Sulfated Iduronic Acid Research Articles

GlycoTorch Vina: Docking Designed and Tested for Glycosaminoglycans.

Glycosaminoglycans (GAGs) are a family of anionic carbohydrates that play an essential role in the physiology and pathology of all eukaryotic life forms. Experimental determination of GAG-protein complexes is challenging due to their difficult isolation from biological sources, natural heterogeneity, and conformational flexibility-including possible ring puckering of sulfated iduronic acid from 1C4 to 2SO conformation. To overcome these challenges, we present GlycoTorch Vina (GTV), a molecular docking tool based on the carbohydrate docking program VinaCarb (VC). Our program is unique in that it contains parameters to model 2SO sugars while also supporting glycosidic linkages specific to GAGs. We discuss how crystallographic models of carbohydrates can be biased by the choice of refinement software and structural dictionaries. To overcome these variations, we carefully curated 12 of the best available GAG and GAG-like crystal structures (ranging from tetra- to octasaccharides or longer) obtained from the PDB-REDO server and refined using the same protocol. Both GTV and VC produced pose predictions with a mean root-mean-square deviation (RMSD) of 3.1 Å from the native crystal structure-a statistically significant improvement when compared to AutoDock Vina (4.5 Å) and the commercial software Glide (5.9 Å). Examples of how real-space correlation coefficients can be used to better assess the accuracy of docking pose predictions are given. Comparisons between statistical distributions of empirical "salt bridge" interactions, relevant to GAGs, were compared to density functional theory (DFT) studies of model salt bridges, and water-mediated salt bridges; however, there was generally a poor agreement between these data. Water bridges appear to play an important, yet poorly understood, role in the structures of GAG-protein complexes. To aid in the rapid prototyping of future pose scoring functions, we include a module that allows users to include their own torsional and nonbonded parameters.

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C4 -2 S0 equilibrium in the unbound saccharide, shift to full 2 S0 and full 1 C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

Binding and structure-kinetic relationship analysis of selective TLR4-targeted immunosuppressive self-assembling heparin nanoparticles

Self-assembling aliphatic heparin derivatives were shown to inhibit the immune system by antagonizing Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2). In the present study, glycol split heparin–d-erythro-sphingosine conjugates (NAHNP) and its regioselectively desulfated derivatives with shortened aliphatic chains were investigated regarding their biophysical properties in the interaction with TLR4/MD2. Two-dimensional nuclear Overhauser effect spectroscopy studies showed that upon glycol splitting, the heparin backbone gains extra adaptability that facilitates binding to proteins. However, unlike native heparin or glycol split non-anticoagulant heparin (NAH), hydrophobic derivatization of NAH forces sulfated iduronic acid residues to change configuration from a 2S0 skew-boat to a 1C4 chair form. Whereas neither heparin nor NAH had any appreciable effect, NAHNP significantly inhibited lipopolysaccharide-induced activation of the NF-κB transcription factor. We showed that NAHNP binds to TLR4/MD2 with an affinity of 62.3 nM. In line with computational studies, biosensor-based structure-kinetic relationship studies demonstrated that 6-O-sulfo groups of d-glucosamine residue were essential in binding to arginines of both TLR4 and MD2 domains of the receptor complex. The desulfation of 6-O-sulfo groups decreases the association kinetics from 4.2 × 104 M−1 s−1 to 3.8 × 103 M−1 s−1, which results in a decreased affinity of 800 nM. Two aliphatic chains of NAHNP bound to the MD2 pocket similarly to lipopolysaccharide. A decrease in chain length resulted in a loss of inhibitory activity on NF-κB transcription and binding affinity to TLR4/MD2. In conclusion, the present study characterizes the immunosuppressive effect of aliphatic heparin derivatives and provides a promising strategy to develop selective immunosuppressants for acute and chronic inflammatory disorders.

Solution Structure of Heparin Pentasaccharide: NMR and DFT Analysis.

