RESUMEN
Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.
Asunto(s)
Vesículas Extracelulares , Galectinas , Proteínas de la Membrana , Polisacáridos , Polisacáridos/química , Polisacáridos/metabolismo , Glicosilación , Galectinas/metabolismo , Galectinas/química , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Humanos , Difusión , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMEN
We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled ß-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to â¼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Membrana Dobles de Lípidos , Antígenos , Adyuvantes Inmunológicos , PéptidosRESUMEN
Targeted alpha therapy (TAT) has garnered significant interest as an innovative cancer therapy. Owing to their high energy and short range, achieving selective α-particle accumulation in target tumor cells is crucial for obtaining high potency without adverse effects. To meet this demand, we fabricated an innovative radiolabeled antibody, specifically designed to selectively deliver 211At (α-particle emitter) to the nuclei of cancer cells. The developed 211At-labeled antibody exhibited a superior effect compared to its conventional counterparts. This study paves the way for organelle-selective drug delivery.
Asunto(s)
Neoplasias , Radioisótopos , Humanos , Radioisótopos/uso terapéutico , Sistemas de Liberación de Medicamentos , Núcleo Celular , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapiaRESUMEN
Antibody dynamics on membranes, such as endocytosis and clustering, are vital in determining antibody functions. In this study, we demonstrated that glycan conjugation can modulate antibody dynamics through the glycan-lectin interaction to regulate its potency. The anti-HER2 antibody, an anti-breast-cancer antibody, was conjugated with galactose-containing N-glycan, and its internalization was suppressed by interaction with galectin-3, leading to enhanced complement-dependent cytotoxic (CDC) activity. This glycan-antibody conjugate is proposed as a new approach to modulate antibody activity and may provide an alternative strategy for redeveloping antibody drugs that do not exhibit sufficient activity.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Lectinas , PolisacáridosRESUMEN
We propose a de novo glycan display approach that combines metabolic labeling and a glycan-caging strategy as a facile editing method for cell-surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α-rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell-surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.
Asunto(s)
Anticuerpos , Polisacáridos , Polisacáridos/metabolismo , Membrana Celular/metabolismo , RamnosaRESUMEN
Asparagine-linked protein glycosylations (N-glycosylations) are one of the most abundant post-translational modifications and are essential for various biological phenomena. Herein, we describe the isolation, structural determination, and chemical synthesis of the N-glycan from the hyperthermophilic archaeon Thermococcus kodakarensis. The N-glycan from the organism possesses a unique structure including myo-inositol, which has not been found in previously characterized N-glycans. In this structure, myo-inositol is highly glycosylated and linked with a disaccharide unit through a phosphodiester. The straightforward synthesis of this glycan was accomplished through diastereoselective phosphorylation and phosphodiester construction by SN 2 coupling. Considering the early divergence of hyperthermophilic organisms in evolution, this study can be expected to open the door to approaching the primitive function of glycan modification at the molecular level.
Asunto(s)
Thermococcus , Inositol/metabolismo , Polisacáridos/metabolismoRESUMEN
We describe the synthesis and characterization of a photoactivated boron-based Lewis acid catalyst based on a cage-shaped triphenolic ligand with three pyrenylmethyl moieties. The obtained cage-shaped borate functioned as a photoactivated Lewis acid catalyst thanks to the flexible three pyrenylmethyl moieties. The deformation of the cage-shaped scaffold driven by intramolecular excimer formations of the pyrenes is a critical factor in realizing the photoactivation. Mannich-type reactions and glycosylations significantly were accelerated under 370â nm light irradiations. It is noteworthy that various glycosyl fluorides, which are not easily activated in photocatalytic systems due to their high C-F bond stability, are activated by the photoimproved catalytic activity of the catalyst.
Asunto(s)
Boratos , Ácidos de Lewis , Ácidos de Lewis/química , Glicosilación , Catálisis , BoroRESUMEN
We have synthesized B-antigen-displaying dendrimers (16-mers) with different sizes and evaluated their affinity to their IgM antibody in order to investigate which design features lead to effective multivalency. Unexpectedly, the smallest dendrimer, which cannot chelate the multiple binding sites of IgM, clearly exhibited multivalency, together with an affinity similar to or higher than those of the larger dendrimers. These results indicate that the statistical rebinding model, which involves the rapid exchange of clustered glycans, significantly contributes to the multivalency of glycodendrimers. Namely, in the design of glycodendrimers, high-density glycan presentation to enhance statistical rebinding should be considered in addition to the ability to chelate multiple binding sites. This notion stands in contrast to the currently prevailing scientific consensus, which prioritizes the chelation model. This study thus provides new and important guidelines for molecular design of glycodendrimers.
Asunto(s)
Dendrímeros , Dendrímeros/química , Polisacáridos , Sitios de UniónRESUMEN
α-Methyl-l-tyrosine (AMT) has a high affinity for the cancer-specific l-type amino acid transporter 1 (LAT1). Therefore, we established an anti-cancer therapy, with 211 At-labeled α-methyl-l-tyrosine (211 At-AAMT) as a carrier of 211 At into tumors. 211 At-AAMT had high affinity for LAT1, inhibited tumor cell growth, and induced DNA double-stranded breaks in vitro. We evaluated the accumulation of 211 At-AAMT in vivo and the role of LAT1. Treatment with 0.4 MBq/mouse 211 At-AAMT inhibited tumor growth in the PANC-1 tumor model and 1 MBq/mouse 211 At-AAMT inhibited metastasis in the lung of the B16F10 metastasis model. Our results suggested that 211 At would be useful for anti-cancer therapy and that LAT1 is suitable as a target for radionuclide therapy.
Asunto(s)
Partículas alfa/uso terapéutico , Astato/administración & dosificación , Portadores de Fármacos/farmacología , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Neoplasias/radioterapia , alfa-Metiltirosina/farmacología , Animales , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de la radiación , Modelos Animales de Enfermedad , Estudios de Factibilidad , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.
Asunto(s)
Polisacáridos/química , Animales , Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos , Glicoproteínas/metabolismo , Glicosilación , Humanos , Lectinas/química , Polisacáridos/metabolismoRESUMEN
The chemical synthesis of a fully sialylated tetraantennary N-glycan has been achieved for the first time by using the diacetyl strategy, in which NHAc is protected as NAc2 to improve reactivity by preventing intermolecular hydrogen bonds. Another key was the glycosylation to the branched mannose in an ether solvent, which promoted the desired glycosylation by stabilizing the oxocarbenium ion intermediate. Furthermore, high α-selectivity of these glycosylation reactions was realized by utilizing remote participation. Two asymmetrically deuterium labeled sialyl N-glycans were also synthesized by the same strategy. The synthesized N-glycans were used to probe the molecular basis of H1N1 neuraminidase recognition. The asymmetrically deuterated N-glycans revealed a difference in the recognition of sialic acid on each branch. Meanwhile, the tetraantennary N-glycan was used to evaluate the effects of multivalency and steric hinderance by forming branching structures.
Asunto(s)
Neuraminidasa/metabolismo , Polisacáridos/síntesis química , Deuterio/química , Glicosilación , Subtipo H1N1 del Virus de la Influenza A/enzimología , Espectrometría de Masas/métodos , Polisacáridos/análisis , Polisacáridos/metabolismo , Ácidos Siálicos/análisis , Ácidos Siálicos/metabolismo , Espectrofotometría UltravioletaRESUMEN
CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells.
Asunto(s)
Simulación de Dinámica Molecular , Polisacáridos/química , Lectina 2 Similar a Ig de Unión al Ácido Siálico/química , Linfocitos B/química , Conformación de Carbohidratos , Galactosa/química , HumanosRESUMEN
C60- and C100-dolichols were synthesized. A Z-selective Wittig reaction was achieved with high selectivity in a microflow system to realize the scalable supply of the Z-isoprene unit. An isoprene chain was efficiently elongated by an SN2-type coupling between allyl sulfone and allyl chloride using t-BuOK. These key reactions enabled the efficient syntheses of dolichols. This study will pave the way for the functional studies of dolichols.
