Publications by authors named "Pedro M Nieto"

54 Publications

Synthesis, self-assembly and Langerin recognition studies of a resorcinarene-based glycocluster exposing a hyaluronic acid thiodisaccharide mimetic.

Org Biomol Chem 2021 Jul;19(29):6455-6467

Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EGA Buenos Aires, Argentina. and CONICET-Universidad de Buenos Aires, Centro de Investigaciones en Hidratos de Carbono (CIHIDECAR), C1428EGA Buenos Aires, Argentina.

Herein, we report the synthesis of an octavalent glycocluster exposing a thiodisaccharide mimetic of the repetitive unit of hyaluronic acid, βSGlcA(1 → 3)βSGlcNAc, constructed on a calix[4]resorcinarene scaffold by CuAAC reaction of suitable precursors. This glycocluster showed a strong tendency toward self-aggregation. DOSY-NMR and DLS experiments demonstrated the formation of spherical micelles of d ≅ 6.2 nm, in good agreement. TEM micrographs showed the presence of particles of different sizes, depending on the pH of the starting solution, thus evidencing that the negative charge on the micelle surface due to ionization of the GlcA residues plays an important role in the aggregation process. STD-NMR and DLS experiments provided evidence of the interaction between the synthetic glycocluster and Langerin, a relevant C-type lectin. This interaction was not observed in the STD-NMR experiments performed with the basic disaccharide, providing evidence of a multivalent effect.
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http://dx.doi.org/10.1039/d1ob00895aDOI Listing
July 2021

GAG Multivalent Systems to interact with Langerin.

Curr Med Chem 2021 Jul 5. Epub 2021 Jul 5.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Av. Américo Vespucio 49, Seville 41092, Spain.

Langerin is a C-type Lectin expressed at the surface of Langerhans cells, which play a pivotal role in protecting organisms against pathogen infections. To address this aim, Langerin presents at least two recognition sites, one Ca2+-dependent and another one independent, capable of recognizing a variety of carbohydrate ligands. In contrast to other lectins, Langerin recognizes sulfated glycosaminoglycans (GAGs), a family of complex and heterogeneous polysaccharides present in the cell membrane and the extracellular matrix at the interphase generated in the trimeric form of Langerin but absent in the monomeric form. The complexity of these oligosaccharides has impeded the development of well-defined monodisperse structures to study these interaction processes. However, in the last few decades, an improvement of synthetic developments to achieve the preparation of carbohydrate multivalent systems mimicking the GAGs has been described. Despite all these contributions, very few examples are reported where the GAG multivalent structures are used to evaluate the interaction with Langerin. These molecules should pave the way to explore these GAG-Langerin interactions.
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http://dx.doi.org/10.2174/0929867328666210705143102DOI Listing
July 2021

Midkine Interaction with Chondroitin Sulfate Model Synthetic Tetrasaccharides and Their Mimetics: The Role of Aromatic Interactions.

Chemistry 2021 Jul 2. Epub 2021 Jul 2.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC, Universidad de Sevilla, C/ Américo Vespucio, 49, 41092, Sevilla, Spain.

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.
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http://dx.doi.org/10.1002/chem.202101674DOI Listing
July 2021

Synthesis, structure and midkine binding of chondroitin sulfate oligosaccharide analogues.

Org Biomol Chem 2021 Jun 28;19(24):5312-5326. Epub 2021 May 28.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

The preparation of chondroitin sulfate (CS) oligosaccharide mimetics, more easily synthesized than natural sequences, is a highly interesting task because these compounds pave the way for modulation of the biological processes in which CS is involved. Herein, we report the synthesis of CS type E analogues which present easily accessible glucose units instead of glucuronic acid (GlcA) moieties. NMR experiments and molecular dynamics simulations showed that the 3D structure of these compounds is similar to the structure of the natural CS-E oligosaccharides. In addition, fluorescence polarization (FP) and saturation transfer difference NMR (STD-NMR) experiments revealed that the synthesized CS-like derivatives were able to interact with midkine, a model heparin-binding growth factor, suggesting that the presence of the GlcA carboxylate groups is not essential for the binding. Overall, our results indicate that the synthesized glucose-containing oligosaccharides can be considered as functional and structural CS mimetics.
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http://dx.doi.org/10.1039/d1ob00882jDOI Listing
June 2021

Second-Generation Dendrimers with Chondroitin Sulfate Type-E Disaccharides as Multivalent Ligands for Langerin.

Biomacromolecules 2020 07 24;21(7):2726-2734. Epub 2020 Jun 24.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

Chondroitin sulfate type-E (CS-E) is a sulfated polysaccharide that shows several interesting biological activities, such as modulation of the neuronal growth factor signaling and its interaction with langerin, a C-type lectin with a crucial role in the immunological system. However, applications of CS-E are hampered by the typical heterogeneous structure of the natural polysaccharide. Well-defined, homogeneous CS-E analogues are highly demanded. Here, we report the synthesis of monodispersed, structurally well-defined second-generation glycodendrimers displaying up to 18 CS-E disaccharide units. These complex multivalent systems have a molecular weight and a number of disaccharide repeating units comparable with those of the natural polysaccharides. In addition, surface plasmon resonance experiments revealed a calcium-independent interaction between these glycodendrimers and langerin, in the micromolar range, highlighting the utility of these compounds as CS-E mimetics.
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http://dx.doi.org/10.1021/acs.biomac.0c00476DOI Listing
July 2020

Synthesis of (1→3) Thiodisaccharides of GlcNAc and the Serendipitous Formation of 2,3-Dideoxy-(1→2)-thiodisaccharides through a Vinyl Azide Intermediate.

J Org Chem 2020 01 19;85(2):306-317. Epub 2019 Dec 19.

Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires , Ciudad Universitaria , Pabellón 2, C1428EGA Buenos Aires , Argentina.

The syntheses of β--GlcA(1→3)GlcNAc and β--Gal(1→3)GlcNAc thiodisaccharides, which can be considered mimetics of the repeating units of hyaluronan and keratan respectively, were achieved by S2 displacement of a triflate group allocated at the 3-position of a convenient 2-azido-4,6--benzylidene-2-deoxy-β-d-allopyranose precursor by the corresponding nucleophilic suitable protected thioaldoses derived from glucuronic acid (GlcA) and galactose (Gal). The study of the reaction led to the finding that the vinyl azide formed by competitive E2 reaction of the mentioned triflate was an interesting precursor of a new kind of 2,3-dideoxy-2-azido-(1→2) thiodisaccharides through an addition reaction. Determination of the stereochemistry of the new stereocenter at C-2 was achieved by NOESY experiments. Final protecting group manipulation of the (1→3) thiodisaccharides led to a family of derivatives that could be used as building blocks for the synthesis of complex glycomimetics.
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http://dx.doi.org/10.1021/acs.joc.9b01883DOI Listing
January 2020

Synthesis of a Fluorous-Tagged Hexasaccharide and Interaction with Growth Factors Using Sugar-Coated Microplates.

Molecules 2019 Apr 22;24(8). Epub 2019 Apr 22.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

Here, we report the synthesis of a sulfated, fully protected hexasaccharide as a glycosaminoglycan mimetic and the study of its interactions with different growth factors: midkine, basic fibroblast growth factor (FGF-2) and nerve growth factor (NGF). Following a fluorous-assisted approach, monosaccharide building blocks were successfully assembled and the target oligosaccharide was prepared in excellent yield. The use of more acid stable 4,6--silylidene protected glucosamine units was crucial for the efficiency of this strategy because harsh reaction conditions were needed in the glycosylations to avoid the formation of orthoester side products. Fluorescence polarization experiments demonstrated the strong interactions between the synthesized hexamer, and midkine and FGF-2. In addition, we have developed an alternative assay to analyse these molecular recognition events. The prepared oligosaccharide was non-covalently attached to a fluorous-functionalized microplate and the direct binding of the protein to the sugar-immobilized surface was measured, affording the corresponding K value.
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http://dx.doi.org/10.3390/molecules24081591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515340PMC
April 2019

Unexpected loss of stereoselectivity in glycosylation reactions during the synthesis of chondroitin sulfate oligosaccharides.

