Unveiling Molecular Recognition of Sialoglycans by Human Siglec-10

29 p.-6 fig.-2 tab.-7 fig. supl.-2 tab. supl.-1 graph. abst.Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality.This study was supported by the project ‘‘GLYTUNES’’ funded by MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2017) (2017XZ2ZBK, 2019–2022) to A.S.; by progetto POR SATIN and Progetto POR CampaniaOncoterapia to A.M.; by the European Commission (H2020-MSCA- 814102-SWEET CROSSTALK project) to A.M., R.M., and A.S.. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement No 851356 to R.M. FSE,PON Ricerca e Innovazione 2014–2020, Azione I.1 ‘‘Dottorati Innovativi con caratterizzazione Industriale’’ is acknowledged for funding the PhD grant to R.E.F. Grants by the Spanish Ministry of Science MICINN (CTQ2017-88353-R and fellowship BES 2015–071588 to J.G.-C.) and Wellcome Trust 103744/Z/14/Z to P.R.C. are acknowledged.Peer reviewe

NMR and computational studies of the molecular recognition of eukaryotic glycans by receptor proteins

All living cells are covered by a layer of glycans at the interface between the environment and the cell membrane, capable of mediating cellular behavior, including critical mechanisms in immunoregulation and pathological processes. The molecular recognition of glycoconjugates from several proteins triggers a plethora of biological functions, especially in the infection process, immune response, and inflammation. Within this frame, interactions between glycans and their binding partners at molecular level have been studied, using a multidisciplinary approach of advanced NMR techniques, including ligand- and protein-based approaches, in combination with biophysical and computational methodologies, such as docking and molecular dynamic simulations. Siglecs (Sialic acid-binding immunoglobulin-type lectins) exploit a major application in the immune system regulation, recognizing glycans containing sialic acid. Indeed, in their cytoplasmic region, Siglecs contain one or multiple tyrosine-based signaling motifs that trigger cellular signaling, inhibiting the immune cell activation. In this thesis, the molecular bindings of different inhibitory Siglecs, in particular Siglec-2 and -7, containing cytosolic immunoreceptor tyrosine-based inhibition motifs (ITIMs), have been investigated with several glycoconjugates. Although the inhibition of immune system plays a fundamental role in some aberrant events, such as the over-reaction of response against self-molecules that often leads to produce autoimmune diseases, it is worth knowing that many pathogens have evolved the ability to cover their surfaces of sialic acids, subverting the immune system and dampening the host immune recognition. Thus, Siglecs have been studied as attractive targets for the design of therapeutic agents, such as antibodies or glycomimetics, for the treatment of inflammatory, autoimmune, and infectious diseases. In the case of Siglec-2, or CD22, the binding mode with complex-type N-glycans has been assessed, showing the possibility to form CD22 homo-oligomers on the B-cell surface, favoring the cis interactions on the same cell. As for Siglec-7, mainly located on NK cells, novel structural insights have been provided on its binding to sialylated lipopolysaccharides on different strains of the oncogenic pathogen F. nucleatum, with the aim to develop therapies for the modulation of both Siglec-7 activity and host-pathogen binding. On the other hand, bacterial adhesins, also implicated in the biology of infection, as in the bacterial pathogenesis, have been studied in interaction with their cognate ligands. In particular, Siglec-like adhesins, similar to Siglecs in the V-set N-terminal domain of sialoglycan recognition, are serine-rich repeat glycoproteins involved in the pathogenesis of infective endocarditis. In this context, the binding site of Siglec-like adhesins expressed on different strains of Streptococci has been investigated in interaction with a variety of sialylated N- and O-glycans. Partially related systems have been also investigated during the PhD, involving the study of the interactions between monoclonal antibodies (mAb) against bacterial glycoconjugates (and mimetics). Therefore, the molecular details of different glycans recognized by mammalian and bacterial proteins, as well as monoclonal antibodies, that play roles in health and disease, or host-pathogen interactions have been unveiled to provide a tool for the design of glycomimetics for therapeutic targets of human diseases

