Glycoblotting enables seamless and straightforward workflow for MALDI-TOF/MS-based sulphoglycomics of N- and O-glycans.

Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings' on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ.

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.

Sodium-Doped 3-Amino-4-hydroxybenzoic Acid: Rediscovered Matrix for Direct MALDI Glycotyping of O-Linked Glycopeptides and Intact Mucins.

3-Amino-4-hydroxybenzoic acid (AHB) was the first matrix identified by glycoprotein glycan analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, compared to commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), AHB is less efficient at glycan ionization and lacks the ability to ionize other molecular species, such as peptides, and thus is no longer used. In this study, we focused on the glycan-selective ionization ability of AHB and its low-noise properties in the low-molecular-weight region, as we expected that these properties could be enhanced by adding sodium to AHB. Sodium-doped AHB (AHB/Na) selectively imparts sodium adduct ions onto O-glycan fragments generated by the in-source decay (ISD) of glycopeptides and glycoproteins containing O-glycans that occurs during intense laser irradiation, enabling direct O-glycan analysis. Furthermore, we demonstrated that it is possible to investigate the internal structure of each O-glycan fragment with pseudo-MS/MS/MS using the sodium adduct ion of the O-glycan-derived ISD fragments from an intact mucin mixture.

BOA/DHB/Na: An Efficient UV-MALDI Matrix for High-Sensitivity and Auto-Tagging Glycomics.

Matrix selection is a critical factor for success in glycomics studies using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). In this study, we evaluated and optimized a new solid ionic matrix-O-benzylhydroxylamine (BOA)/2,5-dihydroxybenzoic acid (DHB)/Na-containing BOA and a small amount of sodium as the counter salt of DHB. The concentration of a mixture of BOA/DHB/Na and glycans on a MALDI target plate led to O-benzyloxy tagging of the reducing ends of the glycans. The BOA/DHB/Na matrix showed excellent aggregation performance and the ability to form a homogeneous solid salt on the MALDI target plate with a water-repellent surface. In addition, the BOA/DHB/Na matrix showed a simple peak pattern with suppressed in-source and post-source decay of the reducing ends of the glycans, as well as improved ionization efficiency of glycans. Utilizing the characteristics of the BOA/DHB/Na matrix, O-glycan analysis of porcine stomach mucin showed excellent detection sensitivity and reproducibility of the peak patterns. This BOA/DHB/Na matrix can accelerate glycomics studies using MALDI-MS and, in combination with other organic salt-type matrices that we have developed, constitutes a valuable tool for glycomics studies.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Click Synthesis of Size- and Shape-Tunable Star Polymers with Functional Macrocyclic Cores for Synergistic DNA Complexation and Delivery.

The architectural perfection and multivalency of dendrimers have made them useful for biodelivery via peripheral functionalization and the adjustment of dendrimer generations. Modulation of the core-forming and internal matrix-forming structures offers virtually unlimited opportunities for further optimization, but only in a few cases this has been made compatible with strict diastereomeric purity over molecularly diverse series, low toxicity, and limited synthetic effort. Fully regular star polymers built on biocompatible macrocyclic platforms, such as hyperbranched cyclodextrins, offer advantages in terms of facile synthesis and flexible compositions, but core elaboration in terms of shape and function becomes problematic. Here we report the synthesis and characterization of star polymers consisting of functional trehalose-based macrocyclic cores (cyclotrehalans, CTs) and aminothiourea dendron arms, which can be efficiently synthesized from sequential click reactions of orthogonal monomers, display no cytotoxicity, and efficiently complex and deliver plasmid DNA in vitro and in vivo. When compared with some commercial cationic dendrimers or polymers, the new CT-scaffolded star polymers show better transfection efficiencies in several cell lines and structure-dependent cell selectivity patterns. Notably, the CT core could be predefined to exert Zn(II) complexing or molecular inclusion capabilities, which has been exploited to synergistically boost cell transfection by orders of magnitude and modulate the organ tropism in vivo.

Exploring serum and immunoglobulin G N-glycome as diagnostic biomarkers for early detection of breast cancer in Ethiopian women.

