RESUMO
Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.
Assuntos
Glicoproteínas/farmacologia , Glicosídeo Hidrolases/metabolismo , Trastuzumab/química , Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/farmacologia , Glicosilação , Humanos , Pichia/metabolismo , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Trastuzumab/metabolismoRESUMO
We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAV-specific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Células A549 , Animais , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proteção Cruzada/imunologia , Cães , Feminino , Células HEK293 , Humanos , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Virulência/imunologiaRESUMO
Fluorinated glycosides are known to resist the glycosidase-catalyzed glycosidic bond cleavage; however, the synthesis of such glycans, especially 3-fluoro-sialic acid (3F-Neu5Ac) containing sialosides, has been a major challenge. Though the enzymatic synthesis of α-2,3-linked 3F-sialosides was reported, until recently there has not been any effective method available for the synthesis of 3F-sialosides in the α-2,6-linkage. In order to understand the biological effect of such modification, we report here a chemical synthesis of 3Fax-Neu5Ac-α2,6-Gal as a building block for the assembly of 3Fax-Neu5Ac-containing sialosides and a representative homogeneous antibody glycoform. Our results showed that the sialosides are stable under sialidase catalysis and the rituximab glycoform with a sialylated complex-type biantennary glycan terminated with 3Fax-Neu5Ac in the α-2,6-linkage (α2,6-F-SCT) has a similar binding avidity as its parent glycoform. These findings open up new opportunities for the development of therapeutic glycoproteins with improved pharmacokinetic parameters.
Assuntos
Anticorpos/química , Neuraminidase/química , Oligossacarídeos/síntese química , Ácidos Siálicos/química , Anticorpos/metabolismo , Biocatálise , Configuração de Carboidratos , Neuraminidase/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Ácidos Siálicos/metabolismo , Especificidade por SubstratoRESUMO
Glycosyl phosphates are known as versatile donors for the synthesis of complex oligosaccharides both chemically and enzymatically. Herein, we report the stereoselective construction of modular building blocks for the synthesis of N-glycan using glycosyl phosphates as donors. We have synthesized four trisaccharide building blocks with orthogonal protecting groups, namely, Manß2GlcNAc(OAc)3ß6GlcNAc (9), Manß2GlcNAc-ß6GlcNAc(OAc)3 (15), Manß2GlcNAc(OAc)3ß4GlcNAc (18) and Manß2GlcNAcß4GlcNAc(OAc) (22) for further selective elongation using glycosyltransferases. The glycosylation reaction using glycosyl phosphate was found to be high yielding with shorter reaction time. Initially, The phthalimide protected glucosamine donor was exploited to ensure the formation of ß-glycosidic linkage and later converted to the N-acetyl group before the enzymatic synthesis. The selective deprotection of O-benzyl group was performed prior to enzymatic synthesis to avoid its negative interference.
RESUMO
Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Polissacarídeos/química , Rituximab/química , Acetilglucosamina/química , Acetilglucosamina/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/enzimologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Engenharia de Proteínas , Receptores de IgG/imunologia , Rituximab/imunologia , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Streptococcus pyogenes/enzimologia , Relação Estrutura-Atividade , Trastuzumab/química , Trastuzumab/imunologia , alfa-L-Fucosidase/metabolismoRESUMO
Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.
Assuntos
Química Click/métodos , Técnicas de Sonda Molecular , Sondas Moleculares/química , Neuraminidase/química , Neuraminidase/ultraestrutura , Alcinos/química , Cromatografia Líquida , Adutos de DNA/metabolismo , Humanos , Influenza Humana/diagnóstico , Estrutura Molecular , Neuraminidase/metabolismo , Proteômica/métodos , Estreptavidina/química , Espectrometria de Massas em TandemRESUMO
Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Fucose/imunologia , Polissacarídeos Fúngicos/imunologia , Reishi/química , Animais , Linfócitos B/imunologia , Quimiocina CCL2/metabolismo , Polissacarídeos Fúngicos/metabolismo , Imunização , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em MicrossériesRESUMO
Globo H-based therapeutic cancer vaccines have been tested in clinical trials for the treatment of late stage breast, ovarian, and prostate cancers. In this study, we explored Globo H analogue antigens with an attempt to enhance the antigenic properties in vaccine design. The Globo H analogues with modification at the reducing or nonreducing end were synthesized using chemoenzymatic methods, and these modified Globo H antigens were then conjugated with the carrier protein diphtheria toxoid cross-reactive material (CRM) 197 (DT), and combined with a glycolipid C34 as an adjuvant designed to induce a class switch to form the vaccine candidates. After Balb/c mice injection, the immune response was studied by a glycan array and the results showed that modification at the C-6 position of reducing end glucose of Globo H with the fluoro, azido, or phenyl group elicited IgG antibody response to specifically recognize Globo H (GH) and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4). However, only the modification of Globo H with the azido group at the C-6 position of the nonreducing end fucose could elicit a strong IgG immune response. Moreover, the antibodies induced by these vaccines were shown to recognize GH expressing tumor cells (MCF-7) and mediate the complement-dependent cell cytotoxicity against tumor cells. Our data suggest a new potential approach to cancer vaccine development.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/imunologia , Animais , Antígenos Glicosídicos Associados a Tumores/farmacologia , Vacinas Anticâncer/química , Vacinas Anticâncer/farmacologia , Configuração de Carboidratos , Proliferação de Células/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oxirredução , Relação Estrutura-AtividadeRESUMO
The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.
Assuntos
Vacinas contra a AIDS/síntese química , Anticorpos Anti-HIV/imunologia , HIV/imunologia , Polissacarídeos/síntese química , Polissacarídeos/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Configuração de Carboidratos , HIV/química , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/isolamento & purificação , Dados de Sequência Molecular , Polissacarídeos/químicaRESUMO
We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Escherichia coli/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Antígenos Glicosídicos Associados a Tumores/química , Clonagem Molecular , Glicosiltransferases/isolamento & purificação , Microbiologia Industrial , Regulação para CimaRESUMO
Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted ß-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.
Assuntos
Dissacarídeos/química , Dissacarídeos/síntese química , Glucosamina/metabolismo , Heparina/análogos & derivados , Heparina/síntese química , Lectinas/metabolismo , Mycobacterium tuberculosis , Dissacarídeos/metabolismo , Glucosamina/química , Glicosilação , Heparina/metabolismo , Lectinas/química , Fragmentos de Peptídeos/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
COVID-19, an infectious disease caused by the SARS-CoV-2 virus, emerged globally in early 2020 and has remained a serious public health issue. To date, although several preventative vaccines have been approved by FDA and EMA, vaccinated individuals increasingly suffer from breakthrough infections. Therapeutic antibodies may provide an alternative strategy to neutralize viral infection and treat serious cases; however, the clinical data and our experiments show that some FDA-approved monoclonal antibodies lose function against COVID-19 variants such as Omicron. Therefore, in this study, we present a novel therapeutic agent, SI-F019, an ACE2-Fc fusion protein whose neutralization efficiency is not compromised, but actually strengthened, by the mutations of dominant variants including Omicron. Comprehensive biophysical analyses revealed the mechanism of increased inhibition to be enhanced interaction of SI-F019 with all the tested spike variants, in contrast to monoclonal antibodies which tended to show weaker binding to some variants. The results imply that SI-F019 may be a broadly useful agent for treatment of COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais/uso terapêutico , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de CoronavírusRESUMO
While the improved treatment of human immunodeficiency virus type 1 (HIV-1) infection is available, the development of an effective and safe prophylactic vaccine against HIV-1 is still an unrealized goal. Encouragingly, the discovery of broadly neutralizing antibodies (bNAbs) from HIV-1 positive patients that are capable of neutralizing a broad spectrum of HIV-1 isolates of various clades has accelerated the progress of vaccine development in the past few years. Some of these bNAbs recognize the N-glycans on the viral surface gp120 glycoprotein. We have been interested in using the glycan epitopes recognized by bNAbs for the development of vaccines to elicit bNAb-like antibodies with broadly neutralizing activities. Toward this goal, we have identified novel hybrid-type structures with subnanomolar avidity toward several bNAbs including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Here, we report the immunogenicity evaluation of a novel hybrid glycan conjugated to carrier DTCRM197, a nontoxic mutant of the diphtheria toxin, for immunization in mice. Our results indicated that the IgG response was mainly against the chitobiose motif with nonspecific binding to a panel of N-glycans with reducing end GlcNAc-GlcNAc (chitobiose) printed on the glass slides. However, the IgM response was mainly toward the reducing end GlcNAc moiety. We further used the glycoconjugates of Man3GlcNAc2, Man5GlcNAc2, and Man9GlcNAc2 glycans for immunization, and a similar specificity pattern was observed. These findings suggest that the immunogenicity of chitobiose may interfere with the outcome of N-glycan-based vaccines, and modification may be necessary to increase the immunogenicity of the entire N-glycan epitope.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoconjugados/imunologia , Anticorpos Anti-HIV/imunologia , Polissacarídeos/imunologia , Acetilglucosamina/imunologia , Animais , Proteínas de Bactérias/química , Sequência de Carboidratos , Dissacarídeos/imunologia , Epitopos , Feminino , Glicoconjugados/síntese química , HIV-1/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos Endogâmicos C57BL , Polissacarídeos/síntese química , Desenvolvimento de VacinasRESUMO
The first systematic investigation of the effect of high mannose, hybrid, and bi- and tri-antennary complex type glycans on the effector functions of antibodies was achieved by the discovery of novel Endo-S2 mutants generated by site-directed mutagenesis as glycosynthases with broad substrate specificity.
Assuntos
Anticorpos/química , Glicosiltransferases/química , Polissacarídeos/química , Anticorpos/metabolismo , Glicosídeo Hidrolases/genética , Glicosilação , Glicosiltransferases/genética , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Receptores de IgG/metabolismo , Streptococcus pyogenes/enzimologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Correction for 'Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies' by Sachin S. Shivatare et al., Chem. Commun., 2018, 54, 6161-6164.
RESUMO
Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-l-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc, and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endoglycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.
Assuntos
Bactérias/enzimologia , Fucose/metabolismo , Glicoproteínas/metabolismo , Imunoglobulina G/metabolismo , alfa-L-Fucosidase/metabolismo , Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Fucose/química , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicoproteínas/química , Humanos , Imunoglobulina G/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Especificidade por SubstratoRESUMO
A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Polissacarídeos/síntese química , Vacinas contra a AIDS/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Ligantes , Polissacarídeos/química , Polissacarídeos/imunologiaRESUMO
Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp.