Research
Biosynthesis and functions of N-glycan branches
Fig. 1 Branched structures of N-glycans, their biosynthetic enzymes and implications in disease.
We work on expression, functions, and disease-involvement of N-glycans attached to proteins. Particularly, we focus on branched structures of glycans.
N-glycosylation is fundamental protein modifications with large structural variations (Fig. 1). Moreover, formation of each branch is protein-selective and occurs on limited proteins. However, it is poorly understood at molecular level how each branch functions and how such protein selectivity is accomplished.
We tackle these questions by focusing on biosynthetic enzymes (glycosyltransferases). We expect that our results will finally lead to development of new therapeutics against glycan-related diseases.
Bisecting GlcNAc, an accelerator of Alzheimer’s disease
Fig. 2. (Upper) Structure and biosynthetic enzyme of bisecting GlcNAc. (Lower) Deposition of amyloid-β (Aβ) in brain of an AD model mouse. The Aβ level is reduced in bisecting GlcNAc-deficient mice.
(Reference：Kizuka et al., EMBO Mol. Med., 2015)
Fig. 3. 3D structure of the upper branch of N-glycan is changed by the presence of bisecting GlcNAc. This suppresses further extension and branching of N-glycans.
(Reference：Nakano et al., Mol. Cell Proteomics, 2019)
Bisecting GlcNAc is one of N-glycan branches (Fig. 2, upper). We have revealed that this glycan structure is profoundly involved in development and progression of Alzheimer’s disease (AD). AD is known to be caused by deposition of Amyloid-β (Aβ) peptide in brain, and we found that bisecting GlcNAc-deficient mice have reduced Aβ deposition and improved AD pathology (Fig. 2, lower). As a mechanism, we found that Aβ-producing enzyme BACE1 is modified with bisecting GlcNAc and that BACE1 function is positively regulated by this glycan modification. Furthermore, the levels of bisecting GlcNAc are increased in AD patients, suggesting its involvement in AD progression in human. However, we still do not know how BACE1 function is regulated by bisecting GlcNAc at a molecular level. We now examine this mechanism and analyze the structure of the biosynthetic enzyme of bisecting GlcNAc, MGAT3 (GnT-III).
We also examine the physiological functions of bisecting GlcNAc. By looking at 3D structure of bisecting GlcNAc-containing glycans (Fig. 3, upper), we found that the upper branch in N-glycan is pointed to the opposite direction by the presence of bisecting GlcNAc, compared with other N-glycans without bisecting GlcNAc (Fig. 3, lower). This results in suppression of further extension and branching of N-glycans which could usually occur in non-bisected glycans. Therefore, bisecting GlcNAc has a suppressive role for N-glycan biosynthesis.
We now investigate the functions of bisecting GlcNAc in neurons and kidney where bisecting GlcNAc is highly expressed and recently found that bisecting GlcNAc plays an important role in maintaining the balance of body fluid in kidney. We also examine the regulation mechanisms of MGAT3 functions.
β1,6-branch, an accelerator of cancer
Fig. 4. (Upper) Structure and biosynthetic enzyme of β1,6 -branch. (Lower left) Crystal structure of MGAT5. (Lower right) Docking model of MGAT5 and a glycoprotein substrate.
(Reference：Nagae et al., Nat. Commun., 2018)
(Reference：Osuka et al., J. Biol. Chem., 2022)
Fig. 5. Selection of substrate glycoproteins of MGAT5 in kidney
(Reference：Osuka et al., iScience, 2025)
We also focus on other branches in N-glycan which are highly involved in cancer. Particularly, β1,6-branch (Fig. 4, upper) synthesized by MGAT5 (GnT-V) enzyme is known to promote cancer growth and metastasis. Therefore, it has been considered as a promising drug target for a long time.
We for the first time revealed the 3D crystal structures of MGAT5 in collaboration with an expert of structural biology (Dr. Masamichi Nagae) (Fig. 4, lower left). This provided us with basic information regarding how MGAT5 recognizes substrate glycans and biosynthesizes β1,6-branch. Furthermore, the clarification of the structure will allow us to design specific inhibitors of MGAT5. We Indeed try to develop MGAT5 inhibitors in collaboration with organic chemists.
However, the detailed mechanisms are still poorly understood how MGAT5 selects target glycoproteins and works in living cells. To solve this question, we recently found that N domain in MGAT5 (Fig. 4, lower right) is important. Mutant MGAT5 lacking N domain almost lacks activity toward glycoproteins while it almost fully retains activity toward glycans. This suggests that MGAT5 recognizes its substrate proteins through N domain.
Recently, to investigate how MGAT5 selects proteins for glycosylation, we examined mouse kidney to identify the proteins modified by MGAT5. We found that two proteins, ANPEP and Meprinα, located on the apical side of proximal tubules are the major substrate proteins of MGAT5 (Fig. 5). Our findings indicate that, in the mouse kidney, whether MGAT5-mediated glycosylation occurs is primarily determined by the transport of proteins to the apical side and by the three-dimensional structures of the substrate proteins.
