RESUMO
Development of novel anti-cancer therapeutics based on Golgi α-mannosidase II (GMII) inhibition is considerably impeded by an undesired co-inhibition of lysosomal α-mannosidase leading to severe side-effects. In this contribution, we describe a fully stereoselective synthesis of (5S)-5-[4-(halo)benzyl]swainsonines as highly potent and selective inhibitors of GMII. The synthesis starts from a previously reported aldehyde readily available from l-ribose, and the key features include an intramolecular reductive amination with substrate-controlled stereoselectivity and a late-stage derivatisation of the benzyl group via ipso-substitution. These novel swainsonine analogues were found to be nanomolar inhibitors of the Golgi-type α-mannosidase AMAN-2 (Ki = 23-75 nM) with excellent selectivity (selectivity index = 205-870) over the lysosomal-type Jack bean α-mannosidase. Finally, molecular docking and pKa calculations were performed to provide more insight into the structure of the inhibitor:enzyme complexes, and a pair interaction energy analysis (FMO-PIEDA) was carried out to rationalise the observed potency and selectivity of the inhibitors.
Assuntos
Inibidores Enzimáticos , Swainsonina , Humanos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Manosidases/antagonistas & inibidores , Manosidases/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Swainsonina/farmacologia , Swainsonina/síntese química , Swainsonina/química , Compostos de Benzil/química , Compostos de Benzil/farmacologiaRESUMO
An engineered cyanovirin-N homologue that exhibits specificity for high mannose N-glycans has been constructed to aid typeâ I α 1,2-mannosidase inhibitor discovery and development. Engineering the lectins C-terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N-glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by typeâ I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of typeâ I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin-N homologue constructs, and highlight their potential in analytical contexts that require high mannose-specific lectins.
Assuntos
Lectinas/química , Manosidases/antagonistas & inibidores , Alcaloides/química , Alcaloides/metabolismo , Motivos de Aminoácidos , Aminoaciltransferases/química , Proteínas de Bactérias/química , Linhagem Celular , Cisteína Endopeptidases/química , Desenho de Fármacos , Corantes Fluorescentes/química , Glicosilação , Humanos , Lectinas/metabolismo , Manose/química , Manose/metabolismo , Manosidases/metabolismo , Microscopia de FluorescênciaRESUMO
Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
Assuntos
Cicloexanóis/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Manosidases/antagonistas & inibidores , Cicloexanóis/síntese química , Cicloexanóis/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Manosidases/metabolismo , Estrutura MolecularRESUMO
Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell-cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/enzimologia , Manosidases/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Manosidases/metabolismo , Estrutura Molecular , Imagem Óptica , Esferoides Celulares/metabolismo , Relação Estrutura-AtividadeRESUMO
N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.
Assuntos
Alcaloides/farmacologia , Engenharia Metabólica/métodos , Nicotiana/metabolismo , Polissacarídeos/metabolismo , Rituximab/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD20/metabolismo , Fucose/metabolismo , Glicosilação/efeitos dos fármacos , Manose/metabolismo , Manosidases/antagonistas & inibidores , Manosidases/metabolismo , Nicotiana/efeitos dos fármacosRESUMO
Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.
Assuntos
Membrana Celular/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Glicômica , Manose/farmacologia , Manosidases/metabolismo , Alcaloides/farmacologia , Células CACO-2 , Sequência de Carboidratos , Membrana Celular/química , Membrana Celular/enzimologia , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Voláteis/metabolismo , Frutose/metabolismo , Frutose/farmacologia , Fucose/metabolismo , Fucose/farmacologia , Galactose/metabolismo , Galactose/farmacologia , Glutamina/metabolismo , Glutamina/farmacologia , Glicosilação/efeitos dos fármacos , Células HT29 , Humanos , Manose/metabolismo , Manosidases/antagonistas & inibidoresRESUMO
Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar "epoxide" mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.
Assuntos
Glucosamina/análogos & derivados , Inibidores de Glicosídeo Hidrolases/farmacologia , Manosidases/antagonistas & inibidores , Configuração de Carboidratos , Cristalografia por Raios X , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Manosidases/metabolismo , Modelos MolecularesRESUMO
Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man9 GlcNAc2 to Man5 GlcNAc2 by the combined actions of assorted α-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific α-1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man8 GlcNAc2 isomers from a structurally defined synthetic Man9 GlcNAc2 substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Manosidases/antagonistas & inibidores , Animais , Inibidores Enzimáticos , Humanos , Manose/metabolismo , Manosidases/metabolismo , Camundongos , Transporte ProteicoRESUMO
A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C4 chair conformation as opposed to the "normal" 4 C1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJâ CkGH47 complex, showing two conformations-3 S1 and 1 C4 -for the DMJ inhibitor.