Beilstein J Org Chem 2019 15;15:137-144. Epub 2019 Jan 15.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

Here, we present an exploratory study on the fluorous-assisted synthesis of chondroitin sulfate (CS) oligosaccharides. Following this approach, a CS tetrasaccharide was prepared. However, in contrast to our previous results, a significant loss of β-selectivity was observed in [2 + 2] glycosylations involving -trifluoroacetyl-protected D-galactosamine donors and D-glucuronic acid (GlcA) acceptors. These results, together with those obtained from experiments employing model monosaccharide building blocks, highlight the impact of the glycosyl acceptor structure on the stereoselectivity of glycosylation reactions. Our study provides useful data about the substitution pattern of GlcA units for the efficient synthesis of CS oligomers.
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http://dx.doi.org/10.3762/bjoc.15.14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350880PMC
January 2019

The Use of NMR to Study Transient Carbohydrate-Protein Interactions.

Authors:
Pedro M Nieto

Front Mol Biosci 2018 11;5:33. Epub 2018 Apr 11.

Glycosystems Laboratory, Instituto de Investigaciones Científicas, cicCartuja, CSIC/USE, Seville, Spain.

Carbohydrates are biologically ubiquitous and are essential to the existence of all known living organisms. Although they are better known for their role as energy sources (glucose/glycogen or starch) or structural elements (chitin or cellulose), carbohydrates also participate in the recognition events of molecular recognition processes. Such interactions with other biomolecules (nucleic acids, proteins, and lipids) are fundamental to life and disease. This review focuses on the application of NMR methods to understand at the atomic level the mechanisms by which sugar molecules can be recognized by proteins to form complexes, creating new entities with different properties to those of the individual component molecules. These processes have recently gained attention as new techniques have been developed, while at the same time old techniques have been reinvented and adapted to address newer emerging problems.
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http://dx.doi.org/10.3389/fmolb.2018.00033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904382PMC
April 2018

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand.

Bioorg Med Chem 2018 03 3;26(5):1076-1085. Epub 2018 Feb 3.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain. Electronic address:

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1 → 4)-Glc-β(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC of 270 nM and 2.4 µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.
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http://dx.doi.org/10.1016/j.bmc.2018.01.022DOI Listing
March 2018

Rational-Differential Design of Highly Specific Glycomimetic Ligands: Targeting DC-SIGN and Excluding Langerin Recognition.

ACS Chem Biol 2018 03 8;13(3):600-608. Epub 2018 Jan 8.

Univ. Grenoble Alpes , CNRS, CEA, Institut de Biologie Structurale , F-38044 Grenoble , France.

At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carbohydrate-based PAMPS or DAMPS (pathogen- or danger-associated molecular patterns, respectively) and promote immune response regulation. However, some CLRs are hijacked by viral and bacterial pathogens. Thus, the design of ligands able to target specifically one CLR, to either modulate an immune response or to inhibit a given infection mechanism, has great potential value in therapeutic design. A case study is the selective blocking of DC-SIGN, involved notably in HIV trans-infection of T lymphocytes, without interfering with langerin-mediated HIV clearance. This is a challenging task due to their overlapping carbohydrate specificity. Toward the rational design of DC-SIGN selective ligands, we performed a comparative affinity study between DC-SIGN and langerin with natural ligands. We found that GlcNAc is recognized by both CLRs; however, selective sulfation are shown to increase the selectivity in favor of langerin. With the combination of site-directed mutagenesis and X-ray structural analysis of the langerin/GlcNS6S complex, we highlighted that 6-sulfation of the carbohydrate ligand induced langerin specificity. Additionally, the K313 residue from langerin was identified as a critical feature of its binding site. Using a rational and a differential approach in the study of CLR binding sites, we designed, synthesized, and characterized a new glycomimetic, which is highly specific for DC-SIGN vs langerin. STD NMR, SPR, and ITC characterizations show that compound 7 conserved the overall binding mode of the natural disaccharide while possessing an improved affinity and a strict specificity for DC-SIGN.
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http://dx.doi.org/10.1021/acschembio.7b00958DOI Listing
March 2018

Interactions between a Heparin Trisaccharide Library and FGF-1 Analyzed by NMR Methods.

