RESUMO
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
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
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 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
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.