RESUMEN
Individual interactions between glycans and their receptors are usually weak, although these weak interactions can combine to realize a strong interaction (multivalency). Such multivalency plays a crucial role in the recognition of host cells by pathogens. Glycodendrimers are useful materials for the reconstruction of this multivalent interaction. However, the introduction of a large number of glycans to a dendrimer core is fraught with difficulties. We herein synthesized antipathogenic glycodendrimers using the self-activating click chemistry (SACC) method developed by our group. The excellent reactivity of SACC enabled the efficient preparation of sialyl glycan and Gb3 glycan dendrimers, which exhibited strong avidity toward hemagglutinin on influenza virus and Shiga toxin B subunit produced by Escherichia coli, respectively. We demonstrated the usefulness of SACC-based glycodendrimers as antipathogenic compounds.
Asunto(s)
Química Clic , Dendrímeros , PolisacáridosRESUMEN
Compared to traditional vaccines that are formulated into mixtures of an adjuvant and an antigen, a self-adjuvanting vaccine consists of an antigen that is covalently conjugated to a well-defined adjuvant. In self-adjuvanting vaccines, innate immune receptor ligands are usually used as adjuvants. Innate immune receptor ligands effectively trigger acquired immunity through the activation of innate immunity to enhance host immune responses to antigens. When a self-adjuvanting vaccine is used, immune cells simultaneously uptake the antigen and the adjuvant because they are covalently linked. Consequently, the adjuvant can specifically induce immune responses against the conjugated antigen. Importantly, self-adjuvanting vaccines do not require co-administration of additional adjuvants or immobilization to carrier proteins, which enables avoidance of the use of highly toxic adjuvants or the induction of undesired immune responses. Given these excellent properties, self-adjuvanting vaccines are expected to serve as candidates for the next generation of vaccines. Herein, we review vaccine adjuvants, with a focus on the adjuvants used in self-adjuvanting vaccines, and then overview recent advances made with self-adjuvanting conjugate vaccines.
Asunto(s)
Adyuvantes de Vacunas , Vacunas , Adyuvantes Inmunológicos , GlicoconjugadosRESUMEN
Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.
Asunto(s)
Adyuvantes Inmunológicos/química , Neoplasias de la Mama/tratamiento farmacológico , Vacunas contra el Cáncer/uso terapéutico , Péptidos/inmunología , Animales , Antígenos/química , Antígenos/inmunología , Neoplasias de la Mama/patología , Vacunas contra el Cáncer/química , Femenino , Galactosilceramidas/química , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Lipopéptidos/química , Ratones , Ratones Endogámicos BALB C , Péptidos/químicaRESUMEN
Monomeric cage-shaped aluminum aryl oxides 1Al were synthesized using tripodal triphenolic ligands. The Lewis acidity and catalytic activity of the obtained 1Al·py were investigated. The Lewis acidity of 1Al·py originates from the flexible change in the coordination number of the aluminum center, allowing the catalytic O-glycosylation to occur with a high efficiency and an unusual stereoselectivity.
RESUMEN
Many circulating cancer-related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans-particularly heparan sulfate-at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF-1 and FGF-2. Taking note of the typical pose of l-iduronic acid, we conceived of per-O-sulfonated analogues of such disaccharides, and devised a single-step procedure for per-O-sulfonation of unprotected sugars with concomitant 1,6-anhydro bridge formation to achieve such compounds through direct use of SO3 â Et3 N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF-1 and FGF-2 mostly at the sub-micromolar level, although the tetrasaccharide analogue achieved low-nanomolar binding with FGF-2.
Asunto(s)
Factores de Crecimiento de Fibroblastos/química , Heparitina Sulfato/química , Azúcares/química , Conformación de CarbohidratosRESUMEN
Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.
Asunto(s)
Anticuerpos/inmunología , Neoplasias/inmunología , Trisacáridos/inmunología , Anticuerpos/química , Conformación de Carbohidratos , Línea Celular Tumoral , Supervivencia Celular/inmunología , Humanos , Neoplasias/patología , Neoplasias/terapia , Trisacáridos/síntesis química , Trisacáridos/químicaRESUMEN
The core fucose, a major modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin-1, a known anti-ß-glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.