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[Display omitted] • The neural network described extracts coupling constants and chemical shifts from a 2D J-resolved spectrum. • Training of the network on spectra simulated using quantum dynamics is demonstrated. • Results on simulated test sets are satisfactory, but experimental spectra present obstacles. • Future work employing pure-phase J-resolved spectra is suggested. Extracting parameters such as chemical shifts and coupling constants from proton NMR spectra is often a first step in using spectra for compound identification and structure determination. This can become challenging when scalar couplings between protons are comparable in size to chemical shift differences (strongly coupled), as is often the case with low-field (bench top) spectrometers. Here we explore the potential utility of AI methods, in particular neural networks, for extracting parameters from low-field spectra. Rather than seeking large experimental sets of spectra for training a network, we chose quantum mechanical simulation of sets, something that is possible with modern software packages and computer resources. We show that application of a network trained on 2-D J-resolved spectra and applied to a spectrum of iduronic acid, shows some promise, but also meets with some obstacles. We suggest that these may be overcome with improved pulse sequences and more extensive simulations. [ABSTRACT FROM AUTHOR]

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2- O -sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2- O -sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2- O -sulfated domains but had an increase in N - and 6- O -sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2- O -sulfated heparan sulfate in human neuronal, skeletal, and renal development. [ABSTRACT FROM AUTHOR]

Due to the all-axial orientation of the OH-groups in the 1C 4 chair conformation considered standard for L-hexapyranosides, including l -iduronopyranoside – a component of many biologically and medically significant sulfated glycans, these monosaccharides can be anticipated to display unusual conformations upon the introduction of bulky and charged substituents. Herein we describe the synthesis of a series of iduronopyranoside derivatives with varying sulfation patterns, which were studied computationally using the DLPNO-MP2 approach and by means of analyzing their chemical shifts to ascertain the effects sulfation has on the conformation of the iduronopyranoside ring. [Display omitted] • A series of iduronosides covering all possible sulfation patterns was completed. • The effects of sulfate location on the iduronoside ring conformation were studied. • High-level DLPNO-MP2 method was used, as opposed to older works employing just DFT. • It emerges that apart from 2S O , 1S 3 may be present in the equilibrium. [ABSTRACT FROM AUTHOR]

L‐Iduronic acid (IdoA) plays a pivotal role in glycosaminoglycan (GAG) protein interactions. However, the structural microheterogeneity of GAG appears to impede the systematic investigation of IdoA functions. Under such conditions, oligo‐Iduronic acid (Oligo‐IdoA) are ideal and straightforward heparin mimetics to unravel the relationship between IdoA structure and functions. Herein, we report for the first‐time linear synthesis of rare oligo‐IdoA precursor utilizing anhydrous β‐L‐idopyranosyl and IdoA thiophenol building block. After screening various synthetic strategies, we have Installed successive IdoA by 5 step reactions with 25–26 % overall yield. These oligo‐IdoA are expected to be excellent probes to understand conformational plasticity of IdoA and fine tune carbohydrate–protein interactions. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice. [ABSTRACT FROM AUTHOR]

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts D-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers. [ABSTRACT FROM AUTHOR]

The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler-Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease. [ABSTRACT FROM AUTHOR]

Dermatan sulfate epimerase 2 (DS-epi2), together with its homolog DS-epi1, transform glucuronic acid into iduronic acid in DS polysaccharide chains. Iduronic acid gives DS increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in the brain. Here, we report the generation and initial characterization of DS-epi2 null mice. DS-epi2-deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin sulfate (CS)/DS isolated from the newborn mutated mouse brains had a 38% reduction in iduronic acid compared with wild-type littermates, and compositional analysis revealed a decrease in 4-O-sulfate and an increase in 6-O-sulfate containing structures. Despite the reduction in iduronic acid, the adult DS-epi2−/− brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2. These results extend previous findings of the functional redundancy of brain extracellular matrix components. [ABSTRACT FROM PUBLISHER]

Abstract: The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine–glucuronic acid (GlcN–GlcA) or iduronic acid (GlcN–IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation. [Copyright &y& Elsevier]

