Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Microcalorimetric investigations of the metabolism of yeasts

Summary The anaerobic growth of the yeast Saccharomyces cerevisiae with six different mono- and disaccharides as energy source was investigated calorimetrically. With mixtures of monosaccharides and disaccharides or disaccharides with each other, biphasic thermograms were obtained. The diauxic growth is discussed in view of constitutive and inducible transport systems and degradation enzymes.     

Use of graphitised carbon negative ion LC-MS to analyse enzymatically digested glycosaminoglycans

Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.     

The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD79.41

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose).Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Disaccharide Composition of Heparan Sulfates: Brain, Nervous Tissue Storage Organelles, Kidney, and Lung

Abstract: We have characterized the structural properties of heparan sulfates from brain and other tissues after de-polymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46–69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17–21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10–14%, together with smaller and approximately equall proportions (5–9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine-and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5–10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71 %) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large densecored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for ～ 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21 % unsulfated disaccharides, 10% mono-and disulfated disaccharides, and 69% trisulfated disaccharides. Our results therefore reveal significant differences among heparan sulfates from different sources, supporting other evidence that structural variations in heparan sulfate may be related to specific biological functions, such as the switching in the neural response from fibroblast growth factor-2 to fibro-blast growth factor-1 resulting from developmental changes in the glycosaminoglycan chains of a heparan sulfate proteoglycan.     

Modified mannose disaccharides as substrates and inhibitors of a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis

A panel of alpha-(1-->6)-linked mannose disaccharides (5-8) in which the 2'-OH group has been replaced, independently, by deoxy, fluoro, amino, and methoxy functionalities has been synthesized. Evaluation of these compounds as potential substrates or inhibitors of a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in mycobacterial LAM biosynthesis demonstrated that the enzyme is somewhat tolerant substitution at this site. The enzyme recognizes the disaccharides with groups similar or smaller in size than the native hydroxyl (6-8), but not the disaccharide with the more sterically demanding methoxy group (5). The 2'-OH appears not form a critical hydrogen bonding interaction with the protein as the 2'-deoxy analog is a substrate for the enzyme.     

Prediction of the anomeric configuration, type of linkage, and residues in disaccharides from 1D (13)C NMR data

A machine learning approach was explored for the prediction of the anomeric configuration, residues, and type of linkages of disaccharides using (13)C NMR chemical shifts. For this study, 154 pyranosyl disaccharides were used that are dimers of the α or β anomers of d-glucose, d-galactose or d-mannose residues bonded through α or β glycosidic linkages of types 1→2, 1→3, 1→4, or 1→6, as well as methoxylated disaccharides. The (13)C NMR chemical shifts of the training set were calculated using the casper (Computer Assisted SPectrum Evaluation of Regular polysaccharides) program, and chemical shifts of the test set were experimental values obtained from the literature. Experiments were performed for (1) classification of the anomeric configuration, (2) classification of the type of linkage, and (3) classification of the residues. Classification trees could correctly classify 67%, 74%, and 38% of the test set for the three tasks, respectively, on the basis of unassigned chemical shifts. The results for the same experiments using Random Forests were 93%, 90%, and 68%, respectively.     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.     

The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans.

Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4), IdoA----ATalR (4-SO4) and GlcA(2-SO4)----ATalR(6-SO4); dermatan sulphate gave IdoA----ATalR(4-SO4), GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4)----IdoA(2-SO4)ATalR(4-SO4) and IdoA----ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)----AManR(6-SO4), Gal----AManR(6-SO4), Gal(6-SO4)----AManR and Gal----AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.     

Acetolysis of disaccharides: Comparative kinetics and mechanism

The initial acetolysis rates of several disaccharides were compared using an assay procedure which involves adding portions of the reaction mixture to an alkaline sodium borohydride solution. After reduction, glycosidically-linked hexose was determined by the phenol-sulfuric acid method. For D-glucose disaccharides, β linkages were cleaved faster than α linkages, suggesting anchimeric assistance from the trans C-2 acetoxyl group. The acetolysis reaction rates for the various β-linked D-glucose disaccharides decreased in the order (1→6) ? (1→3) > (1→2) > (1»4). For the various α-linked disaccharides the order was (1→6) ? (1→4) > (1»3)> (1→2). The acetolysis rates for D-mannose disaccharides were in the order α-(1»6) ? α-(1→3) > β-(1»4) > α-(1»2). Turanose (3-O-α-D-glucopyranosyl-D-fructose) was cleaved at a much faster rate than either D-mannobiose or D-glucobiose with α-(1»2) or α-(1»3) linkages. A reaction mechanism is supported which features an acyclic intermediate, and, for certain -disaccharides, C-2 acetoxyl anchimeric assistance.     

