distributed across the brain. More tools will be needed to further decipher the mechanism controlling glycan expression and to better understand the functional consequences of these processes in the brain.

1. Importance of protein glycosylation in the brain

• N- and O-glycans in brain development

While specific functions of each type of N- and O-glycan in the brain are still being studied, the evidence is clear that these glycans are essential for the proper development of an organism. Dysregulation of N- and O-glycosylation can lead to severe neurological disorders and brain malformations, demonstrated by the majority of Congenital Disorders of Glycosylation (CDGs) (Table 1) (Freeze et al. 2015). In the brain, N-glycans change during brain development and ageing of the organism with an increased abundance of oligomannose in aged rodents and humans (Brunngraber and Webster 1986; Simon et al. 2019; Klarić and Lauc 2022). During embryogenesis and the development of the neocortex, the position of the Golgi apparatus in neural progenitors changes as they undergo cell differentiation, influencing the distribution of N-glycans between the basal and apical side of neurons (Taverna et al. 2016). It is not known, however, whether Golgi movement within the cell is a cause or consequence of the cell differentiation process. The fate of neural stem cells is also influenced by MGAT5, which is the enzyme that adds the β1-6GlcNAc to complex N-glycans to generate large tetra-antennary structures (Fig. 1). Mgat5-deficient neural stem cells showed accelerated neuronal differentiation and the depletion of neural stem and progenitor cells (Yale et al. 2023).

Table 1.

Genes of glycosyltransferases and glycosidases involved in N- and O-glycosylation process and their association to brain disorders.

Associated pathway	Gene name	Enzymatic activity	Associated to brain disorders?	Comments	References
Synthesis of dolichol-linked precursor for N-glycans	DOLK	Adds second phosphate on dolichol-linked precursor	Yes (CDG & ASD)	Infantile spasms at age 4 months, intellectual disability and an autism spectrum disorder were identified. Transcripts are targeted by miRNAs.	(Helander et al. 2013; Mirabella et al. 2025)
Synthesis of dolichol-linked precursor for N-glycans	DPAGT1	Adds first GlcNAc on dolichol linked precursor	Yes (CDG)	Intractable epilepsy, global developmental delay/intellectual disability, and early death are commonly found in those patients	(Ng et al., 2019)
Synthesis of dolichol-linked precursor for N-glycans	ALG13	Adds Second GlcNAc on dolichol-linked precursor	Yes (CDG)	Early infantile epileptic encephalopathy and West syndrome were described	(Ng et al., 2020)
Synthesis of dolichol-linked precursor for N-glycans	ALG14	Adds Second GlcNAc on dolichol-linked precursor	Yes (CDG)	Progressive cerebral atrophy, therapy-refractory epilepsy and other symptoms were described	(Schorling et al. 2017)
Synthesis of dolichol-linked precursor for N-glycans	ALG1	Adds first mannose on dolichol-linked precursor	Yes (CDG)	Has a broad clinical spectrum ranging from mild intellectual disability to death within the first few weeks of life	(Ng et al., 2016)
Synthesis of dolichol-linked precursor for N-glycans	ALG2	Adds second mannose on dolichol-linked precursor	Yes (CDG)	Hypotonia, proximal muscle weakness, decreased tendon reflexes and other symptoms were described	(Martínez Duncker et al. 2024)
Synthesis of dolichol-linked precursor for N-glycans	ALG11	Adds fourth and fifth mannose on dolichol-linked precursor	Yes (CDG)	Developmental delay, mental retardation, strabismus convergens and seizures occur in the first year of life	(Thiel et al. 2012)
Synthesis of dolichol-linked precursor for N-glycans	RFT1	Translocates dolichol linked-linked precursor	Yes (CDG)	Associated with deafness, developmental delay, and non-specific epilepsy	(Aeby et al. 2016)
Synthesis of dolichol-linked precursor for N-glycans	MPDU1	Synthesis of mannose phosphate	Yes (CDG)	Generalized seizures at 15 months have been observed with other severe symptoms	(Schenk et al. 2001)
Synthesis of dolichol-linked precursor for N-glycans	DPM1	Synthesis of Dol-P-Man	Yes (CDG)	Patient was described with non-febrile seizures from the age of 3 weeks, global developmental delay, and severely retarded motor skills.	(Lausmann et al. 2022)
Synthesis of dolichol-linked precursor for N-glycans	DPM2	Synthesis of Dol-P-Man	Yes (CDG)	Patients had profound developmental delay, intractable epilepsy, progressive microcephaly and severe hypotonia	(Barone et al. 2012)
Synthesis of dolichol-linked precursor for N-glycans	ALG9	Addition of the seventh and ninth mannose on dolichol-linked precursor	Yes (CDG)	Failure to thrive, dysmorphic features, seizures are common symptoms found in patients	(Davis et al. 2017)
Synthesis of dolichol-linked precursor for N-glycans	ALG3	Adds sixth mannose on dolichol-linked precursor	Yes (CDG)	Authors describe clinical phenotypes of ALG3-CDG patients consistently severe and encompasses predominantly abnormalities of the brain (e.g. microcephaly, epilepsy, axial hypotonia	(Himmelreich et al. 2019)
Synthesis of dolichol-linked precursor for N-glycans	ALG6	Adds first glucose on the dolichol-linked precursor	Yes (CDG)	All described ALG6-CDG patients have epilepsy, ataxia and proximal muscle weakness	(Morava et al. 2016)
Synthesis of dolichol-linked precursor for N-glycans	ALG8	Adds second glucose on the dolichol-linked precursor	Yes (CDG)	Some patients were observed to have structural brain pathology, psychomental retardation, seizures, and other pathologies	(Höck et al. 2015)