Abstract: A divergent de novo synthesis of six differentially protected l-iduronic acid thioglycosides from a common advanced precursor is described. The key step of this synthetic sequence is the stereoselective elongation of dithioacetal protected C5-dialdehyde 11 via a highly diastereoselective MgBr2·OEt2-mediated cyanation. Orthogonally protected l-iduronic acid building blocks obtained by this synthesis are expected to facilitate access to differentially sulfated heparins for microarray-based structure–activity relationship studies. [Copyright &y& Elsevier]

Abstract: The monosaccharide 2-O-sulfo-α-l-iduronic acid (IdoA2S) is one of the major components of glycosaminoglycans. The ability of molecular mechanics force fields to reproduce ring-puckering conformational equilibrium is important for the successful prediction of the free energies of interaction of these carbohydrates with proteins. Here we report unconstrained molecular dynamics simulations of IdoA2S monosaccharide that were carried out to investigate the ability of commonly used force fields to reproduce its ring conformational flexibility in aqueous solution. In particular, the distribution of ring conformer populations of IdoA2S was determined. The GROMOS96 force field with the SPC/E water potential can predict successfully the dominant skew-boat to chair conformational transition of the IdoA2S monosaccharide in aqueous solution. On the other hand, the GLYCAM06 force field with the TIP3P water potential sampled transitional conformations between the boat and chair forms. Simulations using the GROMOS96 force field showed no pseudorotational equilibrium fluctuations and hence no inter-conversion between the boat and twist boat ring conformers. Calculations of theoretical proton NMR coupling constants showed that the GROMOS96 force field can predict the skew-boat to chair conformational ratio in good agreement with the experiment, whereas GLYCAM06 shows worse agreement. The omega rotamer distribution about the C5–C6 bond was predicted by both force fields to have torsions around 10°, 190°, and 360°. [Copyright &y& Elsevier]

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca2+. Proton–proton and proton–carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of 1C4 and 2So conformers of iduronic acid that agreed well with the analysis based on proton–proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton–carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of 1H-1H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans. [ABSTRACT FROM PUBLISHER]

Chondroitin/dermatan sulfate is a highly complex linear polysaccharide ubiquitously found in the extracellular matrix and at the cell surface. Several of its functions, such as binding to growth factors, are mediated by domains composed of alternating iduronic acid and 4-O-sulfated N-acetylgalactosamine residues, named 4-O-sulfated iduronic acid blocks. These domains are generated by the action of two DS-epimerases, which convert d-glucuronic acid into its epimer l-iduronic acid, in close connection with 4-O-sulfation. In this study, dermatan sulfate structure was evaluated after downregulating or increasing dermatan 4-O-sulfotransferase 1 (D4ST-1) expression. siRNA-mediated downregulation of D4ST-1 in primary human lung fibroblasts led to a drastic specific reduction of iduronic acid blocks. No change of epimerase activity was found, indicating that the influence of D4ST-1 on epimerization is not due to an altered expression level of the DS-epimerases. Analysis of the dermatan sulfate chains showed that D4ST-1 is essential for the biosynthesis of the disulfated structure iduronic acid-2-O-sulfate-N-acetylgalactosamine-4-O-sulfate, thus confirmed to be strictly connected with the iduronic acid blocks. Also the biologically important residue hexuronic acid-N-acetylgalactosamine-4,6-O-disulfate considerably decreased after D4ST-1 downregulation. In conclusion, D4ST-1 is a key enzyme and is indispensable in the formation of important functional domains in dermatan sulfate and cannot be compensated by other 4-O-sulfotransferases. [ABSTRACT FROM PUBLISHER]

Abstract: 2-O-Sulfo-α-l-iduronic acid (IdoA2S) is one of the main components of heparin, an anticoagulant and antithrombotic polysaccharide able to potentiate the inhibitory effect of antithrombin over plasma serine proteases. This monosaccharide unit adopts an equilibrium between chair (1 C 4) and skew-boat (2 S O) forms as a function of heparin sequence size and composition. Although the prevalence of the 1 C 4 chair conformation in monosaccharides is understood, the reasons for the increase in 2 S O contribution in the whole polysaccharide chain are still uncertain. In this context, 0.2μs molecular dynamics simulations of IdoA2S-containing oligosaccharides indicated that stabilization due to intramolecular hydrogen bonds around IdoA2S is highly correlated (p ⩽0.001) with the expected conformational equilibrium for this residue in solution. This behavior explains the known effect of different heparin compositions, at the monosaccharide level, on IdoA2S conformation in biological solutions. [Copyright &y& Elsevier]