Dermatan sulfates of normal and scarred fascia

We evaluated the composition of dermatan sulfates (DS) derived from 23 samples of normal and 23 samples of scarred fascia lata. We analyzed the molecular weight of intact DS chains and the length of chain regions comprising: (1) clusters of L-iduronate-containing disaccharides ("iduronic sections"); (2) clusters of D-glucuronate-containing disaccharides ("glucuronic sections"); and (3) copolymeric sections with both types of disaccharides. A portion of scarred fascia DS chains demonstrated higher molecular weight compared with those from normal tissue. Most disaccharides of DS chains derived from both fascia types form copolymeric segments - heterogeneous in size - with alternatively distributed single disaccharides with glucuronic residues and mainly single ones with iduronate. Only a small number of disaccharides form "glucuronic sections" of heterogeneous size or short "iduronic sections". However, the scarred fascia DS chains demonstrate an increased content of shorter "glucuronic sections" and shorter, often oversulfated, copolymeric segments. It seems that in normal fascia, the DS chain type with a single, long copolymeric region and a single, shorter "glucuronic section" is predominant, while in scarred tissue an increase in multidomain DS chain content may occur.     

Prediction of the anomeric configuration, type of linkage, and residues in disaccharides from 1D C NMR data

A machine learning approach was explored for the prediction of the anomeric configuration, residues, and type of linkages of disaccharides using ¹³C NMR chemical shifts. For this study, 154 pyranosyl disaccharides were used that are dimers of the α or β anomers of d-glucose, d-galactose or d-mannose residues bonded through α or β glycosidic linkages of types 1→2, 1→3, 1→4, or 1→6, as well as methoxylated disaccharides. The ¹³C NMR chemical shifts of the training set were calculated using the casper (Computer Assisted SPectrum Evaluation of Regular polysaccharides) program, and chemical shifts of the test set were experimental values obtained from the literature. Experiments were performed for (1) classification of the anomeric configuration, (2) classification of the type of linkage, and (3) classification of the residues. Classification trees could correctly classify 67%, 74%, and 38% of the test set for the three tasks, respectively, on the basis of unassigned chemical shifts. The results for the same experiments using Random Forests were 93%, 90%, and 68%, respectively.     

Unsaturated disaccharides in the enzymatic digests of heparan sulfates

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Crystal Structure of a Bacterial Unsaturated Glucuronyl Hydrolase with Specificity for Heparin

Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in β-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by x-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and Bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35-Å resolution. In comparison with structures of streptococcal and Bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.     

Quantitation of the sulfated disaccharides of heparin by high performance liquid chromatography

Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boro[³H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-2,5-anhydro-d-mannitol, 4-O-(α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, and 4-O-(β-d-glucopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol. The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 mm KH₂PO₄. The KH₂PO₄ concentration was tehn increased to 400 mm to elute the disulfated disaccharide. Column effluents were collected in $\text{1}\text{2}\text{-}\text{ml}$ fractions, and the recovery of each ³H-labeled product was determined by scintillation counting. When sodium boro-[³H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the ³H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro[³H]hydride-reduced heparin deamination mixtures.     

Conformational and dynamical properties of disaccharides in water: a molecular dynamics study

Explicit-solvent molecular dynamics simulations (50 ns, 300 K) of the eight reducing glucose disaccharides (kojibiose, sophorose, nigerose, laminarabiose, maltose, cellobiose, isomaltose, and gentiobiose) have been carried out using the GROMOS 45A4 force field (including a recently reoptimized carbohydrate parameter set), to investigate and compare their conformational preferences, intramolecular hydrogen-bonding patterns, torsional dynamics, and configurational entropies. The calculated average values of the glycosidic torsional angles agree well with available experimental data, providing validation for the force field and simulation methodology employed in this study. These simulations show in particular that: 1) (1-->6)-linked disaccharides are characterized by an increased flexibility, the absence of any persistent intramolecular hydrogen bond and a significantly higher configurational entropy (compared to the other disaccharides); 2) cellobiose presents a highly persistent interresidue hydrogen bond and a significantly lower configurational entropy (compared to the other disaccharides); 3) persistent hydrogen bonds are observed for all disaccharides (except (1-->6)-linked) and typically involve a hydrogen donor in the reducing residue and an acceptor in the nonreducing one; 4) the probability distributions associated with the glycosidic dihedral angles and psi are essentially unimodal for all disaccharides, and full rotation around these angles occurs at most once or twice for (never for psi) on the 50-ns timescale; and 5) the timescales associated with torsional transitions (except around and psi) range from approximately 30 ps (rotation of hydroxyl groups) to the nanosecond range (rotation of the lactol and hydroxymethyl groups, and around the omega-glycosidic dihedral angle in (1-->6)-linked disaccharides).