We now work on the mechanisms by which MGAT5 function is regulated in cells.
β1,4-branch related to diabetes
Fig. 6. (Upper) Structure and biosynthetic enzyme of β1,4 -branch. (Lower) MGAT4A and B have a glycan-binding lectin domain other than the catalytic domain. Using the lectin domain, MGAT4A and B recognize their substrate proteins. In addition, glycan attached to the enzyme itself self-regulates the activity by binding to and inhibiting the lectin domain.
(Reference：Nagae et al., Commun. Biol., 2022)
(Reference：Osada et al., J. Biol. Chem., 2022)
(Reference：Osada et al., iScience, 2024)
Another topic is β1,4-branch, that is related to diabetes. This branch structure is biosynthesized by either one of two similar enzymes, MGAT4A (GnT-IVa) or MGAT4B (GnT-IVb) (Fig. 6, upper). As MGAT4A-deficient mice show impairments of pancreatic functions, leading to diabetic phenotypes with high blood glucose levels, this branch is expected to be one of drug targets for diabetes. On the other hand, it remains largely unclear how MGAT4A and 4B synthesize glycans in cells and how functions of two similar enzymes, MGAT4A and 4B, are different.
We recently found that MGAT4A and 4B have a unique lectin domain which is not present in other related glycosyltransferases. Moreover, the lectin domain was found to bind with specific glycans, which is required for enzyme activity of the catalytic domain (Fig. 6, lower).Furthermore, we found that MGAT4A and MGAT4B possess their own glycans within the lectin domain. Since these glycans inhibit the function of the lectin domain in a glycan structure-dependent manner, MGAT4 self-regulates its activity through its own glycan. No other N-glycan biosynthetic enzyme has been known to possess such a self-regulatory mechanism, MGAT4A and MGAT4B synthesize glycans in a novel manner.
Furthermore, we recently revealed that MGAT4 family has not only A and B but also C-G members, and that the number of these genes and their enzymatic activity vary among species. Therefore, MGAT4 plays a crucial role in synthesizing species-specific N-glycan branches.
Core fucose, a versatile sugar structure
Fig. 7. (Upper) Structure and biosynthetic enzyme of core fucose. (Lower left) Crystal structure of FUT8. FUT8 has unique SH3 domain and stem region other than catalytic domain. (Lower right) FUT8 is cleaved by SPP and SPPL3 and secreted. This cleavage alters substrate protein selectivity of FUT8.
(Reference：Tomida et al., J. Biol. Chem., 2020)
(Reference：Tomida et al., J. Biol. Chem., 2022)
(Reference：Tomida et al., J. Biol. Chem., 2026)
We also work on “core fucose” that is a fucose branch on the core part of N-glycan. Core fucose is biosynthesized by an enzyme called FUT8 (Fig. 7, upper), and is known to be involved in chronic obstructive pulmonary disease (COPD), as FUT8-deficient mice show emphysema phenotype. In addition, core fucose was reported to promote lung cancer and melanoma. Furthermore, a core fucose-targeted technique is clinically used in which anti-tumor activity of antibody drug is drastically elevated by removing core fucose from antibody drug. Therefore, core fucose is highly related to diagnosis and therapy of these diseases.
On the other hand, the regulation mechanisms of FUT8 functions remain to be clarified. We examine the mechanisms of how FUT8 functions are regulated in cells, by focusing on 3D structures and regulatory proteins. FUT8 3D structure is already known, and it has unique SH3 domain near the catalytic domain (Fig. 7, lower left). We recently found that the SH3 domain is necessary for the enzyme activity of FUT8 and interaction with RPN1 protein. We also clarified that the stem region of FUT8 is required for its oligomerization.
In addition, we discovered that FUT8 is cleaved by SPP and SPPL3 proteases and secreted (Fig. 7, lower right). This cleavage impacts substrate protein selectivity of FUT8, indicating that core fucosylation activity of FUT8 in cells is regulated by its cleavage and secretion.
Projects on other glycosyltransferases
O-Mannose glycans associated with brain functions
Fig. 8. A brain-specific glycosyltransferase, MGAT5B,
forms the branched structure of O-mannose glycans. The branched structure is recognized by the enzymes for keratan sulfate biosynthesis, leading to the efficient synthesis of keratan sulfate in O-mannose glycans in brain.
(Reference：Itoh et al., J. Biol. Chem., 2026)
We work on a brain-specific glycosyltransferase MGAT5B (GnT-IX), which is associated with brain functions and disorders. MGAT5B synthesizes a branch in O-mannose glycans and has been shown to be involved in demyelinating diseases and brain tumors.