Int J Mol Sci 2017 Jun 17;18(6). Epub 2017 Jun 17.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

FGF-1 is a potent mitogen that, by interacting simultaneously with Heparan Sulfate Glycosaminoglycan HSGAG and the extracellular domains of its membrane receptor (FGFR), generates an intracellular signal that finally leads to cell division. The overall structure of the ternary complex Heparin:FGF-1:FGFR has been finally elucidated after some controversy and the interactions within the ternary complex have been deeply described. However, since the structure of the ternary complex was described, not much attention has been given to the molecular basis of the interaction between FGF-1 and the HSGAG. It is known that within the complex, the carbohydrate maintains the same helical structure of free heparin that leads to sulfate groups directed towards opposite directions along the molecular axis. The precise role of single individual interactions remains unclear, as sliding and/or rotating of the saccharide along the binding pocket are possibilities difficult to discard. The HSGAG binding pocket can be subdivided into two regions, the main one can accommodate a trisaccharide, while the other binds a disaccharide. We have studied and analyzed the interaction between FGF-1 and a library of trisaccharides by STD-NMR and selective longitudinal relaxation rates. The library of trisaccharides corresponds to the heparin backbone and it has been designed to interact with the main subsite of the protein.
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http://dx.doi.org/10.3390/ijms18061293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486114PMC
June 2017

Glycodendrimers as Chondroitin Sulfate Mimetics: Synthesis and Binding to Growth Factor Midkine.

Chemistry 2017 Aug 3;23(47):11338-11345. Epub 2017 Aug 3.

Instituto de Investigaciones Químicas (IIQ), CSIC- Universidad de Sevilla, Américo Vespucio 49, 41092, Seville, Spain.

Chondroitin sulfate (CS) is a member of the glycosaminoglycan (GAG) family, a class of polysaccharides implicated in relevant biological functions. The structural complexity of these carbohydrates demands the development of simple glycomimetics as useful tools to study the biological processes in which GAGs are involved. In this work we described the synthesis of the disaccharide unit of the CS-E (GlcA-GalNAc(4,6-di-OSO )), in a multivalent presentation. Using a fluorescence polarization competition assay we have demonstrated that a hexavalent dendrimer of this disaccharide interact with midkine, in the low micromolar range. This result highlights the potency of these disaccharide-displaying multivalent systems as interesting mimetics of longer and synthetically more complex GAG oligosaccharides.
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http://dx.doi.org/10.1002/chem.201701890DOI Listing
August 2017

Flavonoid glycosides from Persea caerulea. Unraveling their interactions with SDS-micelles through matrix-assisted DOSY, PGSE, mass spectrometry, and NOESY.

Magn Reson Chem 2016 Sep 13;54(9):718-728. Epub 2016 Apr 13.

Department of Chemistry and Physics, ceiA3, Universidad de Almería, Almería, Spain.

