Acid-containing Polysaccharides Research Articles

A chemo-enzymatic method for preparation of saturated oligosaccharides from alginate and other uronic acid-containing polysaccharides

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Structural and Biochemical Basis of a Marine Bacterial Glycoside Hydrolase Family 2 β-Glycosidase with Broad Substrate Specificity.

Uronic acids are commonly found in marine polysaccharides and increase structural complexity and intrinsic recalcitrance to enzymatic attack. Glycoside hydrolase family 2 (GH2) includes proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we report a novel GH2, AqGalA from a marine alga-associated Bacteroidetes organism with broad substrate specificity. Biochemical analyses revealed that AqGalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the AqGalA crystal structure in complex with galacturonic acid (GalA) and determined (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the AqGalA active-site pocket was proposed as a key component for broad substrate enzyme selectivity. Our AqGalA structural and functional data augment the current understanding of substrate recognition of GH2 enzymes and provide key insights into the bacterial use of uronic acid-containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformations of organic matter fueling marine food webs and global carbon cycling. However, our knowledge on carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of the Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificities of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, AqGalA from a marine Bacteroidetes organism, to identify the key components responsible for glycuronide recognition and hydrolysis. The broad substrate specificity of AqGalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially driven mechanisms of marine carbohydrate cycling.

Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacterium Catenovulum sp. CCB-QB4.

BackgroundOligosaccharides from polysaccharides containing uronic acids are known to have many useful bioactivities. Thus, polysaccharide lyases (PLs) and glycoside hydrolases (GHs) involved in producing the oligosaccharides have attracted interest in both medical and industrial settings. The numerous polysaccharide lyases and glycoside hydrolases involved in producing the oligosaccharides were isolated from soil and marine microorganisms. Our previous report demonstrated that an agar-degrading bacterium, Catenovulum sp. CCB-QB4, isolated from a coastal area of Penang, Malaysia, possessed 183 glycoside hydrolases and 43 polysaccharide lyases in the genome. We expected that the strain might degrade and use uronic acid-containing polysaccharides as a carbon source, indicating that the strain has a potential for a source of novel genes for degrading the polysaccharides.MethodsTo confirm the expectation, the QB4 cells were cultured in artificial seawater media with uronic acid-containing polysaccharides, namely alginate, pectin (and saturated galacturonate), ulvan, and gellan gum, and the growth was observed. The genes involved in degradation and utilization of uronic acid-containing polysaccharides were explored in the QB4 genome using CAZy analysis and BlastP analysis.ResultsThe QB4 cells were capable of using these polysaccharides as a carbon source, and especially, the cells exhibited a robust growth in the presence of alginate. 28 PLs and 22 GHs related to the degradation of these polysaccharides were found in the QB4 genome based on the CAZy database. Eleven polysaccharide lyases and 16 glycoside hydrolases contained lipobox motif, indicating that these enzymes play an important role in degrading the polysaccharides. Fourteen of 28 polysaccharide lyases were classified into ulvan lyase, and the QB4 genome possessed the most abundant ulvan lyase genes in the CAZy database. Besides, genes involved in uronic acid metabolisms were also present in the genome. These results were consistent with the cell growth. In the pectin metabolic pathway, the strain had genes for three different pathways. However, the growth experiment using saturated galacturonate exhibited that the strain can only use the pathway related to unsaturated galacturonate.

Disentangling the role of extracellular polysaccharides in desiccation tolerance in lichen-forming microalgae. First evidence of sulfated polysaccharides and ancient sulfotransferase genes.

Trebouxia sp. TR9 and Coccomyxa simplex are desiccation-tolerant microalgae with flexible cell walls, which undergo species-specific remodelling during dehydration-rehydration (D/R) due to their distinct ultrastructure and biochemical composition. Here, we tested the hypothesis that extracellular polysaccharides excreted by each microalga could be quantitatively and/or qualitatively modified by D/R. Extracellular polysaccharides were analysed by size exclusion and anion exchange chromatography, specific stains after gel electrophoresis and gas chromatography/mass spectrometry of trimethylsilyl derivatives (to determine their monosaccharide composition). The structure of a TR9-sulfated polymer was deduced from nuclear magnetic resonance (NMR) analyses. In addition, sugar-sulfotransferase encoding genes were identified in both microalgae, and their expression was measured by RT-qPCR. D/R did not alter the polydispersed profile of extracellular polysaccharides in either microalga but did induce quantitative changes in several peaks. Furthermore, medium-low-sized uronic acid-containing polysaccharides were almost completely substituted by higher molecular mass carbohydrates after D/R. Sulfated polysaccharide(s) were detected, for the first time, in the extracellular polymeric substances of both microalgae, but only increased significantly in TR9 after cyclic D/R, which induced a sugar-sulfotransferase gene and accumulated sulfated ß-D-galactofuranan(s). Biochemical remodelling of extracellular polysaccharides in aeroterrestrial desiccation-tolerant microalgae is species-specific and seems to play a role in the response to changes in environmental water availability.