High-resolution NMR and density functional theory (DFT) calculations have been applied to analysis of heparin pentasaccharide 3D structure in aqueous solution. The fully optimized molecular geometry of two pentasaccharide conformations (differing from each other in the form, one (1)C4 and the other (2)S0, of the sulfated iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as explicit water molecules. The presented approach enabled insight into variations of the bond lengths, bond angles, and torsion angles, formations of intra- and intermolecular hydrogen bonds, and ionic interactions in the two pentasaccharide conformations. A rather complex hydrogen bond network is formed, including inter-residue and intraresidue bonds between the NH group in the GlcN,3,6S with oxygens linked to C-2 at the IdoA2S residue and the glycosidic O-1 and the neighboring OSO3(-) group linked to C-3 in the same residue. On the other hand, because the first hydration shell is strongly influenced by strong ion-ion and ion-dipole interactions between sodium ions, sulfates, carboxylates, and -OH groups, ionic interactions play an important role in the stabilization of the 3D structure. The DFT-computed three-bond proton-proton coupling constants also showed that best agreement with experiment was obtained with a weighted average of 15:85 ((1)C4/(2)S0) of the sulfated iduronic acid forms indicating that the ratio is even more shifted toward the (2)S0 form than previously supposed. The DFT-computed pentasaccharide conformation differs from the previously published data, with the main changes at the glycosidic linkages, namely, the ψ1 torsion angles and the ϕ3 angle. The comparison of the glycosidic linkage torsion angle values in solution with the antithrombin-pentasaccharide complex also indicates that the pentasaccharide conformation changes upon binding to antithrombin III. The data supports the assumption that the protein selects the more populated (2)S0 conformer of heparin pentasaccharide and, consequently, the binding process of heparin pentasaccharide with antithrombin III is energetically more favorable than formerly expected.

Importance of IdoA and IdoA(2S) ring conformations in computational studies of glycosaminoglycan–protein interactions

Glycosaminoglycans (GAGs) interact with chemokines and growth factors in the extracellular matrix and, therefore, mediate cell communication processes. Heparin is one of the most studied GAGs, for which many experimental structures of its complexes with proteins are available. One of the monosaccharide components of heparin, sulfated iduronic acid (IdoA(2S)), is observed to adopt both 1C4 and 2S0 ring conformations. Despite the biological relevance of the sugar ring conformations for heparin–protein interactions, it is very challenging to take into account the conformational space of IdoA(2S) sugar ring for computational studies. Therefore, instead of systematically analyzing several ring conformations, which represents a combinatorial problem for a periodic heparin molecule, often only one ring conformation is taken into account. Here, we use docking and molecular dynamics (MD) to estimate how crucial this assumption could be for the conclusions being made in computational studies of heparin–protein interactions. We show that both docking solutions and free energy calculations from MD simulations are significantly affected by the conformations adopted by IdoA and IdoA(2S) rings. Therefore, in the application of computational approaches to heparin–protein systems the ring conformations should be treated properly to avoid misleading conclusions.

Is isolation and characterization of heparan sulfate from marine scallop Amussium pleuronectus (Linne.) an alternative source of heparin?

The present study describes the isolation, purification and structural characterization of heparan sulfate (HS) from marine scallop. Enzymatic depolymerization of the isolated HS, followed by polyacrylamide gradient gel electrophoresis (PAGE), confirmed its presence of HS is by metachromatic activity and agarose gel electrophoresis. The purity of HS is determined by the following treatment with heparin lyases though PAGE. 1H NMR spectroscopy revealed that the marine scallop HS is enriched in both glucuronic and sulfated iduronic acid residues. The result of this analysis clearly demonstrates that the isolated glycosaminoglycan (GAG) is HS. Marine scallop HS has contents of carbon, hydrogen, nitrogen and sulfated disaccharide and an elevated average sulfation level. The activated partial thromboblastin time (APTT) and prothrombin time (PT) of marine scallop HS, are lower than standard bovine intestinal HS. Moreover, marine scallop HS shows equal degree of sulfation to HS standard.

Modulations of Glypican-1 Heparan Sulfate Structure by Inhibition of Endogenous Polyamine Synthesis: MAPPING OF SPERMINE-BINDING SITES AND HEPARANASE, HEPARIN LYASE, AND NITRIC OXIDE/NITRITE CLEAVAGE SITES

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

The Putative Heparin-specific N-AcetylglucosaminylN-Deacetylase/N-Sulfotransferase Also Occurs in Non-heparin-producing Cells