Evaluation of Colostral Immunity Against Equine Herpesvirus Type 1 (EHV-1) in Martina Franca's Foals

Eight Martina Franca pregnant jennies were selected in order to evaluate the transfer of colostral antibodies against equine herpesvirus type 1 in their relative foals after immunization with a commercial inactivated vaccine, compared with an unvaccinated group. Samples of serum and colostrums/milk were collected from jennies and foals under study starting from 10 min before and up to 21 days after the foaling. Specific anti-EHV-1 antibody titers were evaluated by means of a serum neutralization test, and the results obtained from both groups were analyzed. The serological titers in the vaccinated jennies was significantly higher (p < 0.01). No significant differences were found in the specific time-point intervals in both groups examined (p > 0.05). The antibody titers in milk at the time of delivery and subsequent withdrawal (T0 and T1) were very high in both groups, but no significant differences were found between the two groups (p > 0.05). In the foal sera, a significant difference was found between foals in the vaccinated group compared with those in the unvaccinated group (p < 0.05). Finally, a significant correlation (p < 0.05) was observed between the antibody titers found in serum and colostrum of jennies and the foal titers in the first time-point sampling (up to 12 h after foaling). The results confirm a substantial homology in the antibody production compared with other most investigated equids, highlighting the efficacy of the vaccination against EHV-1 of the jennies to ensure the protective immunity to their foals during the first weeks after delivery

Solving the structural puzzle of bacterial glycome

The analysis of the bacterial glycome (glycomics) is among the complex 'omics' analysis owing to the inherent difficulties in structural and functional characterization of glycans. The complexity and variability of bacterial glycans, spanning from simple carbohydrates to complex glycolipids, glycopeptides and glycoproteins, make their study a challenging research area. The last two decades have witnessed tremendous advances and development of highly sophisticated methods, in combination with optimized protocols and hyphenate techniques for the understanding of structure, conformations, dynamics and organization of microbial glycans. We here present an overview of the novel approaches that have massively improved our understanding of the carbohydrate-based world of bacteria

Biophysical and Structural Characterization of the Interaction between Human Galectin-3 and the Lipopolysaccharide from <i>Pseudomonas aeruginosa</i>

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa

Estudio y proyecto para el desarrollo de las Áreas de Retención Temporal de Excedentes Hídricos (ARTEH) en la cuenca del arroyo del Gato y sus afluentes con adecuación para el uso social : Informe componente urbanistica territorial

El análisis del Área de Retención Temporal de Excedentes Hídricos, denominada Nodo ARTEH «Pérez Norte», (Imagen1) se desarrolló a escala de entorno inmediato delimitando dos sectores inmediatos yuxtapuestos a la avenida 143: uno (a) - rectangular- comprendido entre la calle 139, la avenida 143 y las avenida 38 y la calle 37 y, el otro (b) -cuadrado-, entre la avenida 143 y la calle 145 y la avenida 38 y la calle 36. El objetivo principal que se plantea es la construcción del diagnóstico que permita conocer el estado de situación actual de esta zona a fin de, posteriormente, definir lineamientos generales de ordenamiento territorial que permitan recuperar un espacio vacante sobre la subcuenca del A° Pérez brazo Norte, como área de oportunidad para el desarrollo de espacios públicos urbanos que permita, inicialmente, atenuar el riesgo por inundaciones y, de igual modo, mejorar la calidad del entorno en el que se encuentran y de la sociedad que habita en el área.Producto emergente del proyecto de investigación: «Territorios vulnerables y paisajes emergentes. Parte II. Medidas no estructurales para la reducción del riesgo por inundación. Caso: Gran La Plata», cuyo director y codirectora son respectivamente el Dr. Arq. Etulain y la Arq. López.Centro de Investigaciones Urbanas y TerritorialesFacultad de Arquitectura y UrbanismoFacultad de IngenieríaFacultad de Ciencias Agrarias y Forestale