BACKGROUND: Alterations in protein glycosylation patterns have potentially been targeted for biomarker discovery in a wide range of diseases including cancer. Although there have been improvements in patient diagnosis and survival for breast cancer (BC), there is no clinically validated serum biomarker for its early diagnosis. Here, we profiled whole serum and purified Immunoglobulin G (IgG) fraction N-glycome towards identification of non-invasive glycan markers of BC. METHODS: We employed a comprehensive glycomics approach by integrating glycoblotting-based glycan purification with MALDI-TOF/MS based quantitative analysis. Sera of BC patients belonging to stages I-IV and normal controls (NC) were collected from Ethiopian women during 2015-2016. IgG was purified by affinity chromatography using protein G spin plate and further subjected to glycoblotting for glycan release. Mass spectral data were further processed and evaluated rigorously, using various bioinformatics and statistical tools. RESULTS: Out of 35 N-glycans that were significantly up-regulated in the sera of all BC patients compared to the NC, 17 complex type N-glycans showed profound expression abundance and diagnostic potential (AUC = 0.8-1) for the early stage (I and II) BC patients. Most of these glycans were core-fucosylated, multiply branched and sialylated structures, whose abundance has been strongly associated with greater invasive and metastatic potential of cancer. N-glycans quantified form IgG confirmed their abundance in BC patients, of which two core-fucosylated and agalactosylated glycans (m/z 1591, 1794) could specifically distinguish (AUC = 0.944 and 0.921, p ≤ 0.001) stage II patients from NC. Abundance of such structural features in IgG is associated with a decrease in its immunosuppressive potential towards tumor cells, which in part may correlate with the aggressive nature of BC commonly noticed in black population. CONCLUSIONS: Our comprehensive study has addressed for the first time both whole serum and IgG N-glycosylation signatures of native black women suffering from BC and revealed novel glyco-biomarkers with marked overexpression and distinguishing ability at early stage patients. Further studies on direct identification of the intact glycoproteins using a glycoprteomics approach will provide a deeper understanding of specific biomarkers towards their clinical utility.

Synthetic glycopeptides reveal specific binding pattern and conformational change at O-mannosylated position of α-dystroglycan by POMGnT1 catalyzed GlcNAc modification.

Structural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, 372TRGAIIQTPTLGPIQPTRV390. Evanescent-field fluorescence-assisted microarray system illuminated the specific binding pattern of plant lectins that can discriminate the glycan structure of core M1 glycan of the library. The comparative NMR analysis of synthetic glycopeptide having different length of the O-mannosylated glycans revealed a conformational change of the peptide backbone along with core M1 disaccharide formation. No long-range NOE signals of glycan-amino acid nor inter amino acid indicate the conformational change is induced by steric hindrance of core M1, the sole 1,2-O-modified form among protein binding sugar residue found in mammals.

Further applications of classical amide coupling reagents: Microwave-assisted esterification on solid phase.

Ester linkage (s) is a key chemical connector in organic chemistry, including natural products, peptides, and synthetic polymers. We herein describe a straightforward method for the efficient formation of ester linkage (s) on solid-phase. This method simply involves the use of amide coupling reagents under microwave irradiation. The robustness of this method relies on the use of classical solid-phase coupling reagents, heating by microwave irradiation, and a short time period, which results in high yields and the minimization of racemization.

Chemical Synthesis Demonstrates That Dynamic O-Glycosylation Regulates the Folding and Functional Conformation of a Pivotal EGF12 Domain of the Human NOTCH1 Receptor.

