Publications by authors named "Carolina Fontana"

26 Publications

  • Page 1 of 1

Structural insights in galectin-1-glycan recognition: Relevance of the glycosidic linkage and the N-acetylation pattern of sugar moieties.

Bioorg Med Chem 2021 Aug 14;44:116309. Epub 2021 Jul 14.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, 11800 Montevideo, Uruguay. Electronic address:

Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for β-galactosides such as N-acetyllactosamine (β-d-Galp-(1 → 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for β-(1 → 6) galactosides, including β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was evaluated, and their performance was compared to that of β-(1 → 4) and β-(1 → 3) galactosides. To this end, the trisaccharide β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing β-(1 → 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving β-(1 → 4) and β-(1 → 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔG) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔG), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.
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http://dx.doi.org/10.1016/j.bmc.2021.116309DOI Listing
August 2021

Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA.

Nature 2020 07 27;583(7814):139-144. Epub 2020 May 27.

Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA. Here we use R relaxation-dispersion nuclear magnetic resonance and molecular simulations to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.
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http://dx.doi.org/10.1038/s41586-020-2336-3DOI Listing
July 2020

Preparation and mechanistic studies of 2-substituted Bisthiazolidines by imine exchange.

European J Org Chem 2020 Mar 17;2020(9):1084-1092. Epub 2020 Jan 17.

Laboratorio de Química Farmacéutica, Departamento de Química Orgánica, Facultad de Química, Avda. General Flores 2124, CC1157, Universidad de la República (UdelaR), Montevideo, Uruguay.

Bisthiazolidines (BTZ) are bicyclic compounds considered penicillin analogs that inhibit the full range of Metallo-β-Lactamases (MBLs) and potentiate β-lactam activity against resistant bacteria. Herein we present a new methodology to prepare 2-substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranged from 31 to 75%. The reaction is based on imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments and a key imine could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine by i) a N-assisted N-C bond cleavage, and ii) a thiol-mediated 5 -trig cyclization followed by a C-N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of with aldehyde leads to bicycle the iminium ion .
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http://dx.doi.org/10.1002/ejoc.201901677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8442850PMC
March 2020

First report of a canine morbillivirus infection in a giant anteater (Myrmecophaga tridactyla) in Brazil.

Vet Med Sci 2020 08 5;6(3):606-611. Epub 2020 Feb 5.

Laboratory of Veterinary Clinical Pathology, Veterinary Hospital of Federal University of Mato Grosso, Cuiaba, Brazil.

Canine morbillivirus, also known as canine distemper virus (CDV), induces a contagious multisystemic disease caused by an enveloped RNA virus belonging to the genus Morbillivirus within the family Paramyxoviridae. CDV replicates readily in epithelial, nerve and lymphoid tissues; it is excreted in urine, feces, saliva, oral and nasal discharge; and its major route of entry for infection is through the respiratory system. Although the virus was originally believed to infect domestic dogs, new studies have shown that it can also naturally or experimentally infect non-domestic hosts. A recent blood test performed on a giant anteater (Myrmecophaga tridactyla) found Lentz inclusions in the animal's leucocytes. A rapid CDV test, an RT-PCR assay and pathology findings confirmed this report of canine morbillivirus in this species, which corresponds to the second report of CDV infection in the order Pilosa, family Myrmecophagidae in central west Brazil.
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http://dx.doi.org/10.1002/vms3.246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397876PMC
August 2020

Enzymatic synthesis of non-natural trisaccharides and galactosides; Insights of their interaction with galectins as a function of their structure.

Carbohydr Res 2019 Jan 3;472:1-15. Epub 2018 Nov 3.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, Montevideo, Uruguay. Electronic address:

Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of β-d-Galp-(1 → 6)-β-d-Galp-(1 → 4)-d-Glcp(2), a mixture of β-d-Galp-(1 → 6)-β-d-Glcp-(1 → 4)-d-Glcp(5) and β-d-Galp-(1 → 3)-β-d-Glcp-(1 → 4)-d-Glcp(6), and finally benzyl β-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using H and C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable β-Gal linkage seems to be β(1 → 4) followed by β(1 → 3) and β(1 → 6) for hGal-1, and β(1 → 4) followed by β(1 → 6) and β(1 → 3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.
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http://dx.doi.org/10.1016/j.carres.2018.10.011DOI Listing
January 2019

Elucidation of the O-antigen structure of Escherichia coli O63.