(Continued)

Table 1.

Continued

Associated pathway	Gene name	Enzymatic activity	Associated to brain disorders?	Comments	References
Synthesis of dolichol-linked precursor for N-glycans	ALG10	Adds third glucose on the dolichol-linked precursor	No	In drosophila, essential for maintaining appropriate neuronal firing activity, healthy sleep and preventing seizures	(Gill et al. 2024)
N-glycosylation in ER	OSTA	Transfers the dolichol-linked precursor to nascent protein	Yes (CDG)	All patients have developmental delay, intellectual disability, with absent speech and seizures	(Ghosh et al. 2017)
N-glycosylation in ER	OSTB	Transfers the dolichol-linked precursor to nascent protein	Yes (CDG)	Patient was described with microcephaly, intellectual disability, seizures and epilepsy with other severe symptoms	(Shrimal et al. 2013)
Quality Control	MOGS	Removes third glucose from precursor	Yes (CDG)	Seizure was a common symptom to the described patients along with other symptoms	(Abuduxikuer et al. 2022)
Oligoman-nose N-glycans	MAN1B1	Removes first mannose from Man9	Yes (CDG)	Patients were described with intellectual disability and delayed motor and speech development with some with microcephaly	(Van Scherpenzeel et al. 2014)
Hybrid/ complex N-glycans	MGAT1	Adds β2GlcNac to α3 branch	Yes (ALZ)	No known CDG-patient. In drosophila, knock-out in the brain leads to decrease of locomotor activity and life span. Mice with neuronal deletion have locomotor deficits, paralysis and early postnatal death	(Ye and Marth 2004; Sarkar et al. 2010; Tang et al. 2023)
N-glycan processing	MAN2A1	Removes Mannose from hybrid N-glycans	Yes (SCZ)	No known CDG	(Schizophrenia Working Group of the Psychiatric Genomics Consortium 2014)
N-glycan processing	MAN2A2	Removes Mannose from hybrid N-glycans	Yes (CDG)	Speech delay and intellectual disability were described	(Mahajan et al. 2023)
N-glycan biosynthesis	MGAT2	Adds β2GlcNAc to α6 branch	Yes (CDG)	Patients have profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations.	(Poskanzer et al. 2021)
N-glycan biosynthesis	MGAT3	Adds bisected GlcNAc	No	Truncated enzymes induce neurological traits not exhibited by knockout mouse	(Bhattacharyya et al. 2002)
N-glycan biosynthesis	MGAT4A	Adds β4GlcNac on α3 branch	Yes (SCZ)	Decreased protein expression found in 12 patients with SCZ	(Kippe et al. 2015)
N-glycan biosynthesis	MGAT4B	Adds β4GlcNac on α3 branch	Yes (ALZ)	Expression regulated by transcription factors and miRNAs	(Tang et al. 2023)
N-glycan biosynthesis	MGAT5	Adds β6 GlcNAc on α6 branch	No	Loss of MGAT5 favors neuron differentiation	(Yale et al. 2023)
N-glycan biosynthesis	B3GAT1	Synthesis of HNK-1 epitope	Yes (SCZ)	Important for long-term potentiation and synaptic plasticity	(Morita et al. 2009; Schizophrenia Working Group of the Psychiatric Genomics Consortium 2014)
N-glycan biosynthesis	B3GNT8	Role in the elongation of specific branch structures of multi-antennary N-glycans	Yes (SCZ)	Decreased protein expression found in 12 patients with SCZ	(Kippe et al. 2015)
N- and O-glycan biosynthesis	FUT1	Synthesis of H-epitope	No	Knock out leads to impaired development of the olfactory nerve and glomerular layers of the OB	(St John et al. 2006)

(Continued)

Table 1.

Continued

Associated pathway	Gene name	Enzymatic activity	Associated to brain disorders?	Comments	References
N-glycan biosynthesis	FUT8	Core fucose	Yes (CDG)	Microcephaly, intellectual disability and seizures were described in all patients of the study	(Ng et al. 2018)
N- and O-glycan biosynthesis	FUT9	Lewis X epitope	Yes (SCZ)	Causes abnormal neural development in FUT9-deficient mouse	(Schizophrenia Working Group of the Psychiatric Genomics Consortium 2014; Abdullah et al. 2022)
N- and O-glycan biosynthesis	ST3GAL3	Synthesis of sialyl Lewis X and sialyl Lewis a antigen	Yes (CDG & SCZ)	West syndrome with mental retardation described in most patients	(Edvardson et al. 2013; Trubetskoy et al. 2022)
N- and O-glycan biosynthesis	ST3GAL4	Synthesis of sialyl Lewis and Sda antigens	No	Knockout mice have increased anxiety and depression related behaviors	(Okabe et al. 2001; Srimontri et al. 2014)
N- and O-glycan biosynthesis	B4GALT1	Adds β4Gal to GlcNAc	Yes (CDG, ALZ & PD)	Neurological symptoms go from none to Dandy-Walker malformation and mental retardation	(Hansske et al. 2002; Guillard et al. 2011; Schneider and Singh 2022; Tang et al. 2023)
N- and O-glycan biosynthesis	B4GALT2	Adds β4Gal to GlcNAc	No	Impaired spatial learning/memory and motor coordination/learning.	(Yoshihara et al. 2009)
N- and O-glycan biosynthesis	B3GALT2	Adds β3Gal to GlcNAcβ1	Yes (PD)	Gene expression is decreased	(Schneider and Singh 2022)
N- and O-glycan biosynthesis	ST8SIA2	Synthesis of polysialic acid structures	Yes (PD, SCZ, ASD)	Promote axonal targeting in hippocampus	(Angata et al. 2004; Arai et al. 2006; Anney et al. 2010; Schneider and Singh 2022)
O-GalNAc glycans	GALNT2	Synthesis of Tn antigen	Yes (CDG & ASD)	Exome sequencing identified a variant associated with intellectual disability which is a symptom of CDG patients with epilepsy and other symptoms	(Reuter et al. 2017; Zilmer et al. 2020; Mirabella et al. 2025)
O-GalNAc glycans	GALNT9	Synthesis of Tn antigen	Yes (ALZ & ASD)	Gene-network analysis identified GALNT9 associated with ASD. Decreased gene expression was found in ALZ	(van der Zwaag et al. 2009; Tang et al. 2023)
O-GalNAc glycans	GALNT10	Synthesis of Tn antigen	Yes (SCZ and ALZ)	GWAS identified a variant associated with SCZ. Gene expression increased in ALZ	(Schizophrenia Working Group of the Psychiatric Genomics Consortium 2014; Tang et al. 2023)
O-GalNAc glycans	GALNT11	Synthesis of Tn antigen	Yes (ALZ)	Decreased gene expression	(Tang et al. 2023)
O-GalNAc glycans	GALNT13	Synthesis of Tn antigen	Yes (ALZ)	Decreased gene expression	(Chen et al. 2022; Tang et al. 2023)
O-GalNAc glycans	GALNT14	Synthesis of Tn antigen	Yes (ALZ)	Decreased gene expression	(Tang et al. 2023)
O-GalNAc glycans	GALNT15	Synthesis of Tn antigen	Yes (ALZ)	Increased gene expression	(Tang et al. 2023)
O-GalNAc glycans	GALNT17	Synthesis of Tn antigen	Yes (ALZ)	Knockout mice exhibit developmental neuropathology along with abnormal activity, coordination, and social interaction deficits. Decreased gene expression in ALZ	(Tang et al. 2023)

