RESUMO
Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 ß-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.
Assuntos
Celulose 1,4-beta-Celobiosidase , Manose , Celulose 1,4-beta-Celobiosidase/química , Proteínas Fúngicas/metabolismo , Glicosilação , Manose/metabolismo , Manosidases/genética , Manosidases/metabolismo , alfa-Manosidase/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , SARS-CoV-2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/química , Glicosilação , Células HEK293 , Humanos , Fosforilação , SARS-CoV-2/químicaRESUMO
The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.
Assuntos
Anticorpos Antivirais/imunologia , Blastomyces/imunologia , Vacinas Fúngicas/imunologia , Infecções por Orthomyxoviridae/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Celulase/imunologia , Vacinas contra Influenza/imunologiaRESUMO
The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.
Assuntos
Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Polissacarídeos/metabolismo , SARS-CoV-2/fisiologia , COVID-19/virologia , Glicômica , Glicosilação , Células HEK293 , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.
Assuntos
Glicopeptídeos/análise , Glicoproteínas de Membrana/análise , Espectrometria de Massas em Tandem/métodos , Algoritmos , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatografia Líquida , Glicômica , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Células Jurkat , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Proteômica , Espectrometria de Massas em Tandem/estatística & dados numéricosRESUMO
The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.
Assuntos
COVID-19/genética , Polissacarídeos/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , COVID-19/patologia , COVID-19/virologia , Infecções por Coronavirus , Humanos , Pandemias , Polissacarídeos/metabolismo , Ligação Proteica/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Glycoproteins play key roles in various molecular and cellular functions and are among the most difficult to analyze biomolecules on account of their microheterogeneity, non-template-driven synthesis, and low abundances. The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins, including antibodies, vaccines, and biomarkers, are regulated by their glycosylation profile. Thus, there is increasing demand for the qualitative and quantitative characterization and validation of glycosylation on glycoproteins. One of the most important derivatization processes for the structural characterization of released glycans by mass spectrometry (MS) is permethylation. We have recently developed a permethylation strategy in microscale that allows facile permethylation of glycans and permits the processing of large sample sets in nanogram amounts through high-throughput sample handling. Here, we are reporting the wide potential of micropermethylation-based high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells, and purified glycoproteins, through an automated tandem electrospray ionization-mass spectrometry (ESI-MSn) platform. The glycans released from the plasma, cells, and glycoproteins are permethylated in microscale in a 96-well plate or microcentrifuge tube and isolated by a C18 tip-based cleanup through a shorter and simple process. We have developed a workflow to accomplish an in-depth automated structural characterization MS program for permethylated N/O-glycans through an automated high-throughput multistage tandem MS acquisition. We have demonstrated the utility of this workflow using the examples of sialic acid linkages and bisecting GlcNAc (N-acetylglucosamine) on the glycans. This approach can automate the high-throughput screening of glycosylation on large sample sets of glycoproteins, including clinical glycan biomarkers and glycoprotein therapeutics.
Assuntos
Glicômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Microtecnologia , Polissacarídeos/metabolismo , Acetilglucosamina/metabolismo , Automação , Soluções Tampão , Células HEK293 , Humanos , Íons , Isomerismo , Metilação , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/sangue , Polissacarídeos/química , Extração em Fase Sólida , Soluções , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Microbial interaction with the host through sensing receptors, including SIGNR1, sustains intestinal homeostasis against pathogenic inflammation. The newly discovered commensal Propionibacterium strain, P. UF1, regulates the intestinal immunity against pathogen challenge. However, the molecular events driving intestinal phagocytic cell response, including colonic dendritic cells (DCs), by this bacterium are still elusive. Here, we demonstrate that the glycosylation of bacterial large surface layer protein A (LspA) by protein O-mannosyltransferase 1 (Pmt1) regulates the interaction with SIGNR1, resulting in the control of DC transcriptomic and metabolomic machineries. Programmed DCs promote protective T cell response to intestinal Listeria infection and resist chemically induced colitis in mice. Thus, our findings may highlight a novel molecular mechanism by which commensal surface glycosylation interacting with SIGNR1 directs the intestinal homeostasis to potentially protect the host against proinflammatory signals inducing colonic tissue damage.
Assuntos
Proteínas de Bactérias/metabolismo , Moléculas de Adesão Celular/metabolismo , Colite/imunologia , Colo/imunologia , Células Dendríticas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Lectinas Tipo C/metabolismo , Listeria/fisiologia , Listeriose/imunologia , Propionibacterium/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologia , Animais , Proteínas de Bactérias/genética , Moléculas de Adesão Celular/genética , Diferenciação Celular , Células Cultivadas , Colite/induzido quimicamente , Humanos , Lectinas Tipo C/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores de Superfície Celular/genética , SimbioseRESUMO
MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.
