論文 - 藤本　ゆかり

Nitration of GlcCer and related complex lipids enhances CD1d bind-ing affinity and modulates immune responses via lipid antigen presentation to NKT cells

Sueyoshi, K., Kawate-Horiuchi, N., Ueki, K., Nozawa, R., Matsumaru, T., Fujimoto, Y.

ChemistryEurope 2025年09月

研究論文（学術雑誌）, 責任著者, 査読有り

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in press

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Phleum pratense pollen-derived di-galactosyldiacylglycerols promote pro-allergic responses in mice

González Roldán N., Lunding L.P., Fujimoto Y., Düpow S., Schwudke D., Wegmann M., Duda K.A.

Frontiers in Immunology 16 2025年

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Introduction: Grass pollen triggers nearly 30% of bronchial allergic asthma cases. While most Q8 research focuses on pollen allergens, pollen lipids may also influence allergic reactions. Previous studies demonstrated that Timothy grass (TG, Phleum pratense) lipids, such as phytoprostanes, can activate immune cells, promoting pro-allergic responses. However, the role of water-insoluble pollen glycolipids in allergic airway inflammation remains unclear. Thus, this study aimed to isolate and characterize glycolipids from TG pollen and evaluate their bioactivity in allergic airway inflammation. Methods: Lipids were extracted from the water-insoluble pollen fraction, separated by silica gel, and fractionated by HPLC. GC-MS, HR ESI-MS, and NMR confirmed the presence of di-galactosyldiacylglycerol (DGDG). The biological activity of fractions containing DGDG (DGDG-3 and DGDG-4) and synthetic DGDG variants was tested in vitro in murine and human cell systems and in vivo in mice. Results: Fraction 4 induced strong proliferation of murine NKT cells and upregulated CD69 expression in human NKT cells. Synthetic DGDG variants (DGDG-1, DGDG-2, and DGDG-3) with defined acylation profiles stimulated robust NKT-cell proliferation, with DGDG-2 and DGDG-3 increasing IL-13 production, one of the key Th2 cytokines. In vivo, only these variants caused lung inflammation marked by eosinophil infiltration but did not increase airway resistance. Discussion: This study reveals for the first time the structure-dependent role of DGDG of TG pollen grains in immune cell recognition in the context of allergic inflammation. Our data may pave the way for therapies targeting lipid components in combination with protein allergens.

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Solid-Phase Oligosaccharide Synthesis with Highly Complexed Peptidoglycan Fragments

Kadonaga Y., Wang N., Shimoyama A., Fujimoto Y., Fukase K.

Molecules 30 （ 13 ）  2025年07月

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Peptidoglycan (PGN) is a component of bacterial cell walls; its fragments are recognized by the cytoplasmic receptors Nod1 and Nod2, thereby promoting the production of inflammatory cytokines and antibodies. To further elucidate these biological defense mechanisms, a large and stable supply of the PGN fragments via chemical synthesis is essential. However, the synthesis and purification of long PGN fragments are quite challenging due to their low solubility. In this study, we efficiently synthesized PGN fragments via solid-phase oligosaccharide synthesis (SPOS). Using the JandaJel™ Wang resin (JJ-Wang), an octasaccharide glycan chain of PGN was constructed by repeating glycosylation reactions to elongate β-1,4-linked disaccharide units composed of MurNAc and GlcNAc. To enhance reactivity, glycosylation was performed in a mixed solvent comprising C<inf>4</inf>F<inf>9</inf>OEt/CH<inf>2</inf>Cl<inf>2</inf>/THF with the intention of promoting substrate concentration onto the solid support through the fluorophobic effect, affording the PGN octasaccharide in a 19% overall yield (10 steps). Subsequently, after deprotection of the O-Fmoc, N-Troc, and ethyl ester groups, N- and O-acetylation proceeded smoothly, owing to the high swelling property of JJ-Wang. Peptide condensation with L-Ala-D-isoGln(OBn) and carboxylic acids was also achieved. Finally, cleavage of the PGN fragment from the resin with TFA afforded the desired octasaccharide with dipeptides in a 2.3% overall yield (15 steps).

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Lipid structure-dependent CD1d functional stabilization and immunomodulation of endogenous glucosylceramides

Ueki K., Nozawa R., Matsumaru T., Yamasaki S., Fujimoto Y.