Two flavonoid glycosides derived from rhamnopyranoside (1) and arabinofuranoside (2) have been isolated from leaves of Persea caerulea for the first time. The structures of 1 and 2 have been established by H NMR, C NMR, and IR spectroscopy, together with LC-ESI-TOF and LC-ESI-IT MS spectrometry. From the MS and MS/MS data, the molecular weights of the intact molecules as well as those of quercetin and kaempferol together with their sugar moieties were deduced. The NMR data provided information on the identity of the compounds, as well as the α and β configurations and the position of the glycosides on quercetin and kaempferol. We have also explored the application of sodium dodecyl sulfate (SDS) normal micelles in binary aqueous solution, at a range of concentrations, to the diffusion resolution of these two glycosides, by the application of matrix-assisted diffusion ordered spectroscopy (DOSY) and pulse field gradient spin echo (PGSE) methodologies, showing that SDS micelles offer a significant resolution which can, in part, be rationalized in terms of differing degrees of hydrophobicity, amphiphilicity, and steric effects. In addition, intra-residue and inter-residue proton-proton distances using nuclear Overhauser effect build-up curves were used to elucidate the conformational preferences of these two flavonoid glycosides when interacting with the micelles. By the combination of both diffusion and nuclear Overhauser spectroscopy techniques, the average location site of kaempferol and quercetin glycosides has been postulated, with the former exhibiting a clear insertion into the interior of the SDS-micelle, whereas the latter is placed closer to the surface. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/mrc.4434DOI Listing
September 2016

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine.

Chemistry 2016 Feb 19;22(7):2356-69. Epub 2016 Jan 19.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092, Sevilla, Spain.

The biological activity of midkine, a cytokine implicated in neuro- and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well-defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.
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http://dx.doi.org/10.1002/chem.201504440DOI Listing
February 2016

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique.

Chemistry 2015 Oct 21;21(42):15004-12. Epub 2015 Aug 21.

IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.
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http://dx.doi.org/10.1002/chem.201502019DOI Listing
October 2015

Structures of glycans bound to receptors from saturation transfer difference (STD) NMR spectroscopy: quantitative analysis by using CORCEMA-ST.

Methods Mol Biol 2015 ;1273:475-87

Andalusian Centre for Nanomedicine and Biotechnology (BIONAND), C/Severo Ochoa 35, Parque Tecnológico de Andalucía, 29590, Campanillas, Málaga, Spain.

Glycan-receptor interactions are of fundamental relevance for a large number of biological processes, and their kinetics properties (medium/weak binding affinities) make them appropriated to be studied by ligand observed NMR techniques, among which saturation transfer difference (STD) NMR spectroscopy has been shown to be a very robust and powerful approach. The quantitative analysis of the results from a STD NMR study of a glycan-receptor interaction is essential to be able to translate the resulting spectral intensities into a 3D molecular model of the complex. This chapter describes how to carry out such a quantitative analysis by means of the Complete Relaxation and Conformational Exchange Matrix Approach for STD NMR (CORCEMA-ST), in general terms, and an example of a previous work on an antibody-glycan interaction is also shown.
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http://dx.doi.org/10.1007/978-1-4939-2343-4_28DOI Listing
November 2015

Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.

J Am Chem Soc 2015 Apr 23;137(12):4100-10. Epub 2015 Mar 23.

†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.
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http://dx.doi.org/10.1021/ja511529xDOI Listing
April 2015

Importance of the polarity of the glycosaminoglycan chain on the interaction with FGF-1.

Glycobiology 2014 Nov 11;24(11):1004-9. Epub 2014 Jul 11.

Instituto de Investigaciones Químicas, CSIC, Américo Vespucio, 49, Sevilla 41092, Spain

Heparin-like saccharides play an essential role in binding to the fibroblast growth factor (FGF)-1 and to their membrane receptors fibroblast growth factor receptor forming a ternary complex that is responsible of the internalization of the signal, via the dimerization of the intracellular regions of the receptor. In this study, we report the binding affinities between five synthetic hexasaccharides with human FGF-1 obtained by surface plasmon resonance experiments, and compare with the induced mitogenic activity previously obtained. These five oligosaccharides differ in sulfation pattern and in sequence. We have previously demonstrated that all the five hexasaccharides have similar 3D structure of the backbone. Consequently, the differences in binding affinity should have their origin in the substitution pattern. Subsequently, the different capacity for induction of mitogenic activity can be, at least partially, explained from these binding affinities. Interestingly, one of the oligosaccharides lacking axially symmetry ( 3: ) was biologically inactive, whereas the other ( 2: ) was the most active. The difference between both compounds is the order of the FGF-binding motifs along the chain relative to the carbohydrate polarity. We can conclude that the directionality of the GAG chain is essential for the binding and subsequent activation. The relative biological activity of the compounds with regular substitution pattern can be inferred from their values of IC50. Remarkably, the sulfate in position 6 of d-glucosamine was essential for the mitogenic activity but not for the interaction with FGF-1.
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http://dx.doi.org/10.1093/glycob/cwu071DOI Listing
November 2014

Synthesis of hyaluronic acid oligosaccharides and exploration of a fluorous-assisted approach.

Carbohydr Res 2014 Jul 20;394:17-25. Epub 2014 May 20.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

The synthesis of hyaluronic acid oligomers (tri- and tetrasaccharide) is described. We have followed a pre-glycosylation oxidation strategy. Glucuronic acid units were directly employed in coupling reactions with suitably protected glucosamine derivatives. In order to simplify the purification of synthetic intermediates, a fluorous-assisted strategy has been also explored. Using this approach, a hyaluronic acid trisaccharide was prepared.
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http://dx.doi.org/10.1016/j.carres.2014.05.007DOI Listing
July 2014

A STD-NMR study of the interaction of the Anabaena ferredoxin-NADP+ reductase with the coenzyme.

Molecules 2014 Jan 7;19(1):672-85. Epub 2014 Jan 7.

Instituto de Investigaciones Químicas, CSIC, Americo Vespucio, 49, Sevilla 41092, Spain.

Ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD) techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.
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http://dx.doi.org/10.3390/molecules19010672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272016PMC
January 2014

3D structure of a heparin mimetic analogue of a FGF-1 activator. A NMR and molecular modelling study.

Org Biomol Chem 2013 Dec 31;11(47):8269-75. Epub 2013 Oct 31.

Lab. of Glycosystems, Dept. Bioorganic Chemistry, Instituto de Investigaciones Químicas, cicCartuja, CSIC/US, C/Americo Vespucio, 49 Sevilla 41092, Spain.

The motional behaviour of heparin oligosaccharides in solution is best described as a top rotor having two perpendicular rotation axes. This prevents an accurate extraction of interprotonic distances by NOESY/ROESY based methods. In this paper, we describe the solution structure of the hexasaccharide 1 calculated from high exactitude distance data obtained from off-resonance ROESY combined with a long MD simulation of 500 ns. In previous studies, we have found that two synthetic hexasaccharides having the sulphate groups directed towards one side of its central plane have an opposite biological activity, while 1 is unable to activate the FGF-1 signalling pathway, the other (2) is even more active than the regular region derived hexasaccharide (3) that mimics the natural active compound, heparin. From the structural analysis it was concluded that 1 has similar three-dimensional characteristics to 2 or 3 and therefore the differences in the activity should be due to the arrangement of the sulphate groups within the hexasaccharidic sequence.
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http://dx.doi.org/10.1039/c3ob41789aDOI Listing
December 2013

Conformations of the iduronate ring in short heparin fragments described by time-averaged distance restrained molecular dynamics.

Glycobiology 2013 Nov 31;23(11):1220-9. Epub 2013 Jul 31.

Glycosystems Laboratory, Instituto de Investigaciones Quimicas, CSIC - US, Americo Vespucio, 49, E-41092 Sevilla, Spain.

The polyconformational behavior of L-iduronic acid (L-IdoA2S) in heparin derivatives has been previously analyzed in terms of intra-ring proton-proton vicinal coupling constants ((3)JHH) through mathematical fit of experimental and theoretical values (Ferro DR, Provasoli A, Ragazzi M, Casu B, Torri G, Bossennec V, Perly B, Sinay P, Petitou M, Choay J. 1990. Conformer Populations of L-Iduronic Acid Residues in Glycosaminoglycan Sequences. Carbohydr Res. 195:157-167; Muñoz-García JC, López-Prados J, Angulo J, Díaz-Contreras I, Reichardt N, de Paz JL, Martín-Lomas M, Nieto PM. 2012. Effect of the substituents of the neighboring ring in the conformational equilibrium of iduronate in heparin-like trisaccharides. Chemistry. 18:16319-16331.). However, this methodology is subjected to the experimental uncertainties of the J-coupling measurements, the force field deviations and the goodness of the least-squares fit. In the present work, we have used time-averaged distance restrained molecular dynamics (tar-MD) to largely reduce these errors, which enables accurate quantification of the population of conformers, or puckers, of the L-IdoA2S residue, in a set of eight heparin-like trisaccharides following the general sequence d-glucosamine (GlcN)-IdoA-GlcN, directly from the time evolution of the puckering coordinates θ and . Thus, by carrying out tar-MD simulations in explicit water, with the exclusive nuclear overhauser enhancement (NOE)-derived distance H2-H5 of the L-IdoA2S (2)SO conformer as the unique imposed constraint, we have been able to accurately and easily determine the different extents to which the iduronate ring populates the polar ((1)C4 chair) and equatorial ((2)SO skew-boat) areas of the puckering sphere depending on the sulfation pattern of the flanking GlcN residues, under low temperature conditions (278 K). The results indicate that 6-O-sulfation at the reducing-end GlcN residue facilitates the (1)C4 to (2)SO transitions of the iduronate ring by augmenting the flexibility of the C2-C3 torsion, driving the conformational equilibrium toward a majority of equatorial conformers.
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http://dx.doi.org/10.1093/glycob/cwt058DOI Listing
November 2013

Synthesis of chondroitin/dermatan sulfate-like oligosaccharides and evaluation of their protein affinity by fluorescence polarization.

Org Biomol Chem 2013 Jun 17;11(21):3510-25. Epub 2013 Apr 17.

Glycosystems Laboratory, Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain.

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.
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http://dx.doi.org/10.1039/c3ob40306hDOI Listing
June 2013

Structure of a glycomimetic ligand in the carbohydrate recognition domain of C-type lectin DC-SIGN. Structural requirements for selectivity and ligand design.

J Am Chem Soc 2013 Feb 8;135(7):2518-29. Epub 2013 Feb 8.

Institut de Biologie Structurale, Université Grenoble I, 41 rue Jules Horowitz, Grenoble, F-38027, France.

In genital mucosa, different fates are described for HIV according to the subtype of dendritic cells (DCs) involved in its recognition. This notably depends on the C-type lectin receptor, langerin or DC-SIGN, involved in gp120 interaction. Langerin blocks HIV transmission by its internalization in specific organelles of Langerhans cells. On the contrary, DC-SIGN enhances HIV trans-infection of T lymphocytes. Thus, approaches aiming to inhibit DC-SIGN, without blocking langerin, represent attractive anti-HIV strategies. We previously demonstrated that dendrons bearing multiple copies of glycomimetic compounds were able to block DC-SIGN-dependent HIV infection in cervical explant models. Optimization of such ligand requires detailed characterization of its binding mode. In the present work, we determined the first high-resolution structure of a glycomimetic/DC-SIGN complex by X-ray crystallography. This glycomimetic, pseudo-1,2-mannobioside, shares shape and conformational properties with Manα1-2Man, its natural counterpart. However, it uses the binding epitope previously described for Lewis X, a ligand specific for DC-SIGN among the C-type lectin family. Thus, selectivity gain for DC-SIGN versus langerin is observed with pseudo-1,2-mannobioside as shown by surface plasmon resonance analysis. In parallel, ligand binding was also analyzed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol. These studies demonstrate that the complex, defined by X-ray crystallography, represents the unique binding mode of this ligand as opposed to the several binding orientations described for the natural ligand. This exclusive binding mode and its selective interaction properties position this glycomimetic as a good lead compound for rational improvement based on a structurally driven approach.
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http://dx.doi.org/10.1021/ja3053305DOI Listing
February 2013

Effect of the substituents of the neighboring ring in the conformational equilibrium of iduronate in heparin-like trisaccharides.