Assuntos
Compostos Aza/química , Ácidos Carboxílicos/química , Inibidores Enzimáticos/química , Ácidos Hexurônicos/química , Imino Açúcares/química , Manosidases/antagonistas & inibidores , Compostos Aza/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Configuração de Carboidratos , Sequência de Carboidratos , Caulobacter/química , Caulobacter/enzimologia , Inibidores Enzimáticos/síntese química , Ésteres/química , Concentração de Íons de Hidrogênio , Imino Açúcares/síntese química , Manosidases/química , Modelos MolecularesRESUMO
UNLABELLED: The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding of these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (MAbs). However, the V3 MAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IGHV3-53 and IGLV2-28 genes, a pairing rarely used by the other V3 MAbs. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide and showed that the 2424 epitope is located at the tip of the V3 crown ((307)IHIGPGRAFYT(319)), dominated by interactions with His(P308), Pro(P313), and Arg(P315). The binding mode of 2424 is similar to that of the well-characterized MAb 447-52D, although 2424 is more side chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens. IMPORTANCE: HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3, which is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/ultraestrutura , Especificidade de Anticorpos/imunologia , Linhagem Celular , Cristalografia por Raios X , Epitopos/imunologia , Células HEK293 , Antígenos HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Manosidases/antagonistas & inibidores , Dados de Sequência Molecular , Polissacarídeos/imunologia , Estrutura Terciária de ProteínaRESUMO
α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through abâ initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Candida albicans/enzimologia , Inibidores Enzimáticos/síntese química , Proteínas Fúngicas/metabolismo , Manosidases/antagonistas & inibidores , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imino Açúcares/síntese química , Imino Açúcares/química , Imino Açúcares/farmacologia , Manosidases/química , Modelos Moleculares , Conformação ProteicaRESUMO
Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Manose/metabolismo , Manosidases/metabolismo , Proteínas/metabolismo , Fator 6 Ativador da Transcrição/genética , Alcaloides/farmacologia , Animais , Antidepressivos/farmacologia , Linhagem Celular , Galinhas , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Manose/genética , Manosidases/antagonistas & inibidores , Manosidases/genética , Fenelzina/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteínas/genética , Proteólise/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
We report a new classification method for pyranose ring conformations called Best-fit, Four-Membered Plane (BFMP), which describes pyranose ring conformations based on reference planes defined by four atoms. The method is able to characterize all asymmetrical and symmetrical shapes of a pyran ring, is readily automated, easy to interpret, and maps trivially to IUPAC definitions. It also provides a qualitative measurement of the distortion of the ring. Example applications include the analysis of data from crystal structures and molecular dynamics simulations.
Assuntos
Algoritmos , Heparitina Sulfato/química , Manose/química , Piranos/química , Alcaloides/química , Antineoplásicos/química , Configuração de Carboidratos , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Manosidases/antagonistas & inibidores , Manosidases/química , Simulação de Dinâmica MolecularRESUMO
Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including ß-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.
Assuntos
Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Imino Piranoses/farmacologia , Manosidases/antagonistas & inibidores , Termodinâmica , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Imidazóis/síntese química , Imidazóis/química , Imino Piranoses/síntese química , Imino Piranoses/química , Manosidases/química , Manosidases/metabolismo , Modelos Moleculares , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
The role of mannanases is diverse and they are used in many industrial applications, in animal feed, in the food industry and in healthcare. They are also applied in biomass processing, because they play an important role in the breakdown of hemicellulose. Among the mannanase inhibitors, heavy metal ions and general enzyme inhibitors are mainly mentioned. Unfortunately, almost no data are available on carbohydrate-based natural inhibitors of mannanases. According to the literature, carbohydrates do not play an important role in the inhibition of mannanases, so neither do oligosaccharides. This is in contrast to the action and inhibition of other O-glycosyl hydrolases. My hypothesis is that mannanases, like other polysaccharide-degrading enzymes, work in the same way and can be inhibited by oligosaccharides. Evidence from docking and modeling results supports and makes probable the hypothesis that oligosaccharides can inhibit the activity of mannanases, similar to the inhibition of other O-glycosyl hydrolases. Among natural carbohydrate oligomers, several potential mannanase inhibitors have been identified and characterized. In addition to expensive research, it is very important to use research based on cheaper modeling to explore the processes. The results obtained are novel and forward-looking, enabling in-depth and targeted research to be carried out.
