ABSTRACT
Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.
Subject(s)
Mucin-1 , Vaccines , Humans , Mucin-1/chemistry , Galactose/metabolism , Glycopeptides/chemistry , Antigens, Tumor-Associated, Carbohydrate/chemistry , Lectins, C-Type/metabolism , Dendritic Cells , Macrophages/metabolismABSTRACT
Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.
Subject(s)
Legionella pneumophila , Legionella , Legionnaires' Disease , Humans , Legionella/metabolism , Phosphorylcholine/metabolism , Legionella pneumophila/metabolism , Protein Processing, Post-Translational , Bacterial Proteins/metabolism , rab1 GTP-Binding Proteins/metabolismABSTRACT
Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P.â aeruginosa lectin LecB and the C.â difficile toxinâ A.
Subject(s)
Clostridioides difficile , Mucins , Lectins/metabolism , Fucose/metabolism , Glycopeptides , Polysaccharides/metabolismABSTRACT
[This corrects the article DOI: 10.1016/j.isci.2020.101800.].
ABSTRACT
To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.
Subject(s)
Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Protein Processing, Post-Translational , Tetratricopeptide Repeat , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , HEK293 Cells , Heat-Shock Proteins/chemistry , Homeostasis , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Dynamics Simulation , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/genetics , Protein Binding , Protein Conformation , Substrate SpecificityABSTRACT
MUC1 glycopeptides are attractive antigens for anti-cancer vaccine development. One potential drawback in using the native MUC1 glycopeptide for vaccine design is the instability of the O-glycosyl linkage between the glycan and the peptide backbone to glycosidase. To overcome this challenge, a MUC1 glycopeptide mimic has been synthesized with the galactose-galactosamine disaccharide linked with threonine (Thomsen-Friedenreich or Tf antigen) through an unnatural ß-glycosyl bond. The resulting MUC1-ß-Tf had a much-enhanced stability toward a glycosidase capable of cleaving the glycan from the corresponding MUC1 glycopeptide with the natural α-Tf linkage. The MUC1-ß-Tf was subsequently conjugated with a powerful carrier bacteriophage Qß. The conjugate induced high levels of IgG antibodies in clinically relevant human MUC1 transgenic mice, which cross-recognized not only the natural MUC1-α-Tf glycopeptide but also MUC1 expressing tumor cells, supporting the notion that a simple switch of the stereochemistry of the glycan/peptide linkage can be a strategy for anti-cancer vaccine epitope design for glycopeptides.
Subject(s)
Antigens, Tumor-Associated, Carbohydrate/chemistry , Cancer Vaccines/chemistry , Glycopeptides/chemistry , Mucin-1/chemistry , Animals , Cancer Vaccines/immunology , Cell Line, Tumor , Cell Survival/drug effects , Disaccharides/chemistry , Drug Design , Galactosamine/chemistry , Galactose/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/pharmacology , Mice , Mice, Transgenic , Mucin-1/immunologyABSTRACT
Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.
Subject(s)
Adenosine Monophosphate/metabolism , Bacterial Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Legionella pneumophila/pathogenicity , Legionnaires' Disease/pathology , rab1 GTP-Binding Proteins/metabolism , Allosteric Regulation , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/ultrastructure , Binding Sites/genetics , Crystallography, X-Ray , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/isolation & purification , Guanine Nucleotide Exchange Factors/ultrastructure , Guanosine Triphosphate/metabolism , Humans , Legionella pneumophila/metabolism , Legionnaires' Disease/microbiology , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/microbiology , Phagocytosis , Protein Binding , Protein Processing, Post-Translational , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , rab1 GTP-Binding Proteins/genetics , rab1 GTP-Binding Proteins/isolation & purification , rab1 GTP-Binding Proteins/ultrastructureABSTRACT
AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.
ABSTRACT
Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bartonella/metabolism , Biocatalysis , Crystallography, X-Ray , HeLa Cells , Humans , Membrane Proteins/chemistry , Nucleotidyltransferases/chemistry , Pasteurellaceae/metabolism , Protein Processing, Post-Translational , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolismABSTRACT
Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qß carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qß were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qß-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qß-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qß-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.
Subject(s)
Cancer Vaccines/therapeutic use , Glycopeptides/therapeutic use , Immunoconjugates/therapeutic use , Mucin-1/immunology , Peptide Fragments/therapeutic use , Viral Proteins/therapeutic use , Allolevivirus/chemistry , Amino Acid Sequence , Animals , Antigens, Tumor-Associated, Carbohydrate/immunology , Cancer Vaccines/chemical synthesis , Cancer Vaccines/immunology , Cell Line, Tumor , Female , Glycopeptides/chemical synthesis , Glycopeptides/immunology , Humans , Immunoconjugates/immunology , Immunoglobulin G/immunology , Lung Neoplasms/therapy , Male , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Viral Proteins/chemical synthesis , Viral Proteins/immunologyABSTRACT
Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.