(Continued)

Table 1.

Continued

Associated pathway	Gene name	Enzymatic activity	Associated to brain disorders?	Comments	References
O-GalNAc glycans	T-synthase	Synthesis of T antigen	Yes (ALZ)	Associated with blood-brain barrier integrity	(Shi et al. 2025)
O-GalNAc glycans	Cosmc	Folding of T-synthase	Yes (ALZ & CDG)	COSMC-coding mutation associated with reduction in T-synthase activity in ALZ. CDG patients have intellectual disability.	(Gollamudi et al. 2020; Erger et al. 2023)
O-GalNAc glycans	ST6GALNAC2	Adds α2,6-linked sialic acid to GalNAc of core 1 O-glycans	Yes (ALZ)	Upregulated gene	(Tang et al. 2023)
O-GalNAc glycans	ST6GALNAC3	Adds α2,6-linked sialic acid to GalNAc of core 1 O-glycans	Yes (ALZ)	Upregulated gene	(Tang et al. 2023)
N-glycan degradation	NGLY1		Yes (CDDG)	Patients have developmental delay and intellectual disability in the mild to profound range	(Lam et al. 1993)
N-glycan degradation	MAN2C1		Yes (CDDG)	Brain structure abnormalities and variable degrees of intellectual disability were observed in patients	(Maia et al. 2022)
Lysosomal N-glycan degradation	MAN2B2	Cleaves α-1,6-mannose residue during lysosomal degradation of N-glycans	Yes (CDDG)	Patient described with speech delay and other symptoms	(Tian et al. 2023)

A brief description of the known enzymatic activity is provided and the brain disorder they are associated with. CDG: Congenital Disorder of Glycosylation, ALZ: Alzheimer’s disease, SCZ: Schizophrenia, PD: Parkinson’s disease, ASD: Autism spectrum disorder. If the gene is not associated with a human brain disorder, but is associated with a brain phenotype in animal models, we provide a short description of the effect.