Abstract: The synthesis of a bicyclic analogue of the naturally occurring α-l-iduronic acid locked in a biologically active 2 S 0 skewboat conformation is disclosed. The desired 2 S 0 conformation has been obtained by tethering the C-2 and C-5 carbon atoms of the sugar ring with a dimethyloxy bridge and confirmed by NMR and molecular modeling. The new mimic displays the exact hydroxyl pattern of α-l-iduronic acid, a major monosaccharide component of glycosaminoglycans and thus represents a closer mimic of the latter, compared to previously reported bicyclic analogs. Display Omitted [Copyright &y& Elsevier]

Pathogens are able to exploit specific glycosaminoglycans (GAGs), especially iduronic acid (IdoA)-containing GAGs, to invade the host. By analyzing Escherichia coli proteome chip data, we identified the interactomes of three IdoA-containing GAGs: heparin, heparin sulfate (HS), and chondroitin sulfate B (CSB). Using non-IdoA-containing GAG, chondroitin sulfate C, as a negative control, 157 proteins specifically binding with IdoA-containing GAGs were revealed in the present study. These proteins showed functional enrichment in protein synthesis and metabolism. Fifteen proteins which commonly interacts with three IdoA-containing GAGs were further examined. The regular expression for motif showed these common IdoA interactome shared a conserved sequence. Among them, we identified a second flagellar system outer membrane protein, MbhA. The MbhA has Kd values of 8.9 × 10−8 M, 5.3 × 10−7 M, and 1.79 × 10−7 M to interact with heparin, HS, and CSB, respectively. Using flow cytometry, we confirmed that the MbhA protein can bind to human epithelial cells HCT-8. Overexpression of mbhA increased the percentage of invasion in E. coli which lacks a second flagellar system. Moreover, pre-blocking of HCT-8 cells with MbhA inhibited the bacterial invasion, implying the importance of the direct interaction of MbhA and the host cell surface on bacterial invasion. We analyzed the Escherichia coli proteomic data to elucidate the interactomes of three different IdoA-containing GAGs (heparin, HS, and CSB) because these IdoA-containing GAGs can mediate bacterial invasion to the host. Through proteomic and systematic analysis, a second flagellar system outer membrane protein, MbhA, was also identified in the present study. Affinity assay confirmed that MbhA can bind to three IdoA-containing GAGs heparin, HS, and CSB. The result of flow cytometry also showed MbhA can interact with human epithelial cells HCT-8. Results of bacteria invasion assay showed overexpression of mbhA promoted the bacterial invasion. Moreover, pre-blocking of HCT-8 cells with MbhA also reduced the percentage of bacterial invasion. These findings correspond well that MbhA is one of invasion factors. Unlabelled Image • GAGs containing IdoA residues are crucial for bacterial invasion. • The protein interactome of three IdoA-containing GAGs heparin, HS, and CSB were analyzed using E. coli proteome chip data. • Systematic analysis reveals a second flagellar system outer membrane protein, MbhA. • MbhA bind with high affinity to IdoA-containing GAGs and is important for bacterial invasion of host cells. • Study have enhanced understanding of MbhA in host-pathogen interaction. [ABSTRACT FROM AUTHOR]

Abstract: Hunter syndrome (mucopolysaccharidosis-II) is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase. The assay of this sulfatase requires the use of α-l-iduronate glycosides containing a sulfate at the 2-position. We report a simple, three-step procedure for the introduction of sulfate at the 2-position starting with the methyl ester of α-l-iduronate glycosides. The procedure involves protection of the 2- and 4-hydroxyl groups of the iduronate moiety as the dibutyl stannylene acetal, selective sulfation with sulfur trioxide–trimethylamine, and deprotection of the methyl ester to afford the desired 2-sulfate in 61% overall yield. [Copyright &y& Elsevier]