Subject(s)
Antigens, CD/chemistry , Antigens, Differentiation, Myelomonocytic/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Lectins/chemistry , Metalloendopeptidases/chemistry , Mucins/chemistry , Neoplasm Proteins/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins/chemistry , Amino Acid Motifs , Humans , Mass Spectrometry , Substrate SpecificityABSTRACT
Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.
Subject(s)
Breast Neoplasms/therapy , Cancer Vaccines/therapeutic use , Epitopes/immunology , Mucin-1/immunology , Allolevivirus/chemistry , Amino Acid Sequence , Animals , Antibodies/immunology , Antibodies/metabolism , Breast Neoplasms/immunology , Cancer Vaccines/immunology , Female , Gastropoda/chemistry , Hemocyanins/chemical synthesis , Hemocyanins/chemistry , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Mucin-1/chemistry , Mucin-1/genetics , Neoplasm Metastasis/immunology , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Viral Proteins/chemical synthesis , Viral Proteins/chemistryABSTRACT
Distinct structural changes of the α2,3/α2,6-sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high-throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large-scale glycoproteomics, thus allowing the site-specific structural characterization of sialic acid isomers.
Subject(s)
Proteomics , Sialic Acids/chemistry , Carbohydrate Conformation , Chromatography, Liquid , Stereoisomerism , Tandem Mass SpectrometryABSTRACT
Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. In order to generate effective anti-MUC1 immune responses as potential anticancer vaccines, MUC1 peptides and glycopeptides have been covalently conjugated to bacteriophage Qß. Immunization of mice with these constructs led to highly potent antibody responses with IgG titers over one million, which are among the highest anti-MUC1 IgG titers reported to date. Furthermore, the high IgG antibody levels persisted for more than six months. The constructs also elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The unique abilities of Qß-MUC1 conjugates to powerfully induce both antibody and cytotoxic T cell immunity targeting tumor cells bode well for future translation of the constructs as anticancer vaccines.
Subject(s)
Bacteriophages/immunology , Cancer Vaccines/immunology , Immunity, Humoral/immunology , Immunoglobulin G/immunology , Lymphocyte Activation/immunology , Mucin-1/immunology , Amino Acid Sequence , Animals , Antibody Formation/immunology , Bacteriophages/chemistry , Cancer Vaccines/chemical synthesis , Cell Line, Tumor , Humans , Immunization , Lymphoma/immunology , Mice, Inbred C57BL , Microarray Analysis , Mucin-1/chemistry , T-Lymphocytes, Cytotoxic/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunologyABSTRACT
Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain development. Recent advances in glycoproteomics methodology and the "SimpleCell" strategy have enabled rapid identification of glycoproteins and specific glycosylation sites. Despite the enormous progress made, the biological impact of the mammalian O-mannosyl glycoproteome remains largely unknown to date. Tools are still needed to investigate the structure, role, and abundance of O-mannosyl glycans. Although O-mannosyl branching has been shown to be of relevance in integrin-dependent cell migration, and also plays a role in demyelinating diseases, such as multiple sclerosis, a broader understanding of the biological roles of branched O-mannosyl glycans is lacking in part due to the paucity of detection tools. In this work, a glycopeptide vaccine construct was synthesized and used to generate antibodies against branched O-mannosyl glycans. Glycopeptide microarray screening revealed high selectivity of the induced antibodies for branched glycan core structures presented on different peptide backbones, with no cross-reactivity observed with related linear glycans. For comparison, microarray screening of the mannose-binding lectin concanavalin A (ConA), which is commonly used in glycoproteomics workflows to enrich tryptic O-mannosyl peptides, showed that the ConA lectin did not recognize branched O-mannosyl glycans. The binding preference of ConA for short linear O-mannosyl glycans was rationalized in terms of molecular structure using crystallographic data augmented by molecular modeling. The contrast between the ConA binding specificity and that of the new antibodies indicates a novel role for the antibodies in studies of protein O-mannosylation.