The high abundance of Man5 in the mammalian brain might suggest that MGAT1 which modifies Man5 to generate hybrid and complex N-glycans, has only a minor role in the brain (Fig. 1). However, Mgat1-deletion in mice leads to midgestational death, with embryos showing severe growth delay that was particularly noticeable in the neural tissues, suggesting that hybrid/complex N-glycans are essential for brain development (Ioffe and Stanley 1994). Interestingly, Mgat2-KO is not lethal, suggesting that the single α1,3 branch, but not the α1,6 branch, can compensate partly for the loss of complex N-glycans during the brain development (Fig. 1) (Ye and Marth 2004).

As mentioned above, the Tn antigen was detected in the brain of adult mice using glycoproteomics technologies, although its expression is not observed using lectins (Suttapitugsakul et al. 2023; Noel et al. 2025). Its expression has been detected in the developing brain using an anti-Tn antibody up to 3 weeks after birth, after which Tn antigen had essentially disappeared (Akita et al. 2001). Interestingly, preventing extension of the core 1 O-glycan structure (T antigen) by targeted deletion of Cosmc (C1galt1c1) and consequent loss of T-synthase enzyme activity (C1galt1) in neurons did not result in an overt phenotype in adult mice (Fig. 1) (Noel et al. 2025). In drosophila, where the Tn antigen is abundant, with 2 GalNAcTs responsible for its synthesis, it is required for synaptic assembly, neurotransmission strength, and expression of the synaptic Position Specific 2 (αPS2) integrin receptor (Itoh and Nishihara 2021). Expression of core 1 O-glycans has recently been implicated in the integrity of the blood brain barrier of mice, with their expression decreasing in aged mice, and the phenotype could be partly rescued by expression of introduced T-synthase (C1galt1) (Shi et al. 2025). Though most of these glycans appear to be localized to the intraluminal vascular space and expressed by endothelial cells, in contrast to other studies of core 1 O-glycans in the brain parenchyma.

• Glycosylation and neuronal functions

Glycosylation is often studied in the context of neuro-pathologies and major discoveries have already been made. For instance, protein glycosylation is crucial for neurite outgrowth (Gouveia et al. 2012), axonal guidance (Bonfanti 2006), synaptogenesis (Parkinson et al. 2013), neural excitability (Baycin-Hizal et al. 2014), and neuro-transmission (Scott and Panin 2014) and well described in two reviews (Scott and Panin 2014; Conroy et al. 2021).

How does glycosylation of the glycoproteins function within these normal biological processes? Unfortunately, very little mechanistic information is available. For instance, in the development of dendrites, complex N-glycans are essential as shown in zebrafish where deletion of the Mgat1b gene results in a decrease of hybrid and complex N-glycans, which led to motor neurons in the spinal cords with less dendrites compared to WT animals (Hall et al. 2023; Hatchett et al. 2024). In C. elegans, the Golgi α-mannosidase (AMAN-2), essential for the synthesis of complex N-glycans, also leads to abnormal dendrite patterning when individual expressed loss of function alleles (Rahman et al. 2022). However, treatment of neurons with kifunensine, a known inhibitor for the synthesis of complex N-glycans, did not influence neuron morphology in vitro, suggesting that chemical inhibition was less efficient than genetics deletion. However, in this study, tunicamycin, a strong inhibitor of the N-glycosylation pathway, altered dendritic development. Thus, N-glycosylation is important for the formation of dendrites and that complex-type N-glycans are necessary (Hanus et al. 2016).

On a protein level, glycosylation is essential for the proper behavior of proteins in neurons and is proposed to involve several mechanisms. High mannose N-glycans strengthen the cell-cell adhesion and cell migration properties in CHO cells (Hall et al. 2016). Therefore, it is not surprising to find such N-glycans being present in cell-cell adhesion proteins (Hanus et al. 2016). The presence of high-mannose N-glycans at the cell surface, however, has been thought to be uncommon, based on our current knowledge of the secretory pathway (Colley et al. 2022). Interestingly, high mannose N-glycans at the cell surface of neurons may be partially generated through an unconventional pathway that bypasses the Golgi apparatus (Hanus et al. 2016). Nascent glycoproteins can be synthesized and exit the ER through endoplasmic reticulum exit site (ERES) in the dendrites and join the cell surface through a Golgi-independent secretory route via the ERGIC compartment and recycling endosomes (Bowen et al. 2017; Kennedy and Hanus 2019).