Three structurally and functionally distinct β-glucuronidases from the human gut microbe Bacteroides uniformis

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct β-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

Distribution of uronic acid-containing polysaccharides in 5 species of shellfishes

Mollusks are a rich source of uronic acid-containing polysaccharides (UACPs) possessing biological and pharmacological activities. In the present study, UACP compositions of different tissues from 5 species of edible shellfishes, namely Neverita didyma, Natica janthostomoides, Saxidomus purpurata, Cyclina sinensis, and Chlamys farreri were systemically analyzed. Totally 10 UACPs were observed, including chondroitin sulfate, heparin, an identified mannoglucuronan, 3 unknown glycosaminoglycans (GAGs, U1, U3 and U6) with hexuronic acid-hexosamine repeating units, and 4 unknown non-GAGs (U2, U4, U5 and U7) with hexuronic acid-hexose repeating units. U4-7 showed tissue- and species-specificity while the other UACPs prevalently existed in these shellfish tissues. UACP compositions in tissues from the same shellfish species were similar for S. purpurata and C. sinensis but different for C. farreri, N. didyma and N. janthostomoides. And this investigation also indicates that the similarity of UACPs composition between different shellfishes is related to the genetic relationship.

Identification and quantification of uronic acid-containing polysaccharides in tissues of Russian sturgeon (Acipenser gueldenstaedtii) by HPLC–MS/MS and HPLC–MSn

Uronic acid-containing polysaccharides (UACPs) including glycosaminoglycans (GAGs) and some non-GAGs in different tissues of Russian sturgeon were analyzed by detecting characteristic oligosaccharides in their acid hydrolysates after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Disaccharides without substituents were identified and quantified using HPLC–MS/MS with a triple quadrupole mass spectrometer, while other oligosaccharides including sulfated/acetylated disaccharides and tetrasaccharides were characterized by HPLC–MSn with a linear ion trap mass spectrometer. The results showed that the nostril, gill arch and notochord of Russian sturgeon contained high amounts of CS ranging from 1700 to 3660 mg per 100 g of dried tissues. A non-GAG UACP, named AGSP, with a backbone of →4)-GlcA(1 → 2)-Man(1→, was found in the gill arch, caudal fin and notochord of sturgeon with the contents from 37 to 227 mg per 100 g of dried tissue. Another two unknown non-GAGs, UP1 and UP2, existed in intestine and in body muscles and gill arch, respectively. And an unidentified GAG (UP3) was found in testes, ovaries, swim bladder, intestine and skin. This investigation was the first effort to determine the overall UACP species in sturgeon after extraction of the eggs and caviar production.

Quick characterization of uronic acid-containing polysaccharides in 5 shellfishes by oligosaccharide analysis upon acid hydrolysis

Uronic acid-containing polysaccharides (UACPs) including well-known glycosaminoglycans (GAGs) and some non-GAGs exist widely in animal kingdom. Although numerous methods have been established to analyze GAGs, few methods are available for non-GAG UACPs. In the present study, a protocol to identify all kinds of UACPs with repeating disaccharide units of hexosamine and uronic acid was demonstrated, and UACP components in five shellfishes, namely Turritella fortilirata Sowerby (GTF), Batillaria zonalis (GBZ), Nassarius variciferus (GNV), Monodonta labio Linnaeus (GML), and Argopecten irradians Lamarck (BAI) were primarily revealed. After a simple isolation procedure, crude polysaccharides were depolymerized by controlled acid hydrolysis, and then the resulting oligosaccharides were detected by HPLC coupled with mass spectrometer after 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling. According to chromatograms using the triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) mode, chondroitin sulfate (CS) was found in GNV and GML, a non-GAG named abalone gonad sulfated polysaccharide (AGSP) with a backbone of →4)-β-GlcA-(1 → 2)-α-Man-(1→ repeating units in GBZ, and both of AGSP and CS in BAI and GTF. Further characterization of tetrasaccharides and sulfated/acetylated disaccharides by HPLC combined with an ion trap mass spectrometer confirmed the structural identification of CS and AGSP, and indicated CS in GTF and BAI was Type C. These results suggest the 5 mollusks as potential resources for CS and AGSP. And the analysis protocol presented in this study was powerful and effective for quick characterization of UACPs including GAGs as well as non-GAGs in complicated matrix.