Subject(s)
Antibodies/immunology , Concanavalin A/immunology , Glycopeptides/immunology , Mannose/immunology , Amino Acid Sequence , Amino Acids/chemistry , Binding Sites , Glycosylation , Lectins/chemistry , Mannose/chemistry , Nanoparticles , Polysaccharides/chemistry , Protein Array Analysis/methods , Protein Binding , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases. In the current work, three MUC1 antitumor vaccine candidates conjugated with different immune stimulants are evaluated immunologically. For assessment of the influence of the immune stimulant on antibody recognition, a comprehensive library of mucin 1 glycopeptides (>100 entries) is synthesized and employed in antibody microarray profiling; these range from small tumor-associated glycans (TN , STN , and T-antigen structures) to heavily extended O-glycan core structures (type-1 and type-2 elongated core 1-3 tri-, tetra-, and hexasaccharides) glycosylated in variable density at the five different sites of the MUC1 tandem repeat. This is one of the most extensive glycopeptide libraries ever made through total synthesis. On tumor cells, the coreâ 2 ß-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched coreâ 2 structures, which favor formation of linear coreâ 1 or coreâ 3 structures, and in particular, truncated tumor-associated antigen structures. The coreâ 2 structures are commonly found on healthy cells and the elucidation of antibody cross-reactivity to such epitopes may predict the tumor-selectivity and safety of synthetic vaccines. With the extended mucin core structures in hand, antibody cross-reactivity toward the branched coreâ 2 glycopeptide epitopes is explored. It is observed that the induced antibodies recognize MUC1 peptides with very high glycosylation site specificity. The nature of the antibody response is characteristically different for antibodies directed to glycosylation sites in either the immune-dominant PDTR or the GSTA domain. All antibody sera show high reactivity to the tumor-associated saccharide structures on MUC1. Extensive glycosylation with branched coreâ 2 structures, typically found on healthy cells, abolishes antibody recognition of the antisera and suggests that all vaccine conjugates preferentially induce a tumor-specific humoral immune response.
Subject(s)
Cancer Vaccines/immunology , Glycopeptides/immunology , Mucin-1/immunology , Neoplasms/immunology , Polysaccharides/immunology , Vaccines, Synthetic/immunology , Animals , Antibody Formation , Cancer Vaccines/chemistry , Glycopeptides/chemistry , Humans , Immunity, Humoral , Mice , Mucin-1/chemistry , Neoplasms/therapy , Polysaccharides/chemistry , Protein Array Analysis , Vaccines, Synthetic/chemistryABSTRACT
Post-translational glycosylation of proteins play key roles in cellular processes and the site-specific characterisation of glycan structures is critical to understanding these events. Given the challenges regarding identification of glycan isomers, glycoproteomic studies generally rely on the assumption of conserved biosynthetic pathways. However, in a recent study, we found characteristically different HexNAc oxonium ion fragmentation patterns that depend on glycan structure. Such patterns could be used to distinguish between glycopeptide structural isomers. To acquire a mechanistic insight, deuterium-labelled glycopeptides were prepared and analysed. We found that the HexNAc-derived m/z 126 and 144 oxonium ions, differing in mass by H2 O, had completely different structures and that high-mannose N-glycopeptides generated abundant Hex-derived oxonium ions. We describe the oxonium ion decomposition mechanisms and the relative abundance of oxonium ions as a function of collision energy for a number of well-defined glycan structures, which provide important information for future glycoproteomic studies.
Subject(s)
Glycopeptides/chemistry , Onium Compounds/chemistry , Polysaccharides/chemistry , Glycopeptides/metabolism , Glycosylation , Isomerism , Mass Spectrometry , Tandem Mass SpectrometryABSTRACT
Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcß1-O-, Galß3GalNAcα1-O-, Galß4GlcNAcß-O-, and Galß3GlcNAcß-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
Subject(s)
Chromatography, Liquid/methods , Glycopeptides/metabolism , Oligosaccharides/metabolism , Onium Compounds/metabolism , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Carbohydrate Sequence , Glycopeptides/analysis , Glycosylation , Molecular Sequence Data , Molecular Structure , Oligosaccharides/chemistry , Onium Compounds/analysis , Polysaccharides/chemistry , Polysaccharides/metabolism , Proteome/analysis , Proteome/metabolism , Proteomics/methodsABSTRACT
Mucins are a class of highly O-glycosylated proteins found on the surface of cells in epithelial tissues. O-Glycosylation is crucial for the functionality of mucins and changes therein can have severe consequences for an organism. With that in mind, the elucidation of interactions of carbohydrate binding proteins with mucins, whether in morbidly altered or unaltered conditions, continue to shed light on mechanisms involved in diseases like chronic inflammations and cancer. Despite the known importance of type-1 and type-2 elongated mucin cores 1-4 in glycobiology, the corresponding type-1 structures are much less well studied. Here, the first chemical synthesis of extended mucin type-1 O-glycan core 1-3 amino acid structures based on a convergent approach is presented. By utilizing differentiation in acceptor reactivity, shared early stage Tn- and T-acceptor intermediates were elongated with a common type-1 [ß-D-Gal-1,3-ß-D-GlcNAc] disaccharide, which allows for straightforward preparation of diverse glycosylated amino acids carrying the type-1 mucin core 1-3 saccharides. The obtained glycosylated 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid building blocks were employed in synthesis of type-1 mucin glycopeptides, which are useful in biological applications.