Chemical Communications 61 （ 27 ） 5154 - 5157 2025年03月

ISSN  13597345

　概要を見る

Newly developed synthetic methods for monoglycosylceramides enabled representative brain glycolipid synthesis. Immunomodulatory activities of the glycolipids were examined via CD1d and Mincle. CD1d strongly bound endogenous GlcCer with longer unsaturated fatty acids (e.g., C24:1), partly inhibiting other antigens and stabilizing CD1d's immunological role in presenting lipid antigens to NKT cells.

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Highly Selective Cytokine Induction of Nitrated Lipid-Modified α-GalCer Derivatives Demonstrating High Binding Affinity to the Lipid Antigen Presenting Molecule CD1d

Sueyoshi K., Kishi J., Inuki S., Matsumaru T., Fujimoto Y.

Chemistry A European Journal 31 （ 6 ）  2025年01月

ISSN  09476539

　概要を見る

Glycolipid antigens are presented by CD1d on antigen-presenting cells to T cell receptors (TCRs) of natural killer T (NKT) cells, leading to immune responses via cytokine induction. Although various lipid antigens have been found, there are only a limited number of glycolipid antigens having selective cytokine induction. In this study, we identified the glycolipids (α-GalCer nitro-type) that exhibit highly selective induction of Th2 and Th17 type cytokines, with very high binding affinity to CD1d, by introducing nature-inspired nitro-modified fatty acyl groups. The natural nitroalkene moiety of fatty acyl groups in the glycolipids effectively enhances the affinity to CD1d through presumed hydrogen-bonding and NO<inf>2</inf>–π interactions, leading to the distinctive function in cytokine induction and selectivity.

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Synthesis and application of immunomodulatory natural product.

Fujimoto, Y., Shiokawa, Z., Inuki, S., Aiba, T., Arai, Y.

J. Synth. Org. Chem. Jpn. 81 （ 9 ） 867 - 876 2023年09月

研究論文（学術雑誌）

Chemical synthesis and molecular interaction analysis of -galactosyl ceramide derivatives as CD1d ligands

Ueki, K., Sueyoshi, K., Inuki, S., Fujimoto

Methods Mol Biol.  2613   13 - 22 2023年

共著, 責任著者, 査読有り

Trehalose diesters containing a polar functional group-modified lipid moiety: Synthesis and evaluation of Mincle-mediated signaling activity.

Matsumaru, T., Sueyoshi, K., Okubo, K., Fujii, S., Sakuratani, K., Saito, R., Ueki, K., Yamasaki, S., Fujimoto, Y.

Bioorg. Med. Chem. 75   117045 2022年12月

研究論文（学術雑誌）, 査読有り

Precise immunological evaluation rationalizes the design of a self-adjuvanting vaccine composed of glycan antigen, TLR1/2 ligand, and T-helper cell epitope.

Chang, T. C., Manabe, Y., Ito, K., Yamamoto, R., Kabayama, K., Ohshima, S., Kametani, Y., Fujimoto, Y., Lin, C. C., Fukase, K.

RSC Advances （The Royal Society of Chemistry）  12   18985 - 18993 2022年06月

研究論文（学術雑誌）, 査読有り

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Fungal β-Mannosyloxymannitol Glycolipids and Their Analogues: Synthesis and Mincle-Mediated Signaling Activity

Matsumaru T., Sakuratani K., Yanaka S., Kato K., Yamasaki S., Fujimoto Y.

European Journal of Organic Chemistry 2022 （ 20 ）  2022年05月

ISSN  1434193X

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Mincle, a C-type lectin receptor (CLR), senses certain lipid conjugates, leading to the activation of the innate immune system. The lipid conjugate ligands include glyco-, glycero-, and mannitol lipids. Recently, a fungal β-mannosyloxymannitol glycolipid demonstrating a novel architecture, “44-2,” was isolated from the fungus Malassezia and displayed potent Mincle-mediated signaling activity. For this study, we synthesized the diastereomeric pair of mannosyloxymannitol glycolipids and their analogues and analyzed the structure-activity relationships of Mincle-mediated signaling. The results suggest that the dimannosyl-fatty acid moiety in the mannosyloxymannitol glycolipid-type structure plays a major role in the molecular recognition of Mincle.

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β-mannosyloxymannitol glycolipids and their analogues: synthesis and Mincle-mediated signaling activity.