Assuntos
Inibidores Enzimáticos , Manosidases , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Manosidases/antagonistas & inibidores , Manosidases/metabolismo , Manosidases/química , Simulação de Acoplamento Molecular , Oligossacarídeos/química , Oligossacarídeos/farmacologia , HidróliseRESUMO
The activity of α-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that α-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c(+) dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-L(low) and PHA-L(high) subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-L(high). Interestingly, all PHA-L(high) DCs displayed a strongly reduced responsiveness to TNF-α-induced p38-MAPK activation compared with PHA-L(low) DCs, indicating differences in PHA-L-binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing α-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-α-induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). α-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-γ-producing T cells in vivo. In contrast, the activity of α-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs' general capability for phenotypic and functional maturation. Systemic application of an α-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by α-1,2-mannosidase I for alloantigen presentation and T cell priming.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Sobrevivência de Enxerto/imunologia , Polissacarídeos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Apresentação de Antígeno/imunologia , Separação Celular , Transplante de Córnea , Células Dendríticas/química , Células Dendríticas/imunologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Ativação Linfocitária/imunologia , Masculino , Manosidases/antagonistas & inibidores , Manosidases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Legumes belonging to the Astragalus, Oxytropis, and Swainsona genera have been noted by ranchers in the Americas, Asia, and Australia to cause a neurologic disease often referred to as locoism or peastruck. The toxin in these legumes is swainsonine, an α-mannosidase and mannosidase II inhibitor. Recent research has shown that in Astragalus and Oxytropis species swainsonine is produced by a fungal endophyte belonging to the Undifilum genus. Here Swainsona canescens is shown to harbor an endophyte that is closely related to Undifilum species previously cultured from locoweeds of North America and Asia. The endophyte produces swainsonine in vitro and was detected by PCR and culturing in S. canescens. The endophyte isolated from S. canescens was characterized as an Undifilum species using morphological and phylogenetic analyses.
Assuntos
Alcaloides/isolamento & purificação , Fabaceae/química , Swainsonina/farmacologia , Alcaloides/análise , Alcaloides/química , Alcaloides/farmacologia , Endófitos/química , Fabaceae/genética , Manosidases/antagonistas & inibidores , Estrutura Molecular , Oxytropis/química , Análise de Sequência de DNA , Swainsonina/análise , Swainsonina/químicaRESUMO
Removal of α-glucose residues from nascent glycoproteins in the early secretory pathway is a requirement for further N-glycan maturation. Although deglucosylation is a stepwise process mediated by endoplasmic reticulum-associated glucosidases I and II for most glycoproteins, Golgi endo-α-mannosidase provides a backup mechanism for glycoprotein deglucosylation. Although conserved in mammals, in certain cell lines, endomannosidase activity in vitro appears to differ from its activity in cells following glucosidase inhibition. Here, we show that in bovine cells this is explained by restricted substrate specificity allowing processing of Glc(1)Man(7)GlcNAc(1/2) and Glc(1)Man(5)GlcNAc(1/2) but not fully glucosylated glycans that build up when glucosidases are inhibited. Our data further demonstrate that such specificity is determined genetically rather than post-translationally. We also demonstrate that the bovine endomannosidase is transcriptionally upregulated by comparison with glucosidase II in Madin-Darby bovine kidney cells and speculate that this is to compensate for the reduced catalytic activity as measured in the recombinant form of the enzyme.
Assuntos
Manosidases/metabolismo , Polissacarídeos/metabolismo , Animais , Biocatálise , Bovinos , Linhagem Celular , Glicosilação , Manosidases/antagonistas & inibidores , Manosidases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
N-linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N-linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N-glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis-tris, and 1-amino-1-methyl-1,3-propanediol), contain one similar molecular structure: 2-amino-1,3-propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost-effective, and stable at room temperature. In addition, tris and bis-tris provide multiple low-cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.
Assuntos
Alcaloides/farmacologia , Anticorpos Monoclonais/biossíntese , Manose/metabolismo , Manosidases/antagonistas & inibidores , Polissacarídeos/química , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , GlicosilaçãoRESUMO
INTRODUCTION: Vascular smooth muscle cells (VSMCs)-based foam cell formation is a crucial factor in the atherosclerosis process. We aimed to explore the mechanism of Golgi a-mannosidase II (GMII) effects on the VSMCs-based foam cell formation. MATERIAL AND METHODS: VSMCs were exposed to different concentrations of low-density lipoproteins (LDLs), lipopolysaccharide (LPS), and/or GMII inhibitor (swainsonine). The qRT-PCR and western blot were used for expression analysis. Oil Red O staining was used to verify changes of lipid droplets in VSMCs. The translocation of the SCAP from the endoplasmic reticulum (ER) to Golgi was detected by immunofluorescence (IF). RESULTS: LPS disrupted the LDLs-mediated regulation of LDL receptor (LDLr) and increased intracellular cholesterol ester, which was inversely inhibited by swainsonine. The activity of a-mannosidase II and GMII expression were decreased by LDLs but increased by the addition of LPS. Conversely, LPS-induced enhancement was reversed by swainsonine. Additionally, swainsonine reversed the LPS-induced increase of intracellular lipid droplets in the presence of LDLs. Expression analysis demonstrated that LDLr, SCAP, and SREBP2 were up-regulated by LPS, but reversed by swainsonine in LDLs-treated cells. IF staining revealed that swainsonine inhibited the translocation of SCAP to Golgi under inflammatory stress. CONCLUSIONS: Collectively, swainsonine restrained LDLr expression to suppress the formation of VSMCs-based foam cells by reducing SREBP2 and SCAP under inflammatory stress conditions, suggesting that GMII contributes to the formation of VSMCs-based foam cells under inflammatory stress.