Glycobiology 2019 02;29(2):179-187

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden.

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-β-d-Quip3N(d-allo-ThrAc)-(1→2)-β-d-Ribf-(1→4)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element β-d-Galp-(1→3)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.
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http://dx.doi.org/10.1093/glycob/cwy098DOI Listing
February 2019

New heteroleptic oxidovanadium(V) complexes: synthesis, characterization and biological evaluation as potential agents against Trypanosoma cruzi.

J Biol Inorg Chem 2018 12 8;23(8):1265-1281. Epub 2018 Sep 8.

Área Química Inorgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.

Searching for prospective vanadium-based agents against Trypanosoma cruzi, the parasite causing Chagas disease, four new [VO(8HQ-H)(L-2H)] compounds, where 8HQ is 8-hydroxyquinoline and L are tridentate salicylaldehyde semicarbazone derivatives L1-L4, were synthesized and characterized in the solid state and in solution. The compounds were evaluated on T. cruzi epimastigotes (CL Brener) as well as on VERO cells, as mammalian cell model. Compounds showed activity against T. cruzi (IC 6.2-10.5 μM) of the same order than Nifurtimox and 8HQ, and a four- to sevenfold activity increase with respect to the free semicarbazones. For comparison, [VO(L-H)] series was prepared and the new [VO(L3-H)] was fully characterized. They showed negligible activity and low selectivity towards the parasite. The inclusion of 8HQ as ligand in [VO(8HQ-H)(L-2H)] compounds led to good activities and increased selectivity towards the parasite with respect to 8HQ. V NMR experiments, performed to get insight into the nature of the active species, suggested partial decomposition of the compounds in solution to [VO(L-H)] and 8HQ. Depending on the dose, the compounds act as trypanocide or trypanostatic. A high uptake of vanadium in the parasites (58.51-88.9% depending on dose) and a preferential accumulation in the soluble protein fraction of the parasite was determined. Treated parasites do not seem to show a late apoptotic/necrotic phenotype suggesting a different cell death mechanism. In vivo toxicity study on zebrafish model showed no toxicity up to a 25 µM concentration of [VO(8HQ-H)(L1-2H)]. These compounds could be considered prospective anti-T. cruzi agents that deserve further research.
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http://dx.doi.org/10.1007/s00775-018-1613-1DOI Listing
December 2018

Structural studies of a polysaccharide from Vibrio parahaemolyticus strain AN-16000.

Carbohydr Res 2016 Sep 13;432:41-9. Epub 2016 Jun 13.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Electronic address:

The structure of a polysaccharide from Vibrio parahaemolyticus strain AN-16000 has been investigated. The sugar and absolute configuration analysis revealed d-Glc, d-GalN, d-QuiN and l-FucN as major components. The PS was subjected to dephosphorylation with aqueous 40% HF to obtain an oligosaccharide that was analyzed by (1)H and (13)C NMR spectroscopy. The HR-MS spectrum of the oligosaccharide revealed a pentasaccharide composed of two Glc residues, one QuiNAc and one GalNAc, one FucNAc, as well as a glycerol moiety. The structure of the PS was determined using (1)H, (13)C, (15)N and (31)P NMR spectroscopy; inter-residue correlations were identified by (1)H,(13)C-heteronuclear multiple-bond correlation, (1)H,(1)H-NOESY and (1)H,(31)P-hetero-TOCSY experiments. The PS backbone has the following teichoic acid-like structure: →3)-d-Gro-(1-P-6)-β-d-Glcp-(1→4)-α-l-FucpNAc-(1→3)-β-d-QuipNAc-(1→ with a side-chain consisting of α-d-Glcp-(1→6)-α-d-GalpNAc-(1→ linked to the O3 position of the FucNAc residue.
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http://dx.doi.org/10.1016/j.carres.2016.06.004DOI Listing
September 2016

Negligible elongation of mucin glycans with Gal β1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

Int J Parasitol 2016 05 15;46(5-6):311-21. Epub 2016 Feb 15.

Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) and Instituto de Química Biológica (Facultad de Ciencias), Universidad de la República, Montevideo, Uruguay. Electronic address:

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.
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http://dx.doi.org/10.1016/j.ijpara.2015.12.009DOI Listing
May 2016

Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence.

J Biol Chem 2016 Apr 11;291(14):7727-41. Epub 2016 Feb 11.

From the Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden,

The structures of the lipooligosaccharides fromBrucella melitensismutants affected in the WbkD and ManBcoreproteins have been fully characterized using NMR spectroscopy. The results revealed that disruption ofwbkDgives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)[β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcpresidues (48 and 17%, respectively). These structures were identical to those of the R-LPS fromB. melitensisEP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption ofmanBcoregives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcpresidue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcoreproteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion ofB. melitensis wadCremoves the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential inB. melitensisvirulence, the core deficiency in thewadCmutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.
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http://dx.doi.org/10.1074/jbc.M115.701540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817197PMC
April 2016

Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181.

ChemistryOpen 2015 Feb 1;4(1):47-55. Epub 2014 Dec 1.

Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University S-106 91 Stockholm (Sweden).

Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The (1)H and (13)C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR (13)C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.
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http://dx.doi.org/10.1002/open.201402068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380953PMC
February 2015

Structural studies of the exopolysaccharide from Lactobacillus plantarum C88 using NMR spectroscopy and the program CASPER.

Carbohydr Res 2015 Jan 18;402:87-94. Epub 2014 Sep 18.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Electronic address:

Some lactic acid bacteria, such as those of the Lactobacillus genus, have the ability to produce exopolysaccharides (EPSs) that confer favorable physicochemical properties to food and/or beneficial physiological effects on human health. In particular, the EPS of Lactobacillus plantarum C88 has recently demonstrated in vitro antioxidant activity and, herein, its structure has been investigated using NMR spectroscopy and the computer program CASPER (Computer Assisted Spectrum Evaluation of Regular polysaccharides). The pentasaccharide repeating unit of the O-deacetylated EPS consists of a trisaccharide backbone, →4)-α-D-Galp-(1→2)-α-D-Glcp-(1→3)-β-D-Glcp-(1→, with terminal D-Glc and D-Gal residues (1.0 and 0.8 equiv per repeating unit, respectively) extending from O3 and O6, respectively, of the →4)-α-D-Galp-(1→ residue. In the native EPS an O-acetyl group is present, 0.85 equiv per repeating unit, at O2 of the α-linked galactose residue; thus the repeating unit of the EPS has the following structure: →4)[β-D-Glcp-(1→3)][β-D-Galp-(1→6)]α-D-Galp2Ac-(1→2)-α-D-Glcp-(1→3)-β-D-Glcp-(1→. These structural features, and the chain length (∼10(3) repeating units on average, determined in a previous study), are expected to play an important role in defining the physicochemical properties of the polymer.
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http://dx.doi.org/10.1016/j.carres.2014.09.003DOI Listing
January 2015

Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O42.

Carbohydr Res 2015 Feb 17;403:174-81. Epub 2014 May 17.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Electronic address:

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O42 has been investigated by NMR spectroscopy as the main method, which was complemented with sugar analysis, mass spectrometry, and analysis of biosynthetic information. The O-specific chain of the O-deacylated lipopolysaccharide (LPS-OH) consists of branched tetrasaccharide-glycerol repeating units joined by phosphodiester linkages. The lipid-free polysaccharide contains 0.8equiv of O-acetyl groups per repeating unit and has the following teichoic acid-like structure: Based on biosynthetic aspects, this should also be the biological repeating unit. This O-antigen structure is remarkably similar to that of E. coli O28ac, differing only in the presence or absence, respectively, of a glucose residue at the branching point. The structural similarity explains the serological cross-reactivity observed between strains of these two serogroups, and also their almost identical O-antigen gene cluster sequences.
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http://dx.doi.org/10.1016/j.carres.2014.05.003DOI Listing
February 2015

NMR structure analysis of uniformly 13C-labeled carbohydrates.

