Fully Synthetic Self‐Adjuvanting Thioether‐Conjugated Glycopeptide?Lipopeptide Antitumor Vaccines for the Induction of Complement‐Dependent Cytotoxicity against Tumor Cells

Glycopeptides of tumor‐associated mucin MUC1 are promising target structures for the development of antitumor vaccines. Because these endogenous structures were weakly immunogenic, they were coupled to immune‐response‐stimulating T‐cell epitopes and the Pam₃Cys lipopeptide to induce strong immune responses in mice. A new thioether‐ligation method for the synthesis of two‐ and three‐component vaccines that contain MUC1 glycopeptides as the B‐cell epitopes, a T‐cell epitope peptide, and the Pam₃CSK₄ lipopeptide is described. The resulting fully synthetic vaccines were used for the vaccination of mice, either in a liposome with Freund′s adjuvant or in aqueous PBS buffer. The three‐component vaccines that contained the Tetanus Toxoid P2 T‐cell epitope peptide induced strong immune responses, even when administered just in PBS. By activation of the complement‐dependent cytotoxicity (CDC) complex, the antisera induced the killing of tumor cells.     

β-Mannosylation of N-acetyl glucosamine by propargyl mediated intramolecular aglycon delivery (IAD): synthesis of the N-glycan core pentasaccharide

An efficient and completely stereocontrolled synthesis of the N-glycan Manβ(1-4)GlcNAc disaccharide is achieved by propargyl mediated intramolecular aglycon delivery (IAD). Isomerisation of the 2-O-progargyl group of a manno thioglycoside to an allene is followed by iodonium ion mediated mixed acetal formation with the 4-OH of a protected GlcNAc derivative, and subsequent intramolecular glycosylation with complete control of anomeric stereochemistry. Access to this key disaccharide intermediate allows completion of the total synthesis of the core N-glycan pentasaccharide.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewis^x epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery. Graphical abstract

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Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation

Endoglycosidase is a class of glycosidases that specifically cleaves the glycosidic bond between two proximal residues of GlcNAc in the pentasaccharide core of N-glycan, leaving the innermost GlcNAc still attached to its parent protein, which provides a different diagnostic maker for N-glycosylation site assignment. This study aims to validate the use of endoglycosidase for high throughput N-glycosylation analysis. An endoglycosidase of Endo H and the conventional PNGase F were employed, with a similar accessible procedure, for large-scale assignment of N-glycosylation sites and then N-glycoproteome for rat liver tissue. ConA affinity chromatography was used to enrich selectively high-mannose and hybrid glycopeptides before enzymatic deglycosylation. As a result, a total of 1063 unique N-glycosites were identified by nano liquid chromatography tandem mass spectrometry, of which 53.0% were unknown in the Swiss-Prot database and 47.1% could be assigned only by either of the methods, confirmed the possibility of large-scale glycoproteomics by use of endoglycosidase. In addition, 11 glycosites were assigned with core-fucosylation by Endo H. A comparison between the two enzymatic deglycosylation methods was also investigated. Briefly, Endo H provides a more confident assignment but a smaller dataset compared with PNGase F, showing the complementary nature of the two N-glycosite assignment methods.     

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets.     

Rational design of hyper-glycosylated human luteinizing hormone analogs (a bioinformatics approach)

Glycoengineering is a recently used approach to extend serum half-life of valuable protein therapeutics. One aspect of glycoengineering is to introduce new N-glycosylation site (Asn-X-Thr/Ser, where X ≠ Pro) into desirable positions in the peptide backbone, resulting in the generation of hyper-glycosylated protein. In this study, human luteinizing hormone (LH) was considered for identification of the suitable positions for the addition of new N-linked glycosylation sites. A rational in silico approach was applied for prediction of structural and functional alterations caused by changes in amino acid sequence. As the first step, we explored the amino acid sequence of LH to find out desirable positions for introducing Asn or/and Thr to create new N-glycosylation sites. This exploration led to the identification of 38 potential N-glycan sites, and then the four acceptable ones were selected for further analysis. Three-dimensional (3D) structures of the selected analogs were generated and examined by the model evaluation methods. Finally, two analogs with one additional glycosylation site were suggested as the qualified analogs for hyper-glycosylation of the LH, which can be considered for further experimental investigations. Our computational strategy can reduce laborious and time-consuming experimental analyses of the analogs.     

