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

Microwave Effect for Glycosylation Promoted by Solid Super Acid in Supercritical Carbon Dioxide

The effects of microwave irradiation (2.45 GHz, 200 W) on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule

An efficient protocol for the solid-phase synthesis of glycopeptides under microwave irradiation

A standardized and smooth protocol for solid-phase glycopeptides synthesis under microwave irradiation was developed. Double activation system was proved to allow for highly efficient coupling of Tn-Ser/Thr and bulky core 2-Ser/Thr derivatives. Versatility and robustness of the present strategy was demonstrated by constructing a Mucine-1 (MUC1) fragment and glycosylated fragments of tau protein. The success of this approach relies on the combination of microwave energy, a resin consisting totally of polyethylene glycol, a low excess of sugar amino acid and the "double activation" method. © 2012 The Royal Society of Chemistry

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic a-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 a-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 a-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of a-dystroglycanopathy

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.We thank the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R, UNLR13-4E-1931, CTQ2013-44367-C2-2-P, CTQ2015-64597-C2-1P, and BFU2016-75633-P). I.A.B. thanks the Asociación Española Contra el Cancer en La Rioja for a grant. I.S.A. and G.J.L.B. thank FCT Portugal (Ph.D. studentship and FCT Investigator, respectively) and EPSRC. G.J.L.B. holds a Royal Society URF and an ERC StG (TagIt). F.C. and G.J.L.B thank the EU (Marie-Sklodowska Curie ITN, Protein Conjugates). R.H-G. thanks Agencia Aragonesa para la Investigación y Desarrollo (ARAID) and the Diputación General de Aragón (DGA, B89) for financial support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment mx10121-19). The Hokkaido University group acknowledges JSPS KAKENHI grant no. 25220206 and JSPS Wakate B KAKENHI grant no. 24710242. We also thank CESGA (Santiago de Compostela) for computer support.Peer reviewedPeer Reviewe

Aniline derivative/DHB/alkali metal matrices for reflectron mode MALDI-TOF and TOF/TOF MS analysis of unmodified sialylated oligosaccharides and glycopeptides

Sialylglycoconjugates play biologically important roles in nature. The labile nature of the sialic acid residue in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis demands additional modification processes of the carboxylate group to avoid in-source or post-source decay. This paper reports a versatile and high-sensitivity matrix system, aniline derivative/2,5-dihydroxybenzoic acid/alkali metal salt, for MALDI-MS analysis of sialyloligosaccharides and sialylglycoconjugates without any modifications. A simple adduct ion signal was obtained from the femtomole range of the analytes tested with the new matrix system by MALDI-MS in reflectron mode. Besides, the laser power-dependent in-source decay and post-source decay pattern of the sialylated analytes with this matrix system allows a pseudo MS3 strategy for in-depth sequencing studies of the analytes. (C) 2019 The Author. Published by Elsevier B.V

Direct MALDI Glycotyping of Glycoproteins toward Practical Subtyping of Biological Samples

The rapid analysis of glycan patterns (glycoforms) of glycoproteins can accelerate their quality control and biomarker discovery. We have focused on the direct analysis of glycoprotein glycoforms using matrix-assisted laser desorption/ionization in source decay mass spectrometry (MALDI-ISDMS), called MALDI glycotyping. Our results show that the 1,5-diaminonaphthalene (DAN)/2,5-dihydroxybenzoic acid (DHB)/Na matrix can directly analyze the glycoforms in the femtomolar range of intact glycoproteins. The addition of DAN improved the morphology of the solid matrix due to the mixture of DAN and DHB, which significantly contribute to the high sensitivity of this direct analysis. Adding DAN significantly improved the sensitivity of the glycan precursor ions in the TOF/TOF analysis because of its enhanced fragmentation effect as an efficient UV-MALDI matrix. Further, practical glycoform analysis (glycotyping) of diluted biological samples containing glycoproteins, such as egg whites, was also successfully achieved

Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the KI value with little change in the t_[1/2] value of the enzyme activity relative to the basic skeleton (2)