J Biomol NMR 2014 Jun 26;59(2):95-110. Epub 2014 Apr 26.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, 106 91, Stockholm, Sweden.

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.
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http://dx.doi.org/10.1007/s10858-014-9830-6DOI Listing
June 2014

Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: application to the O-antigen of Escherichia coli O59.

Glycobiology 2014 May 19;24(5):450-7. Epub 2014 Feb 19.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, →6)[α-d-GalpA3Ac/4Ac-(1 → 3)]-α-d-Manp-(1 → 3)-α-d-Manp-(1 → 3)-β-d-Manp-(1 → 3)-α-d-GlcpNAc-(1→. The identification of the O-acetylation positions was efficiently performed by comparison of the (1)H,(13)C HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.
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http://dx.doi.org/10.1093/glycob/cwu011DOI Listing
May 2014

Facile Structural Elucidation of Glycans Using NMR Spectroscopy Data and the Program CASPER: Application to the O-Antigen Polysaccharide of Escherichia coli O155.

Chempluschem 2013 Nov 15;78(11):1327-1329. Epub 2013 Aug 15.

Department of Organic Chemistry, Stockholm University, Arrhenius Laboratory, 106 91 Stockholm (Sweden), Fax: (+46) 815-49-08.

Time saver: The program CASPER was successfully employed to rapidly elucidate a new O-antigen polysaccharide structure (obtained from a strain of Escherichia coli serogroup O155), using solely unassigned NMR spectroscopy data as input information. Thus, what is considered the most tedious and time-consuming part of the structural elucidation process has been reduced from several hours (or even days) of manual interpretation to about four minutes of automated analysis.
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http://dx.doi.org/10.1002/cplu.201300273DOI Listing
November 2013

Complete (1)H and (13)C NMR chemical shift assignments of mono- to tetrasaccharides as basis for NMR chemical shift predictions of oligosaccharides using the computer program CASPER.

Carbohydr Res 2013 Oct 10;380:156-66. Epub 2013 Jul 10.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

(1)H and (13)C NMR chemical shift data are used by the computer program CASPER to predict chemical shifts of oligo- and polysaccharides. Three types of data are used, namely, those from monosaccharides, disaccharides, and trisaccharides. To improve the accuracy of these predictions we have assigned the (1)H and (13)C NMR chemical shifts of eleven monosaccharides, eleven disaccharides, twenty trisaccharides, and one tetrasaccharide; in total 43 compounds. Five of the oligosaccharides gave two distinct sets of NMR resonances due to the α- and β-anomeric forms resulting in 48 (1)H and (13)C NMR chemical shift data sets. In addition, the pyranose ring forms of Neu5Ac were assigned at two temperatures, due to chemical shift displacements as a function of temperature. The (1)H NMR chemical shifts were refined using total line-shape analysis with the PERCH NMR software. (1)H and (13)C NMR chemical shift predictions were subsequently carried out by the CASPER program (http://www.casper.organ.su.se/casper/) for three branched oligosaccharides having different functional groups at their reducing ends, namely, a mannose-containing pentasaccharide, and two fucose-containing heptasaccharides having N-acetyllactosamine residues in the backbone of their structures. Good to excellent agreement was observed between predicted and experimental (1)H and (13)C NMR chemical shifts showing the utility of the method for structural determination or confirmation of synthesized oligosaccharides.
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http://dx.doi.org/10.1016/j.carres.2013.06.026DOI Listing
October 2013

Conformational preferences of the O-antigen polysaccharides of Escherichia coli O5ac and O5ab using NMR spectroscopy and molecular modeling.

Biomacromolecules 2013 Jul 18;14(7):2215-24. Epub 2013 Jun 18.

Centre de Recherches sur les Macromolécules Végétales-CNRS, affiliated with Université Grenoble and ICMG, BP 53 X, 38041 Grenoble Cedex, France.

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton-proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models.
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http://dx.doi.org/10.1021/bm400354yDOI Listing
July 2013

Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof.