We demonstrated that the MGAT5B-produced branch is recognized by other glycosylation enzymes, leading to the efficient formation of a glycan structure called keratan sulfate in the branched O-mannose glycans (Fig. 8). Currently, we analyze MGAT5B-deficient mice to investigate the physiological functions of branched O-mannose glycans in the brain.
LacdiNAc, a regulator of protein stability in blood
Fig. 9. B4GALNT3 is a glycosyltransferase synthesizing a glycan called LacdiNAc. We recently unveiled that this enzymes has a lectin domain required for the enzyme activity.
(Reference：Tokoro et al., J. Biol. Chem., 2024)
(Reference：Tokoro et al., J. Biol. Chem., 2026)
We investigate the unique structure and functions of a glycosyltransferase, B4GALNT3. It synthesizes a glycan designated as LacdiNAc, which regulates the stability of proteins in the blood. Structural analysis of B4GALNT3 revealed that, in addition to a catalytic domain, it possesses a lectin domain that recognizes the certain glycan structures. We found that this lectin domain is necessary for the action of B4GALNT3 toward glycoproteins and that it binds to sulfated glycans (Fig. 9). Furthermore, we found that once the LacdiNAc structure is formed by B4GALNT3, the biosynthesis of terminal structures such as sialic acid and HNK-1 is suppressed. This indicates that B4GALNT3 functions to inhibit glycan maturation and simplify the glycan structures. We currently examine possible associations of B4GALNT3 with several diseases.
Regulation mechanisms of glycosyltransferase activity
We also work on various mechanisms by which glycosyltransferase activity is regulated in cells. In more detail, we focus on subcellular localization, posttranslational modifications, complex formation, degradation, and secretion, of glycosyltransferases.
Comprehensive analysis of activity of all human glycosyltransferases (HGA)
Our group participates in the Human Glycome Atlas Project (HGA), one of the national projects funded by MEXT’s Program for Promoting Large-scale Academic Frontier Projects. In this project, we are collecting all human glycosyltransferases and acquiring data on their enzymatic activities as foundational information for constructing a glycan biosynthesis atlas. By combining the obtained enzyme activity data with other information within the project, we aim to improve the accuracy of simulators that predict glycan expression from gene expression in cells.
Other research projects
New glycan probe and inhibitor
Fig. 10. Detection of glycans with a new fucose analog and development of a new fucosylation inhibitor.
(Reference：Kizuka et al., Cell Chem. Biol., 2016)
(Reference：Kizuka et al., Cell Chem. Biol., 2017)
It is essential for a glycobiology study to detect a glycan of interest. Although common detection methods use antibodies or lectins, we focus on a different method using a combination of sugar analog and click chemistry (Fig. 10, left). We aim at developing new drug targets and disease biomarkers by using these new probes.
In addition, for therapeutic purpose, we develop inhibitory compounds for glycan functions. So far, we have developed a fucosylation inhibitor which suppressed cancer cell invasion (Fig. 10, right). Furthermore, in collaboration with Dr. Hidenori Tanaka in Gifu Univ., we developed inhibitor candidates for MGAT5 (Vibhute et al., BBA Gen. Subj., 2022) and an inhibitor for glycosaminoglycan biosynthesis.
Small extracellular vesicle (sEV) and glycan
Collaborative work with Kenichi Suzuki Lab (iGCORE) on expression and functions of glycans in sEVs, including exosomes. sEVs draw attentions of many researchers, as they function for cell-cell communications. We have demonstrated that a caner-associated glycosyltransferase MGAT5 is present in sEVs derived from cancer cells and is taken up by recipient cells (Reference：Hirata et al., J. Biol. Chem., 2023). Furthermore, we found that HNK-1, a brain-specific glycan, and its biosynthetic enzyme B3GAT1 (GlcAT-P) are also present in sEVs, and that HNK-1 glycan is similarly transferred between cells via sEVs (Reference：Tokoro et al., J. Biol. Chem., 2026).
GPI (Glycosyl Phosphatidyl Inositol) anchor
We work on biosynthesis and functions of GPI, a unique glycolipid attached to proteins, particularly focusing on prion and mental diseases (Reference: Hirata et al., J. Biol. Chem., 2022). We also work on intracellular trafficking of GPI anchored proteins.
Arginine methylation and glycan
Collaborative work with Assistant Dr. Misuzu Hashimoto (Faculty of Applied Biol. Sci., Gifu Univ.). We work on relationships between glycans/glycoproteins and arginine methylation which is also known as a posttranslational modification of proteins (Reference: Hashimoto et al., BBA Gen. Subj., 2020).
Biosynthetic regulation of HNK-1 (Human Natural Killer-1) glycan
We study on regulation mechanisms of HNK-1 glycan that is specifically expressed in brain and required for learning and memory functions. We have found that MGAT3 suppresses HNK-1 biosynthesis in tissue-dependent manners (Reference: Kawade et al., Molecules, 2021).