Recent Advances in the Chemical Biology of N-Glycans

Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.     

Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N‐GlcNAcylation

As a unique and unappreciated protein posttranslational modification, arginine N‐glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N‐glycosylation, we report the first general strategy for a rapid and cost‐effective synthesis of glycopeptides carrying single or multiple arginine N‐GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N‐GlcNAcylated peptides or proteins in a sequence‐independent manner.     

From individual proteins to proteomic samples: characterization of <Emphasis Type="Italic">O</Emphasis>-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography–tandem mass spectrometry (LC–MS–MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC–MS–MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.     

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.     

Propargyl mediated intramolecular aglycon delivery (IAD): applications to the synthesis of core N-glycan oligosaccharides

The use of propargyl mediated intramolecular aglycon delivery (IAD) for the synthesis of the key Manβ(1→4)GlcNAc linkage of N-glycan oligosaccharides, including the core N-glycan pentasaccharide, is investigated. Isomerisation of a 2-O-progargyl group of manno thioglycoside donors to an allene is followed by iodonium ion mediated mixed acetal formation with the 4-OH of protected GlcNAc acceptors, and subsequent intramolecular glycosylation occurs with complete control of anomeric stereochemistry to form the Manβ(1→4)GlcNAc linkage. A variety of linear and convergent approaches (1+2, 3+1, 3+2) to the core pentasaccharide are investigated as means of probing the generality and limitations of this type of intramolecular aglycon delivery for the formation of β-mannoside linkages in complex oligosaccharides.     

Synthesis and Characterisation of Self‐Assembled and Self‐Adjuvanting Asymmetric Multi‐Epitope Lipopeptides of Ovalbumin

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host‐cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP‐based vaccine candidates that contained multiple CD4⁺ and CD8⁺ T‐cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave‐assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide–alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self‐assemble in aqueous media. Changes in lipid length and position induced self‐assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism.     

Site-Specific <Emphasis Type="Italic">N</Emphasis>-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

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Computational Modeling of O-Linked Glycan Biosynthesis in CHO Cells

Glycan biosynthesis simulation research has progressed remarkably since 1997, when the first mathematical model for N-glycan biosynthesis was proposed. An O-glycan model has also been developed to predict O-glycan biosynthesis pathways in both forward and reverse directions. In this work, we started with a set of O-glycan profiles of CHO cells transiently transfected with various combinations of glycosyltransferases. The aim was to develop a model that encapsulated all the enzymes in the CHO transfected cell lines. Due to computational power restrictions, we were forced to focus on a smaller set of glycan profiles, where we were able to propose an optimized set of kinetics parameters for each enzyme in the model. Using this optimized model we showed that the abundance of more processed glycans could be simulated compared to observed abundance, while predicting the abundance of glycans earlier in the pathway was less accurate. The data generated show that for the accurate prediction of O-linked glycosylation, additional factors need to be incorporated into the model to better reflect the experimental conditions.     

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

A molecular basis for glycosylation-induced conformational switching

Background: Asparagine-linked glycosylation has the capacity to greatly influence the structure and function of glycoproteins. In most cases, however, it is unclear specifically how the carbohydrate moiety interacts with the protein to influence its conformation.Results: A series of glycopeptides based on the critical A285 glycosylation site of the hemagglutinin glycoprotein from influenza virus was used as a model system to study the effects of asparagine-linked glycosylation. Derivatization of this peptide with a family of short carbohydrates reveals that subtle changes in the structure of the carbohydrate have a dramatic impact on peptide conformation. Modification of the hemagglutinin glycopeptide with a truncated version of the native carbohydrate induces a β-turn structure similar to the structure found in the native protein. Replacement of the C2 and C2′ N-acetyl groups of the carbohydrates with hydroxyl moieties results in a less well-ordered peptide conformation.Conclusions: It is likely that the N-acetyl groups of the carbohydrates have a critical role in promoting the more compact β-turn conformation through steric interactions with the peptide. This study has demonstrated that relatively small changes in carbohydrate composition can have dramatic ramifications on glycopeptide conformation.     