Glycobiology 2013 Mar 28;23(3):354-62. Epub 2012 Nov 28.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[β-L-Rhap-(1 → 4)]-β-D-GlcpNAc-(1 → 4)-α-D-GalpA-(1 → 3)-α-D-Manp-(1 → 3)-β-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a β-L-Rhap-(1 → 4)-β-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-D-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of the l-Rhap residue (∼0.70 per repeating unit) as well as at O-2 and O-3 of the D-GalpA residue (∼0.03 and ∼0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286-290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.
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http://dx.doi.org/10.1093/glycob/cws161DOI Listing
March 2013

Glycoengineering of host mimicking type-2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces.

Mol Microbiol 2013 Jan 30;87(1):112-31. Epub 2012 Nov 30.

ETH Zurich, Institute of Microbiology, Wolfgang-Pauli-Str. 10, HCI F 406, CH- 8093 Zurich, Switzerland.

Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1-4(Fucα1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Galβ1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly-LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.
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http://dx.doi.org/10.1111/mmi.12086DOI Listing
January 2013

Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O174.

Carbohydr Res 2012 Jun 3;354:102-5. Epub 2012 Mar 3.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with (1)H and (13)C NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H,(13)C-heteronuclear multiple-bond correlation and (1)H,(1)H-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [formula see text] Cross-peaks of low intensity were present in the NMR spectra consistent with a β-D-GlcpNAc-(1→2)-β-D-GlcpA(1→ structural element at the terminal part of the polysaccharide, which on average is composed of ∼15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.
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http://dx.doi.org/10.1016/j.carres.2012.02.020DOI Listing
June 2012

Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase.

J Am Chem Soc 2012 Mar 1;134(10):4521-4. Epub 2012 Mar 1.

School of Chemistry, Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester M1 7DN, UK.

O-mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a "one-pot" enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.
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http://dx.doi.org/10.1021/ja211861mDOI Listing
March 2012

Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy.

Biomacromolecules 2011 Nov 30;12(11):3851-5. Epub 2011 Sep 30.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (-)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic (1)H and (13)C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user's point of view only the unknown sample has to be derivatized for use in CASPER.
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http://dx.doi.org/10.1021/bm201169yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215282PMC
November 2011

The major surface carbohydrates of the Echinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures.

Biochemistry 2009 Dec;48(49):11678-91

Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) e Instituto de Química Biológica (Facultad de Ciencias), UdelaR, Montevideo, Uruguay.

The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.
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http://dx.doi.org/10.1021/bi901112qDOI Listing
December 2009

Conformational analysis of phorboxazole bis-oxazole oxane fragment analogs by NMR spectroscopy and molecular modeling simulations.

Magn Reson Chem 2008 Jan;46(1):36-41

Cátedra de Química Farmacéutica, Departamento de Química Orgánica, Facultad de Química, Universidad de la República, Avenida General Flores 2124, Montevideo 11800, Uruguay.

We present a detailed conformational study of a simplified synthetic analog of the bis-oxazole oxane fragment found in the cytostatic agents phorboxazole A and B based on results from NMR spectroscopy and molecular modeling simulations. Complete 1H and 13C resonance assignments for the bis-oxazole oxane system were carried out through the use of COSY, HSQC, HMBC, TOCSY, and HSQC-TOCSY experiments, and its conformational preferences in solution were investigated by analysis of 3J(HH) coupling constants and NOE enhancements obtained from 1D and 2D NOESY experiments. In order to solve inconsistencies from our preliminary structural studies, simulated annealing studies were performed to thoroughly sample the phase space available to the molecule. Our results reveal that the six-membered oxane ring, which constitutes the most important moiety regarding the three-dimensional (3D) structure and flexibility of the analog, exists in rapid equilibrium between its two accessible chair conformers in an approximate ratio of 70:30. The information gathered from these studies will be of critical importance in our efforts to prepare novel compounds with phorboxazole-like structure and activity.
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http://dx.doi.org/10.1002/mrc.2120DOI Listing
January 2008

Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus.

FEBS J 2006 Jul 19;273(14):3192-203. Epub 2006 Jun 19.

Cátedra de Inmunología, Facultad de Química/Ciencias, Universidad de la República, Montevideo, Uruguay.

The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05328.xDOI Listing
July 2006
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