Chemistry 2012 Dec 9;18(51):16319-31. Epub 2012 Nov 9.

Glycosystems Laboratory, Instituto de Investigaciones Quimicas, CSIC-US, Americo Vespucio, 49, 41092 Sevilla, Spain.

Based on the structure of the regular heparin, we have prepared a smart library of heparin-like trisaccharides by incorporating some sulfate groups in the sequence α-D-GlcNS- (1-4)-α-L-Ido2S-(1-4)-α-D-GlcN. According to the 3D structure of heparin, which features one helix turn every four residues, this fragment corresponds to the minimum binding motif. We have performed a complete NMR study and found that the trisaccharides have a similar 3D structure to regular heparin itself, but their spectral properties are such that allow to extract very detailed information about distances and coupling constants as they are isotropic molecules. The characteristic conformational equilibrium of the central iduronate ring has been analyzed combining NMR and molecular dynamics and the populations of the conformers of the central iduronate ring have been calculated. We have found that in those compounds lacking the sulfate group at position 6 of the reducing end glucosamine, the population of (2)S(0) of the central iduronate residue is sensitive to the temperature decreasing to 19% at 278 K. On the contrary, the trisaccharides with 6-O-sulfate in the reducing end glucosamine keep the level of population constant with temperature circa 40% of (2)S(0) similar to that observed at room temperature. Another structural feature that has been revealed through this analysis is the larger flexibility of the L-IdoAS- D-GlcN glycosidic linkage, compared with the D-GlcNS-L-IdoA. We propose that this is the point where the heparin chain is bended to form structures far from the regular helix known as kink that have been proposed to play an important role in the specificity of the heparin-protein interaction.
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http://dx.doi.org/10.1002/chem.201202770DOI Listing
December 2012

Insights into the glycosaminoglycan-mediated cytotoxic mechanism of eosinophil cationic protein revealed by NMR.

ACS Chem Biol 2013 Jan 9;8(1):144-51. Epub 2012 Oct 9.

Departamento de Química Física Biológica, Instituto de Química Física Rocasolano, CSIC, Madrid, Spain.

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP's toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin's inhibition of ECP's ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP's toxicity implicated in eosinophil pathologies.
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http://dx.doi.org/10.1021/cb300386vDOI Listing
January 2013

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition.

Chemistry 2012 Jul 4;18(27):8527-39. Epub 2012 Jun 4.

Instituto de Investigaciones Químicas (IIQ), CSIC-Universidad de Sevilla, Avda. Americo Vespucio 49, 41092, Sevilla, Spain.

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.
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http://dx.doi.org/10.1002/chem.201200279DOI Listing
July 2012

A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins.

Chemistry 2012 Mar 29;18(13):3872-8. Epub 2012 Feb 29.

Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla-C.S.I.C. Avda. Américo Vespucio, 49, 41092 Sevilla, Spain.

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to (1)H-(15)N heteronuclear single quantum coherence(HSQC) spectra to identify the (3)J(NH) correlation between a (15)N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the (1)H-(13)C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.
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http://dx.doi.org/10.1002/chem.201103413DOI Listing
March 2012

Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate-protein interactions in microtiter plates.

Org Biomol Chem 2012 Mar 31;10(10):2146-63. Epub 2012 Jan 31.

Glycosystems Laboratory, Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

The synthesis of well-defined oligosaccharides is crucial for the establishment of structure-activity relationships for specific sequences of heparin, contributing to the understanding of the biological role of this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal functional group that allows selective conjugation of the probes and expands their use as chemical tools in glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to produce efficiently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps, avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the basic fibroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the amount of sugar required for attachment to the solid support. Using this approach we quantified heparin-protein interactions, and surface dissociation constants for the synthetic heparin derivatives were determined.
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http://dx.doi.org/10.1039/c2ob06607fDOI Listing
March 2012
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