Exploring multivalent carbohydrate–protein interactions by NMR

Nuclear Magnetic Resonance (NMR) has been widely employed to assess diverse features of glycan–protein molecular recognition events. Different types of qualitative and quantitative information at different degrees of resolution and complexity can be extracted from the proper application of the available NMR-techniques. In fact, affinity, structural, kinetic, conformational, and dynamic characteristics of the binding process are available. Nevertheless, except in particular cases, the affinity of lectin-sugar interactions is weak, mostly at the low mM range. This feature is overcome in biological processes by using multivalency, thus augmenting the strength of the binding. However, the application of NMR methods to monitor multivalent lectin–glycan interactions is intrinsically challenging. It is well known that when large macromolecular complexes are formed, the NMR signals disappear from the NMR spectrum, due to the existence of fast transverse relaxation, related to the large size and exchange features. Indeed, at the heart of the molecular recognition event, the associated free-bound chemical exchange process for both partners takes place in a particular timescale. Thus, these factors have to be considered and overcome. In this review article, we have distinguished, in a subjective manner, the existence of multivalent presentations in the glycan or in the lectin. From the glycan perspective, we have also considered whether multiple epitopes of a given ligand are presented in the same linear chain of a saccharide (i.e., poly-LacNAc oligosaccharides) or decorating different arms of a multiantennae scaffold, either natural (as in multiantennae N-glycans) or synthetic (of dendrimer or polymer nature). From the lectin perspective, the presence of an individual binding site at every monomer of a multimeric lectin may also have key consequences for the binding event at different levels of complexity.We thank generous funding by the European Research Council (RECGLYCANMR, Advanced Grant No. 788143), the Agencia Estatal de Investigación (Spain) for grant PDI2021-1237810B-C21, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank Marie-Skłodowska-Curie actions (TN BactiVax, under grant agreement No. 860325)

Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by X-ray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the N-terminal tail and the unique G–G′ loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the N-linked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.This work was supported by operating grant PID2019-107770RA-I00 (J.E.-O.) from the Agencia Estatal Investigación of Spain and by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B.). We also thank the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement no. 956758 to J.E.-O and ITN BactiVax under grant agreement no. 860325 to U.A.). Additional funding was provided by CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank the Ikerbasque Basque Foundation of Science and the Spanish Ministry of Economy, Industry and Competitiveness (for the postdoctoral contract Juan de la Cierva Incorporación to J.E-O). X-ray diffraction experiments described in this paper were performed using the XALOC synchrotron beamline at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland)

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Publisher Copyright: © 2023, The Author(s).Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.publishersversionpublishe

New glucosamine-based TLR4 agonists: design, synthesis, mechanism of action, and in vivo activity as vaccine adjuvants

20 p.-15 fig.-1 graph. abst.We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.The authors acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie, project BactiVax (www.bactivax.eu) grant agreement no. 860325; the consortium CINMPIS; the project of excellence CHRONOS, CHRonical multifactorial disorders explored by NOvel integrated Strategies of the Department of Biotechnology and Biosciences; the Agencia Estatal de Investigacion (Spain) for project PID2021-126130OB-I00 (N.G.A.A.), PID2020-113588RB-I00 (S.M.-S.), PRE2018-086249 (A.M.-R), PRE2021-097247 (M.M.-T.); and project FEDER MINECO, the EM-platform at the CIC bioGUNE for support in cryo-EM imaging. J.J.-B. also thanks funding by CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Perkin-Elmer Italia is also acknowledged for providing the cell imaging reagents.Peer reviewe

Unravelling molecular recognition events between glycans and proteins using advanced NMR Techniques

315 p.Glycans are ubiquitous molecules in nature. They participate in a plethora of functions, they cover the surface of cells as integral constituents of complex glycoconjugates (glycoproteins, and glycolipids) forming the glycocalyx. The molecular recognition of glycans by lectins is a finely tuned process where even the most minimal change within the recognized glycan structure can lead to drastic changes within the meticulously orchestrated symphony of life. For that reason, understanding the structure, dynamics, and interactions of glycans is of paramount importance for being able to modulate biological events related to health and disease. In this Thesis, I have employed a synergic combination of advanced NMR spectroscopy and X-ray crystallography methods with biophysical techniques, assisted by computational chemistry tools to elucidate the intricate process of molecular recognition of glycans

Current Status on Therapeutic Molecules Targeting Siglec Receptors

The sialic acid-binding immunoglobulin-type of lectins (Siglecs) are receptors that recognize sialic acid-containing glycans. In the majority of the cases, Siglecs are expressed on immune cells and play a critical role in regulating immune cell signaling. Over the years, it has been shown that the sialic acid-Siglec axis participates in immunological homeostasis, and that any imbalance can trigger different pathologies, such as autoimmune diseases or cancer. For all this, different therapeutics have been developed that bind to Siglecs, either based on antibodies or being smaller molecules. In this review, we briefly introduce the Siglec family and we compile a description of glycan-based molecules and antibody-based therapies (including CAR-T and bispecific antibodies) that have been designed to therapeutically targeting Siglecs

Derivatives of (R)-3-(5-Furanyl)carboxamido-2-aminopropanoic Acid as Potent NMDA Receptor Glycine Site Agonists with GluN2 Subunit-Specific Activity

NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of (R)-3-(5-fyranyl)carboxamido-2-aminopropanoic acid analogs 8a-s as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogs display high variation in potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound 8p is identified as a potent partial agonist at GluN1/2C (EC(50) = 0.074 μM) with agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents