Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling

IF/ 00780/2015 PTDC/BIA-MIB/31028/2017 UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020 SFRH/BD/140394/2018 2020.03261. CEECIND CA18132 PID 2019-107770RA-I00 H2020-MSCA-ITN-2020 agency for the Glytunes project (956758).All cells are decorated with a highly dense and complex structure of glycan chains, which are mostly attached to proteins and lipids. In this context, sialic acids are a family of nine-carbon acidic monosaccharides typically found at the terminal position of glycan chains, modulating several physiological and pathological processes. Sialic acids have many structural and modulatory roles due to their negative charge and hydrophilicity. In addition, the recognition of sialic acid glycans by mammalian cell lectins, such as siglecs, has been described as an important immunological checkpoint. Furthermore, sialic acid glycans also play a pivotal role in host–pathogen interactions. Various pathogen receptors exposed on the surface of viruses and bacteria are responsible for the binding to sialic acid sugars located on the surface of host cells, becoming a critical point of contact in the infection process. Understanding the molecular mechanism of sialic acid glycans recognition by sialic acid-binding proteins, present on the surface of pathogens or human cells, is essential to realize the biological mechanism of these events and paves the way for the rational development of strategies to modulate sialic acid-protein interactions in diseases. In this perspective, nuclear magnetic resonance (NMR) spectroscopy, assisted with molecular modeling protocols, is a versatile and powerful technique to investigate the structural and dynamic aspects of glycoconjugates and their interactions in solution at the atomic level. NMR provides the corresponding ligand and protein epitopes, essential for designing and developing potential glycan-based therapies. In this review, we critically discuss the current state of knowledge about the structural features behind the molecular recognition of sialic acid glycans by different receptors, naturally present on human cells or pathogens, disclosed by NMR spectroscopy and molecular modeling protocols.publishersversionpublishe

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View

[EN] The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly C-13-labelled trisaccharides at the sialic acid and galactose moieties. STD-H-1,C-13-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the alpha 2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.This research was funded by the European Research Council (ERC-2017-AdG, project number 788143-RECGLYCA NMR to J.J.B.) and Agencia Estatal de Investigacion (Spain), projects RTI2018-094751-B-C21 to J.J.B. & A.A. and PID2019-107770RA-I00 to J.E.O., and by the Human Frontier Science Program (HFSP; grant LT000747/2018-C to L.U.) and CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spai

Targeting the Spike: Repurposing Mithramycin and Dihydroergotamine to Block SARS-CoV-2 Infection

: The urgency to find complementary therapies to current SARS-CoV-2 vaccines, whose effectiveness is preserved over time and not compromised by the emergence of new and emerging variants, has become a critical health challenge. We investigate the possibility of jamming the opening of the Receptor Binding Domain (RBD) of the spike protein of SARS-CoV-2 with small compounds. Through in silico screening, we identified two potential candidates that would lock the Receptor Binding Domain (RBD) in a closed configuration, preventing the virus from infecting the host cells. We show that two drugs already approved by the FDA, mithramycin and dihydroergotamine, can block infection using concentrations in the μM range in cell-based assays. Further STD-NMR experiments support dihydroergotamine's direct interaction with the spike protein. Overall, our results indicate that repurposing of these compounds might lead to potential clinical drug candidates for the treatment of SARS-CoV-2 infection

Hypoxia Reduces Cell Attachment of SARS-CoV-2 Spike Protein by Modulating the Expression of ACE2, Neuropilin-1, Syndecan-1 and Cellular Heparan Sulfate

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1αdependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), the European Research Council (ERC) (grant numbers: ERC-2018-StG 804236-NEXTGEN-IO to A.P and ERC-2017-AdG 788143-RECGLYCANMR to J.J-B.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the FERO Foundation. Personal fellowships: E.P. (Juan de la Cierva-Formación, FJC2018-035449-I), L.V. (Juan de la Cierva-Formación, FJCI-2017-34099), A.B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), A.G. (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), A.A (La Caixa Inphinit, LCF/BQ/DR20/11790022), B.J. (Basque Government, PRE_2019_1_0320), L.M. (Juan de la Cierva-Formación, FJC2019-039983-I), E.B. (MINECO, BFU2016-76872-R; Excellence Networks, SAF2017-90794-REDT) and A.P. (Ramón y Cajal, RYC2018-024183-I; Proyectos I+D+I, PID2019-107956RA-I00; and Ikerbasque Research Associate)

N-Linked Glycosylation Regulates CD22 Organization and Function

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners

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 Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins

info:eu-repo/grantAgreement/WT/Physiological Sciences/095700 ERC‐2017‐AdG, 788143‐RECGLYCANMR grant 200077 grant RTI2018‐094751‐B‐C21 GC2018‐098996‐B‐I00 RTI2018‐099592‐B‐C22 RTI2018‐101269‐B‐I00 SEV‐2016‐0644 IF/00780/2015 PTDC/BIA‐MIB/31028/2017 UCIBIO UIDB/04378/2020 Infrastructure project 22161 PD/BD/142847/2018The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.publishersversionpublishe

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

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