Synthesis of Asparagine Derivatives Harboring a Lewis X Type DC-SIGN Ligand and Evaluation of their Impact on Immunomodulation in Multiple Sclerosis

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post-translational N-glycosylation of Asn₃₁ of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis-type glycan structures in the N-glycan of MOG with the DC-SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (Le^X)-containing Fmoc-SPPS-compatible asparagine building block (SPPS=solid-phase peptide synthesis), as well as asparagine building blocks containing two Le^X-derived oligosaccharides: LacNAc and Fucα1-3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG_31-55) and analyzed with respect to their ability to bind to DC-SIGN in different biological setups, as well as their ability to inhibit the citrullination-induced aggregation of MOG_31-55. Finally, a cytokine secretion assay was carried out on human monocyte-derived DCs, which showed the ability of the neoglycopeptide decorated with a single Le^X to alter the balance of pro- and anti-inflammatory cytokines, inducing a tolerogenic response.     

Do Toxoplasma gondii apicoplast proteins have antigenic potential? An in silico study

Toxoplasma gondii, one of the extensively studied Apicomplexan parasites, is prevalent worldwide in animals and humans. Apart from its nuclear genome, T. gondii contains an apicoplast genome in 35 kb length which is originated from a secondary endosymbiotic event. In this study, we aimed to investigate the antigenic potential of apicoplast genome encoded proteins (n:28) of T. gondii using in silico analysis. For this purpose, proteins were primarily predicted to reveal antigenic probability and then, several bioinformatics analyses were applied for all predicted antigenic apicoplast proteins to analyze physico-chemical parameters, subcellular localization and transmembrane domain. Also, further prediction analyses including structural, B cell and MHC-I/II epitope sites as well as post-translational modifications were performed for antigenic proteins that have a signal peptide or a high antigenicity value. Of the 28 apicoplast proteins, 19 were predicted as probable antigen. Among antigenic proteins, ribosomal protein S5, L11 and S2 were predicted to have signal peptide whereas ribosomal protein L36 and S17 were predicted to have a significantly high antigenicity value (P < 0.05). In addition, ribosomal protein S5, L11, S2, L36 and S17 were predicted to have a lot of epitopes which have low IC50 and percentile rank value indicating a strong binding among epitopes and MHC-I/II alleles, and post-translational modifications such as N-linked glycosylation, acetylation and phosphorylation. To the best of authors’ knowledge this is the first study to show the antigenic potential and other properties of apicoplast-derived proteins of T. gondii.     

The Use of Reverse Vaccinology and Molecular Modeling Associated with Cell Proliferation Stimulation Approach to Select Promiscuous Epitopes from <Emphasis Type="Italic">Schistosoma mansoni</Emphasis>

Schistosomiasis remains an important parasitic disease that affects millions of individuals worldwide. Despite the availability of chemotherapy, the occurrence of constant reinfection demonstrates the need for additional forms of intervention and the development of a vaccine represents a relevant strategy to control this disease. With the advent of genomics and bioinformatics, new strategies to search for vaccine targets have been proposed, as the reverse vaccinology. In this work, computational analyses of Schistosoma mansoni membrane proteins were performed to predict epitopes with high affinity for different human leukocyte antigen (HLA)-DRB1. Ten epitopes were selected and along with murine major histocompatibility complex (MHC) class II molecule had their three-dimensional structures optimized. Epitope interactions were evaluated against murine MHC class II molecule through molecular docking, electrostatic potential, and molecular volume. The epitope Sm141290 and Sm050890 stood out in most of the molecular modeling analyses. Cellular proliferation assay was performed to evaluate the ability of these epitopes to bind to murine MHC II molecules and stimulate CD4+ T cells showing that the same epitopes were able to significantly stimulate cell proliferation. This work showed an important strategy of peptide selection for epitope-based vaccine design, achieved by in silico analyses that can precede in vivo and in vitro experiments, avoiding excessive experimentation.