N-Deacetylation and N-sulfation of N-acetylglucosamine of heparin and heparan sulfate are hypothesized to be mediated by different tissue-specific N-acetylglucosaminyl N-deacetylases/N-sulfotransferases, which in turn lead to the higher L-iduronic acid and sulfate content of heparin versus heparan sulfate. Furthermore, the putative heparin-specific N-acetylglucosaminyl N-deacetylase/N-sulfotransferase has been reported to require auxiliary proteins for its N-acetylglucosaminyl N-deacetylase activity in vivo based on its requirement of polycations in vitro. We have now found that cells derived from embryonic bovine trachea, a tissue that does not synthesize heparin, has a N-acetylglucosaminyl N-deacetylase/N-sulfotransferase, which has 95% amino acid sequence identity to the above enzyme postulated to be involved in the biosynthesis of heparin. Both enzymes also have very similar affinity for their substrates. The trachea enzyme does not require additional effectors for its N-acetylglucosaminyl N-deacetylase activity in vitro even though its biochemical characteristics are virtually the same as the enzyme previously isolated from cells of a heparin-producing mastocytoma tumor. The trachea enzyme, which is encoded by an abundant 4.6-kilobase mRNA, like mastocytoma cells, has 70% amino acid sequence identity with the corresponding enzyme from rat liver postulated to participate in the biosynthesis of heparan sulfate. Heparan sulfate synthesized by trachea cells has a higher content of sulfated iduronic acid than from other tissues. Together, the above results strongly suggest that the above enzymes from mastocytoma, liver, and trachea, per se, are not solely responsible for the selective tissue-specific synthesis of heparin or heparan sulfate; more likely cellular factors, additional enzymes, and availability of substrates in the Golgi lumen also play important roles in the differential synthesis of the above proteoglycans.

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units.

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.

Structural characteristics of heparan sulfates with varying sulfate contents.

Structural properties of heparan sulfate preparations from hog mucosa and beef lung sources were obtained by application of Smith degradation and nitrous acid reactions. Products formed by these reactions indicated that most of the iduronic acid present in these mucopolysaccharides is ester sulfated, whereas N-sulfated glucosamine residues are ester sulfated much less frequently. Repeating units with sulfated iduronic acid found to occur almost entirely in single sequences. Futhermore, the iduronic acid moieties may be bound to either N-acetylated or N-sulfated glucosamine units, with these occuring at either end of the uronic acid unit.

The distribution of sulfated uronic acid and hexosamine residues in heparin and heparan sulfate.

Heparins from various sources and heparan sulfate from umbilical cords have been subjected to Smith-degradation and reaction with nitrites. These procedures were effective for providing data relating to the distribution of sulfated iduronic acid residues in the molecule. Results indicated that heparins may have, as a prominent structural feature of the molecule, non-sulfated uronic acid distributed in single sequences, much as had been shown previously for N-acetylglucosamine residues. Sulfated uronic acid, however, may occur in multiple sequences of up to 5 or 6 residues. Heparan sulfate was found to have a major proportion of its ester sulfate on iduronic acid rather than hexosamine units, thereby having sections similar to those in heparins, though in considerably lower proportion.

The defect in the Hunter syndrome: deficiency of sulfoiduronate sulfatase.

Skin fibroblasts cultured from patients affected with the Hunter syndrome are deficient in the activity of a protein, named the "Hunter corrective factor," that is required for degradation of dermatan and heparan sulfates. We now show that this factor, purified from human urine, removes about 2% of the sulfate residues from [(35)S]mucopolysaccharide accumulated within Hunter fibroblasts; these groups are derived from "oversulfated" regions of the polymer. Acetone-powder extracts of fibroblasts derived from patients with the Hunter syndrome are deficient in this sulfatase, in contrast to similar extracts from fibroblasts of individuals of other genotype. Hunter corrective factor coupled to alpha-L-iduronidase (or alternatively, mixed extracts from Hurler and Hunter fibroblasts) release iduronic acid from 4-O-alpha-L-sulfoiduronosyl-D-sulfoanhydromannose. We conclude that the Hunter corrective factor is a sulfatase for sulfated iduronic acid residues.

Structure of pig skin dermatan sulfate. 2. Demonstration of sulfated iduronic acid residues.

The presence of sulfated uronic acid residues in dermatan sulfate has been known for some time. In view of the copolymeric nature of pig skin dermatan sulfate, it was of interest to see whether the extra sulfate groups were attached to iduronic acid or glucuronic acid residues.Hyaluronidase‐degraded dermatan sulfate was fractionated on Dowex‐1 to yield fractions of varying sulfate content. It was observed that the degree of sulfation was invesely proportional to the glucuronic acid content. Oversulfated dermatan sulfate was subsequently treated with chondroitinase‐AC to yield a completely glucuronic acid‐free polymeric product still bearing all the extra sulfate groups. More direct evidence for the presence of sulfated induronic acid moieties was obtained by Smith degradation of oversulfated, chondroitinse‐AC treated dermatan sulfate. A fast‐moving component isolated by preparative electrophoresis had the molar ratio sulfate; hexosamine 1.8:1 and contained exclusively L‐iduronic acid.