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Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward β-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.

contract DL-57/2016 WT108430/Z/15/Z WT218304/Z/19/Z 22-FY18-821 A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health. publishersversion published

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

Fundação para a Ciência e Tecnologia (FCT) -PTDC/BII-BIO/28878/2017; co-financed by ERDF under the PT2020 Partnership Agreement LISBOA-01-0145-FEDER-028878; PD/BD/128251/2016 from MIT-PT PhD Bioengineering Systems to MJBM; SFRH/BPD/112543/2015 to AJMB; Applied Molecular Biosciences Unit – UCIBIO (UIDP/04378/2020 ; UIDB/04378/2020); CICECO-Aveiro Institute of Materials (UIDB/50011/2020; UIDP/50011/2020), financed by national funds from FCT. Antibody technologies are the most representative success-case in the biopharmaceutical industry. Widely available purification technologies fail in providing a dedicated universal purification platform that can accommodate antibodies structural diversity, namely antibodies from non-human sources, as chicken IgY, and antigen-binding fragments. In this work, we took inspiration from natural and engineered antibody-binding ligands, to rationally design affinity adsorbents able to capture full-length antibodies and fragments. The one-pot Petasis and Ugi combinatorial reactions were sequentially employed to rapidly generate a library of putative solid-phase adsorbents. The best performing adsorbent yielded a single-step recovery, under mild conditions, of human and chicken whole antibodies, antigen-binding fragments and engineered single-domain antibodies from different complex feedstocks. Due to its simple preparation, the lead antibody adsorbent finds broad applicability as a universal purification platform to increase the availability of antibody technologies in research and development. authorsversion published

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment., We thank Gilberto Fronza (from Mutagenesi e Prevenzione Oncologica, Ospedale Policlinico San Martino, Genova, Italy), for providing us with the pLS76 vector. We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities and access to beamline ID30B. This work received financial support from the European Union (FEDER, Fundo Europeu de Desenvolvimento Regional, funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade–COMPETE) and National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, and projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014–POCI-01-0145-FEDER-016581 and RECI/BBB-BEP/0124/2012. FCT fellowships: PD/BD/114046/2015 (Ana Sara Gomes) and SFRH/BD/96189/2013 (Sara Gomes) (thanks FCT PhD Doctoral Programme BiotechHealth), and SFRH/BPD/110640/2015 (Carla Oliveira)., info:eu-repo/semantics/publishedVersion

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90 % of adenocarcinomas. Additionally, the binding of the TF antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction have the potential to prevent cancer metastasis. A multidisciplinary approach combining the optimized synthesis of a TF antigen mimetic with NMR, X-ray crystallography methods, and isothermal titration calorimetry assays was used to unravel the molecular structural details that govern the Gal-3/TF mimetic interaction. The TF mimetic has a binding affinity for Gal-3 similar to that of the TF natural antigen and retains the binding epitope and bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective, a decrease in the enthalpic contribution was observed for the Gal-3/TF mimetic complex; however, this behavior is compensated by a favorable gain in entropy. From a structural perspective, these results establish our TF mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and for likely use in hampering Gal-3-mediated cancer cell adhesion and metastasis.