The composition of the glycan composition at the cell surface can also be modulated by neuronal excitation. This has been demonstrated for sialylation that is modulated after 5 seconds of depolarization of the neurons, with an increase or decreased sialylation dependent of the protein after seizures in a mouse model (Okabe et al. 2001; Boll et al. 2020). What causes the rapidity of such changes? Could neosynthesized proteins carry more sialylated glycans after exiting the Golgi apparatus? To understand this process, one study has focused on the nicotinic receptor α4β2R. Under basal conditions, the nicotinic receptor carries high mannose N-glycans at the cell surface, but when the neurons are incubated with nicotine to promote depolarization, the receptor is internalized, transported into Golgi satellites, where the glycan is converted to complex N-glycan carrying sialic acid and brought back to the cell surface (Torre and Steward 1996; Mikhaylova et al. 2016; Govind et al. 2021). This is a prime example of the use of an unconventional pathway to directly modulate the composition of the glycans attached to proteins at the cell surface by neuronal activity (Mikhaylova et al. 2016; Kennedy and Hanus 2019). In return, this glycan modification increased the amplitude of the current generated after nicotine treatment (Fig. 3).

Fig. 3.

Internalization and modification of receptor glycans on dendritic surface during increased neuronal activity. During basal activity, dendrites display a large amount of high-mannose glycans on their cell surface receptors. The activation of a neuron by a stimulus leads to the internalization of the protein from the cell surface. The resulting endosome fuses with a Golgi satellite containing the necessary glycosylation enzymes. Within the fused vesicle, the glycans are modified into complex sialylated forms, and the receptor, now carrying complex N-glycans, is transported back to the cell membrane.

O-glycans can also be regulated by neuronal excitation. PNA, the lectin with high affinity towards the core 1 O-glycan disaccharide, exhibits increased staining after incubation of a brain tissue with potassium, which depolarizes neurons (Minami et al. 2017). O-glycoproteomics analysis of secreted glycoproteins have been performed on a model cell line (SH-SY5Y) after depolarization. This approach showed that glycoproteins associated with dense core granules, key organelle for secretion of hormones and neuropeptides, were significantly more O-glycosylated after stimulation (Madsen et al. 2025), but the underlying mechanism is not known. Interestingly, the internalization and modifications of glycans on proteins is also applicable to O-glycoproteins such as APP in endothelial cells, which can be internalized from the cell surface to be sialylated (Tachida et al. 2023).

• Glycosylation and neuro-disorders

Many deficiencies in glycosylation lead to CDG and neurological phenotypes. Therefore, these diseases can be used to understand the importance of N- and O-glycans in the brain (Sparks and Krasnewich 1993; Freeze et al. 2015; Paprocka et al. 2021). In addition, there is an increasing number of studies demonstrating that glycans are also involved in the development of more common neuro-disorders such as schizophrenia, Alzheimer’s disease and Parkinson’s disease (Mealer et al. 2020b; Yang et al. 2023) (Table 1), although most of the studies focus on the change of glycosylation in the serum or cerebrospinal fluid, but not directly within the brain. It would be interesting to study the glycome of the post-mortem brain of patients with brain disorders, as done already with schizophrenia (Bauer et al. 2010; Tucholski et al. 2013; Williams et al. 2020).

It is interesting to note that most enzymes involved in the first step of N-glycosylation are associated with strong phenotypes such as the development of CDG, and the genes involved in the synthesis of complex N-glycans also appear to be associated with schizophrenia, Alzheimer’s disease and Parkinson’s disease (Table 1). However, despite an abundant literature describing the importance of complex N-glycans in neuronal functions, few describe the importance of high mannose N-glycans. It would therefore be an interesting field of research to understand the functions of high mannose N-glycans in the brain, beside their essential role for creating complex N-glycans.