Matsumaru, T., Sakuratani, K., Yanaka, S., Kato, K., Yamasaki, S., Fujimoto, Y. Fungal

European journal of Organic Chemistry    e202200109 2022年04月

研究論文（学術雑誌）, 責任著者, 査読有り

Alkyne-Tagged Dopamines as Versatile Analogue Probes for Dopaminergic System Analysis

Nuriya M., Ashikari Y., Iino T., Asai T., Shou J., Karasawa K., Nakamura K., Ozeki Y., Fujimoto Y., Yasui M.

Analytical Chemistry 93 （ 27 ） 9345 - 9355 2021年07月

ISSN  00032700

　概要を見る

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter’s small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

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Alkyne-Tagged Dopamines as Versatile Analog Probes for Dopamin-ergic System Analysis.

Nuriya, M., Ashikari, Y., Shou, J., Iino, T., Asai, T., Karasawa, K., Nakamura, K., Ozeki, Y., Fujimoto, Y.

Analytical Chemistry （ACS）  93 （ 27 ） 9345 - 9355 2021年07月

研究論文（学術雑誌）, 査読有り

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Lipopolysaccharide derived from the lymphoid-resident commensal bacteria Alcaligenes faecalis functions as an effective nasal adjuvant to augment IgA antibody and Th17 cell responses.

Wang, Y., Hosomi, K., Shimoyama, A., Yoshii, K., Nagatake, T., Fujimoto, Y., Kiyono, H., Fukase, K., Kunisawa, J.

Frontiers in immunology 12   699349 2021年07月

研究論文（学術雑誌）, 査読有り

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Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid A.

Shimoyama A, Di Lorenzo F, Yamaura H, Mizote K, Palmigiano A, Pither MD, Speciale I, Uto T, Masui S, Sturiale L, Garozzo D, Hosomi K, Shibata N, Kabayama K, Fujimoto Y, Silipo A, Kunisawa J, Kiyono H, Molinaro A, Fukase K.

Angew Chem Int Ed Engl 60 （ 18 ） 10023 - 10031 2021年01月

研究論文（学術雑誌）, 査読有り

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Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis

Inuki S., Hirata N., Kashiwabara E., Kishi J., Aiba T., Teratani T., Nakamura W., Kojima Y., Maruyama T., Kanai T., Fujimoto Y.

Scientific Reports （Scientific Reports）  10 （ 1 ）  2020年12月

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The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure–activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

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Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system

Kusaka H., Kita S., Tadokoro T., Yoshida K., Kasai Y., Niiyama H., Fujimoto Y., Hanashima S., Murata M., Sugiyama S., Ose T., Kuroki K., Maenaka K.

Protein Expression and Purification （Protein Expression and Purification）  172 2020年08月

ISSN  10465928

　概要を見る

© 2020 Elsevier Inc. CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

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The key entity of a DCAR agonist, phosphatidylinositol mannoside Ac ₁PIM ₁: Its synthesis and immunomodulatory function

Arai Y., Torigoe S., Matsumaru T., Yamasaki S., Fujimoto Y.

Organic and Biomolecular Chemistry 18 （ 19 ） 3659 - 3663 2020年05月

査読有り,  ISSN  14770520

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© The Royal Society of Chemistry 2020. Ac1PIM1is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) fromMycobacterium tuberculosis. We achieved the first synthesis of Ac1PIM1by utilizing an allyl-type protecting group strategy and regioselective phosphorylation of inositol. A very potent agonist of an innate immune receptor DCAR, which is better than previously known agonists, is demonstrated.

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Design and Discovery of Covalent α-GalCer Derivatives as Potent CD1d Ligands

Kishi J., Inuki S., Kashiwabara E., Suzuki T., Dohmae N., Fujimoto Y.

ACS Chemical Biology 15 （ 2 ） 353 - 359 2020年02月

査読有り,  ISSN  15548929

　概要を見る

© 2020 American Chemical Society. CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A′ pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.

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Synthetic Studies on FNC-RED and Its Analogues Containing an All syn-Cyclopentanetetrol Moiety

Inuki, S., Sato, K., Zui, N., Yamaguchi, R., Matsumaru, T., Fujimoto, Y.

Journal of Organic Chemistry （Journal of Organic Chemistry）  84 （ 19 ） 12680 - 12685 2019年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00223263

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© 2019 American Chemical Society. FNC-RED exhibits innate immune receptor Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD2) stimulatory activity. We have developed a divergent synthetic route to FNC-RED derivatives containing various alkyl side chains. Key features of the synthetic study include stepwise palladium catalyzed cross-coupling reactions and the construction of an all syn-cyclopentanetetrol moiety.

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