The interaction of the human NOTCH1 receptor and its ligands is a crucial step in initiating the intracellular signal transductions, in which O-glycosylation of the extracellular EGF-like domain strongly affects multiple aspects of cell differentiation, development, and cancer biology. However, consequences of biosynthetic O-glycosylation processes in the endoplasmic reticulum (ER) and Golgi on the folding of EGF domains remain unclear. Synthetic human NOTCH1 EGF12 modules allow for new insight into the crucial roles of O-glycosylation in the folding and conformation of this pivotal domain. Here, we show for the first time that predominant O-glucosylation at Ser458 facilitates proper folding of the EGF12 domain in the presence of calcium ion, while the nonglycosylated linear EGF12 peptide affords large amounts of misfolded products (>50%) during in vitro oxidative folding. Strikingly, O-fucosylation at Thr466 prior to O-glucosylation at Ser458 totally impedes folding of EGF12 independent of calcium ion, whereas modification of the Fucα1→ moiety with ß-linked GlcNAc dramatically enhances folding efficiency. In addition, we elicit that extension of the Glcß1→ moiety with xyloses is a negative-regulation mechanism in the folding of EGF12 when synthesis of a trisaccharide (Xylα1→3Xylα1→3Glcß1→) dominates over the posttranslational modification at Thr466. Comprehensive nuclear magnetic resonance studies of correctly folded EGF12 modules demonstrate that noncovalently bonded bridges between sugars and peptide moieties, namely sugar bridges, contribute independently to the stabilization of the antiparallel ß-sheet in the ligand-binding region. Our results provide evidence that the dynamic O-glycosylation status of the EGF12 domain elaborated in the ER and Golgi strongly affects folding and trafficking of the human NOTCH1 receptor.

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

BACKGROUND: Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. METHODS: We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). RESULTS: The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. CONCLUSION: Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. GENERAL SIGNIFICANCE: The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

An Efficient Glycoblotting-Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein.

Although widely occurring lipid oxidation, which is triggered by reactive oxygen species (ROS), produces a variety of oxidized lipids, practical methods to efficiently analyze oxidized lipids remain elusive. Herein, it is shown that the glycoblotting platform can be used to analyze oxidized lipids. Analysis is based on the principle that lipid aldehydes, one of the oxidized lipid species, can be captured selectively, enriched, and detected. Moreover, 3-methyl-1-p-tolyltriazene (MTT) methylates phosphoric and carboxylic acids, and this MTT-mediated methylation is, in combination with conventional tandem mass spectrometry (MS/MS) analysis, an effective method for the structural analysis of oxidized lipids. By using three classes of standards, liposomes, and a lipoprotein, it is demonstrated that glycoblotting represents a powerful approach for focused lipidomics, even in complex macromolecules.

Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region.

The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a ß-D-glucopyranose-initiated glycan (Xylα1 → 3Xylα1 → 3Glcß1 →) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2 → 3Galß1 → 4GlcNAcß1 → 3Fucα1 →) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel ß-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.

A comprehensive glycome profiling of Huntington's disease transgenic mice.

BACKGROUND: Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. METHODS: Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. RESULTS: We estimated the structure and expression levels of 87 and 58N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. CONCLUSION: Total glycome expression levels are significantly different between HD transgenic and control group mice. GENERAL SIGNIFICANCE: Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era.

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases.

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides.

BACKGROUND: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. METHODS: A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. RESULTS: Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galß1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. CONCLUSION: We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. GENERAL SIGNIFICANCE: The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.

The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

Fast epitope mapping for the anti-MUC1 monoclonal antibody by combining a one-bead-one-glycopeptide library and a microarray platform.

Anti-MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer-related MUC1 molecules, the O-glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) "one-bead-one-compound"-based preparation of bilayer resins carrying glycopeptides on the shell and mass-tag tripeptides coding O-glycan patterns in the core, ii) on-resin screening with an anti-MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass-tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O-glycosylations against anti-MUC1 mAb clone VU-3C6. Qualitative mass-tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray-based assay. Our screening provides valuable information on O-glycosylations of epitopes leading to high affinity with mAb.

Synthesis of neoglycosphingolipid from methoxyamino-functionalized ceramide.

An efficient approach for the synthesis of a methoxyamino-functionalized ceramide was established from phytosphingosine using specific Nß→Nα acyl migration of the octadecanoyl group during the removal of Nα-Fmoc protective group. One step glycoblotting reaction of the ceramide mimic with lactose afforded a neoglycosphingolipid showing potent inhibitory activity against recombinant endoglycoceramidase II from Rhodococcus sp.