Sialylated N-glycans are important for brain functions (Sato and Kitajima 2013; Klaus et al. 2020), but the relationship between sialic acid linkage and brain abnormalities still needs to be deciphered. St6gal1-KO in mouse led to a loss of SNA binding, but no phenotype was described in a mouse model (Hennet et al. 1998; Makarava et al. 2022). This is probably due to the highest abundance of α2,3 sialic acid in the adult brain (Klarić et al. 2023). Targeting this sialic acid linkage in the brain leads to more severe brain phenotypes (Sturgill et al. 2012), but most of these phenotypes are probably due to impacts on ganglioside biosynthesis, making difficult to understand the role of α2,3 sialic acid on glycoproteins. One of the most studied sialic acid linkages in the brain is the α2,8 sialylation which is generated by the enzymes ST8SIA2 and ST8SIA4 (Ong et al. 1998). This linkage is important for brain development and learning, mainly through the NCAM protein and the enzyme ST8SIA2, which has been associated with hippocampal axonal targeting and the development of schizophrenia (Becker et al. 1996; Angata et al. 2004; Arai et al. 2006; Gascon et al. 2010).

In CDG, the most common symptoms are neurodevelopmental delays, brain malformation, epilepsy and intellectual disability (Freeze et al. 2015), indicating the essential role of glycosylation for proper formation of the brain and proper functions of neurons. Although glycosylation is also described on other cell types, they are not reviewed here.

Another family of diseases that also impact glycosylation are the Congenital disorders of deglycosylation (CDDG) (Lam et al. 2017). They are characterized by a deficiency in the degradation of free oligosaccharides (fOS) found in the cytosol (Maia et al. 2022). The fOS are usually generated during the endoplasmic reticulum-associated degradation (ERAD pathway) and/or the hydrolysis of lipid-linked oligosaccharide (LLO pathway) and later degraded by a series of enzymes (Fig. 4). Patients with a deficiency in the NGLY1, enzyme that separates the N-glycans from the protein, also leads to strong neurological disorders (Lam et al. 1993). The following enzymes involved in their degradation are also associated with neuro-disorders such as MAN2C1, and MAN2B1, two mannosidases present either in the cytosol or in lysosome, respectively. Patients with MAN2C1-CDG exhibit strong brain malformation and patients with MAN2B1-CDG show reduced myelination and ganglioside accumulation in lysosomes, highlighting the importance of fOS catabolism in the development of neuro-disorders (Crawley and Walkley 2007; Maia et al. 2022) (Fig. 4) (Table 1). However, the link between fOS degradation and brain disorders is still unclear.

Fig. 4.

Biosynthesis and degradation of N-glycans in eukaryotic cells. The precursor of N-glycans is transferred from dolichol pyrophosphate to the nascent protein. Later, the glycoprotein with the Man9 interacts with the proteins Calnexin and calreticulin to determine if it is properly folded. If not, the glycoprotein is sent for degradation via the ERAD pathway where the N-glycans is dissociated from the protein and further degraded by the successive action of several glycosidases in the cytosol and lysosomes. On the opposite, the glycan is further processed and the glycoprotein transported into the Golgi apparatus to create Man5 in the cis-Golgi. Man5 is then further processed by a series of glycosyltransferases throughout the secretory pathway to create hybrid and complex N-glycans. O-GalNAc glycans biosynthesis generally starts in the cis-Golgi for further elongation in the different saccules of the Golgi apparatus. At the end, most N- and O-glycoproteins reach the cell surface to perform their biological functions.