Assuntos
Anticorpos/imunologia , Epitopos Imunodominantes/imunologia , Mucina-1/imunologia , Conformação Proteica , Antígenos Glicosídicos Associados a Tumores/imunologia , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/ultraestrutura , Modelos Moleculares , Mucina-1/genética , Mucina-1/ultraestrutura , Ressonância Magnética Nuclear BiomolecularRESUMO
Few approaches exist for the stable and controllable synthesis of customized mucin glycoproteins for glycocalyx editing in eukaryotic cells. Taking advantage of custom gene synthesis and a biology-by-parts approach to cDNA construction, we build a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and newly designed domains for synthetic mucins. We construct a library of over 50 mucins, each with unique chemical, structural, and optical properties and describe how additional permutations could readily be constructed. We apply the library to explore sequence-specific effects on glycosylation for engineering of mucins. We find that the extension of the immature α-GalNAc Tn-antigen to Core 1 and Core 2 glycan structures depends on the underlying peptide backbone sequence. Glycosylation could also be influenced through recycling motifs on the mucin cytoplasmic tail. We expect that the mucin parts inventory presented here can be broadly applied for glycocalyx research and mucin-based biotechnologies.
Assuntos
Glicocálix/genética , Mucinas/genética , Antígenos Glicosídicos Associados a Tumores/genética , Linhagem Celular , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , HumanosRESUMO
The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.
Assuntos
Neoplasias do Colo/metabolismo , Glicopeptídeos/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Neoplasias do Colo/patologia , Epitopos , Humanos , Ligantes , Análise em Microsséries , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Células Tumorais CultivadasRESUMO
Permethylation is an essential technique for the detailed structural characterization of glycans by mass spectrometry. However, it requires specialized skills and equipment and is labor-intensive. While this limits glycan analysis to a handful of experts, the increasing awareness of the tremendous importance of glycans in physiological processes of health and disease has drastically raised the demand for detailed structural characterization of glycans. We have developed a simple microplate permethylation method that relies upon solid phase extraction using C18 tips to purify the permethylated glycans. The procedure is easy to perform, making it accessible to nonexperts, and fast, promising to accelerate glycan research. A 96-well plate of released glycan samples can be permethylated in less than an hour. The procedure can be carried out without discontinuous steps in an automatic pipet and integrated seamlessly with glycan release and mass spectrometry.
Assuntos
Polissacarídeos/química , Extração em Fase Sólida/métodos , Metilação , Estrutura Molecular , Polissacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC classâ II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.
Assuntos
Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Mucina-1/imunologia , Vacinas Sintéticas/imunologia , Animais , Vacinas Anticâncer/química , Glicopeptídeos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Conformação Molecular , Mucina-1/química , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/químicaRESUMO
In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)-a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.
Assuntos
Celulase/metabolismo , Polissacarídeos/metabolismo , Celulase/química , Ativação Enzimática , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Glicosilação , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Proteólise , Temperatura de TransiçãoRESUMO
To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MSn system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by ß elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.
Assuntos
Glicopeptídeos/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Aminoácidos/química , Animais , Sequência de Carboidratos , Bovinos , Cromatografia Líquida de Alta Pressão , Fetuínas/metabolismo , Glicosilação , Humanos , Metilação , Polissacarídeos/química , Ribonucleases/metabolismo , Transferrina/metabolismoRESUMO
In cancer cells, the glycoprotein Mucin 1 (MUC1) undergoes abnormal, truncated glycosylation. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. We investigated the role of tumor-specific glycosylation on antigen recognition by the therapeutic antibody AR20.5. We explored the affinity of AR20.5 to a synthetic cancer-specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than the naked peptide. Given these results, we postulated that AR20.5 must specifically bind the carbohydrate as well as the peptide. Using X-ray crystallography, we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific polar contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts support a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide recognized by the antibody. Since high affinity binding of AR20.5 to the MUC1 glycopeptide may not driven by specific antibody-antigen contacts, but rather evidence suggests that glycosylation alters the conformational equilibrium of the antigen, which allows the antibody to select the correct conformation. This study suggests a novel mechanism of antibody-antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Mucina-1/imunologia , Processamento de Proteína Pós-Traducional , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Glicosilação , Humanos , Hibridomas , Simulação de Acoplamento Molecular , Mucina-1/química , Mucina-1/metabolismo , Ligação ProteicaRESUMO
It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.
Assuntos
Vacinas Anticâncer/uso terapêutico , Mucina-1/genética , Mucina-1/imunologia , Neoplasias Experimentais/prevenção & controle , Peptídeos/uso terapêutico , Animais , Antígenos/química , Antígenos/imunologia , Antígenos/uso terapêutico , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Glicosilação , Humanos , Camundongos , Camundongos Transgênicos , Mucina-1/metabolismo , Neoplasias Experimentais/imunologia , Peptídeos/química , Peptídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Evasão TumoralRESUMO
The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc-ß-OMe with Ka ≈20,000 m(-1), whereas the other one binds an O-GlcNAcylated peptide with Ka ≈70,000 m(-1). These values substantially exceed those usually measured for GlcNAc-binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl-peptide complex can be explained by extra-cavity interactions, raising the possibility of a family of complementary receptors for O-GlcNAc in different contexts.
Assuntos
Acetilglucosamina/metabolismo , Receptores Artificiais/metabolismo , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Receptores Artificiais/químicaRESUMO
A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines.
RESUMO
A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T-cells primed by such a vaccine candidate could be restimulated by tumor-associated MUC1.