Recently, genes of glycosylation are being increasingly identified as associated with the development of schizophrenia, Alzheimer’s disease and Parkinson’s disease. Although different studies do not always identify the same genes associated with their development, making difficult the comprehension of the pathways associated with the different diseases difficult. However, the involvement of glycosylation in the development of these diseases is clearly evident (Table 1). One area under intensive study is the development of schizophrenia. Schizophrenia is a chronic mental disorder severely impacting how an individual behaves, thinks and perceives his environment. It is generally characterized by an excessive degradation of the synapses called over-pruning (Yilmaz et al. 2021; Chafee and Averbeck 2022). Although genes of glycosylation have been unequivocally associated with the development of the disease, the mechanisms by which glycosylation modulates the development of the disease is not clear (Mealer et al. 2020b). There is evidence that sialylated glycans are important for synaptic pruning (Puigdellívol et al. 2020; Klaus et al. 2021) and modifications of glycosylation profile in patients could exert pathological phenotypes. This has been demonstrated with the variant rs13107325 of the SLC39A8 gene associated with the development of schizophrenia (Schizophrenia Working Group of the Psychiatric Genomics Consortium 2014). SLC39A8 is a manganese transporter that is known to impact the concentration of manganese in the brain of the patients (Mealer et al. 2020a). Manganese is an essential co-factor for numerous glycosyltransferases containing the DxD motif (Aspartate-X-Aspartate) (Wiggins and Munro 1998; Durin et al. 2023). Absence of this motif results in an absence of activity from several enzymes and sometimes to the development of CDG (Foulquier and Legrand 2020), highlighting the importance of manganese in the glycosylation pathway. It is therefore not surprising to observe a different N-glycan profile in the brain of mice with a variant of Slc39a8 (Mealer et al. 2022). However, the molecular mechanisms leading to the development of the disease, in the context of changes of glycosylation, still need to be clarified.

Modifications in O-GalNAc glycosylation have been mostly associated with the development of Alzheimer’s disease. Cosmc, unique chaperone of the enzyme T-synthase and essential to elongate the Tn antigen, has been associated with the development of the disease (Gollamudi et al. 2020) as well as the gene expression of several GALNTs responsible for synthesizing the Tn antigen (Tang et al. 2023). One current hypothesis is that O-glycosylation sites on the APP (Amyloid-beta precursor protein) protect it from proteolytic cleavage, preventing the formation of amyloid plaques and the development of Alzheimer’s disease (Akasaka-Manya and Manya 2020).

In the context of genetic evidence linking gene variants to the development of a disease, it is important to note that not only the glycosyltransferases, but also the glycoproteins enzymatically modified by such variants, can also be associated with the development of the disease. This is particularly relevant in the context of glycosylation which cannot be purely predicted by genetics, and the modified glycoproteins are typically not detected through Genome-Wide Association Studies (GWAS) (Stanley 2024). One way to address this issue is to perform glycoproteomics to identify the changes occurring on certain proteins compared to healthy patients on post-mortem brains (Suttapitugsakul et al. 2022). The other approach is to produce and purify the mutated version of the enzyme and exogenously label the proteins that are modified by the enzyme using a biotinylated donor monosaccharide (Noel et al. 2024). This would allow the identification of the glycoproteins modified by the enzyme and better understand the glycosylation changes occurring on these glycoproteins in the context of disorders. These can be supported by spatial multiplexing using tissue RNA, labeled antibodies targeting glycans, as well as multiplexing using MALDI-MSI to profile glycans combined with co-detecting by indexing (CODEX) to provide cell identification from the exact same tissue section where the glycans are detected (Veličković et al. 2025). Altogether, these approaches can give a better overview of the glycosylation changes occurring on the proteins when a variant leading to a disease is expressed in a patient, opening a better understanding of the development of brain disorders.

Conclusion

Glycans in the brain are becoming more studied and revealed unexpected features regarding their structures, localization in the brain and their involvement in numerous brain functions. Glycosylation is emerging as a crucial factor to understanding the development of brain disorders. However, the comprehension of its role is still lacking. The functional understanding of the roles of glycans in the brain represents one of the next challenges in the field.