Comparison of polysaccharides of Haliotis discus hannai and Volutharpa ampullacea perryi by PMP-HPLC-MSn analysis upon acid hydrolysis

Haliotis discus hannai Ino (Haliotis) is a highly valued marine shellfish, and it is sometimes replaced by another cheaper Gastropoda mollusk, Volutharpa ampullacea perryi (Volutharpa). Polysaccharides from pleopods, viscera and gonads of these two gastropods were compared by analyzing the mono- and di-saccharides in their acid hydrolysates using high performance liquid chromatography-mass spectrometry (HPLC-MSn) after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Disaccharide analysis revealed the distribution of uronic acid-containing polysaccharides (UACPs) in the biological samples. GlcA-(1→2)-Man, GlcA-(1→3)-GalN, and another disaccharide consisting of a hexuronic acid linked to a hexose were found in the hydrolysates, which indicated the existence of AGSP (abalone gonad sulfated polysaccharide) with the backbone composed of →2)-α-Man(1→4)-β-GlcA(1→ repeating unit, AAP (abalone glycosaminoglycan-like polysaccharide) with the backbone of →3)-GalNAc-(1→2)-GlcA-(1→3)-GalNAc-(1→4)-GlcA-(1→ repeating unit, and unidentified DS1P containing a hexuronic acid linked to a hexose unit, respectively. As shown by extracted ion chromatograms (XICs), AAP was the only UACP found in pleopods of the two gastropods; gonads and viscera of Haliotis contained DS1P and AGSP, while those of Volutharpa contained DS1P, AGSP as well as AAP. Monosaccharides in the acid hydrolysates were demonstrated in XICs by extracting their corresponding PMP derivative quasi-molecular ions one by one, and the results indicated the similar conclusion to the disaccharide analysis. Therefore, it could be concluded that polysaccharides from pleopods of the two gastropods are very similar, while those from their viscera and gonads differ greatly.

Characterization of acidic polysaccharides from the mollusks through acid hydrolysis

Uronic acid-containing polysaccharides (UACPs) including glycosaminoglycans (GAGs) exist widely in nature. Herein we propose an elegant methodology to identify UACPs by analyzing their disaccharides produced from the acid hydrolysis using HPLC–MSn upon 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Based on the optimization of experimental conditions by the single factor experiment and orthogonal test design, the combination of 1.3M TFA at 105°C for 3h is found to be the optimum. Subsequently, these conditions were applied to investigate the distribution of UACPs in 20 selected species of edible Bivalvia and Gastropoda. PMP-disaccharides derived from UACPs in mollusks were identified by comparing the retention time and mass spectra with those of the reference PMP-disaccharides from hyaluronic acid (HA), chondroitin sulfate (CS), heparin (HP), and AGSP with →4)-GlcA(1→2)-Man(1→ repeating units. The analysis reveals the prevalence of CS in the shellfishes as well as the HP, and existence of three non-GAG UACPs in 7 mollusks.

An Anionic Polymer Incorporating Low Amounts of Hydrophobic Residues Is a Multifunctional Surfactant. Part 1: Emulsifying, Thickening,Moisture-Absorption and Moisture-Retention Abilities of a FattyAcid-Containing Anionic Polysaccharide

We have been studying the function and structure of fatty acid-containing extracellular polysaccharides (FACEPS) produced by bacteria belonging to the genus Rhodococcus. In this study, we examined the relationships between the structure and emulsifying, thickening, moisture-absorption, and moisture-retention capabilities of rhodococcal FACEPS using S-2 EPS produced by R. rhodochrous strain S-2. We prepared chemically deacylated S-2 EPS (DeAcyl S-2 EPS) and palmitoylated DeAcyl S-2 EPS (ReAcyl S-2 EPS), and compared them with native S-2 EPS. All of the properties were attenuated and recovered by deacylation and reacylation of S-2 EPS, respectively. These results suggest that the fatty acid moiety of rhodococcal FACEPS is involved in such functional properties. We also showed that palmitoylation improved the emulsifying, moisture-ab-sorption, and moisture-retention abilities of other acidic polysaccharides that are commercially available. These results suggest that the acidity of the polysaccharide backbone is at least partly responsible for the observed functionality of fatty acid-containing polysaccharides. To our knowledge, this is the first report on multifunctional property of an anionic polymer incorporating low amounts of hydrophobic residues. The present findings could be useful for the creation of new multifunctional surfactants from renewable raw materials for use in various industries, e.g., in cosmetics.

An Anionic Polymer Incorporating Low Amounts of Hydrophobic Residues Is a Multifunctional Surfactant. Part 2: Emulsification, Moisture Absorption, and Moisture Retention of Alkyl Esterified Poly-γ-Glutamic Acid

In the accompanying paper (Part 1), we showed that the fatty acid moiety and the acidity of the polysaccharide-backbone of a fatty acid-containing polysaccharide might be involved in its emul-sification, moisture absorption, and moisture retention abilities. In this study, we synthesized alkyl esterified poly-γ-glutamic acid (PGA) with various chain lengths and degrees of substitution of the alkyl moieties to examine how hydrophobic groups incorporated in the anionic polymer contribute to enhanced emulsification, moisture absorption, and moisture retention. With a low degree of alkylation of PGA, these abilities were drastically improved. To improve the moisture absorption of PGA, alkylation with a short chain length is effective in forming interspaces between PGA chains to trap water molecules. Hydrophobic-hydrophilic balance may also be important to improve the emulsification and moisture retention abilities of PGA alkylates. To the best of our knowledge, this is the first report of the relationship between the structure and the multifunctional abilities of an anionic polymer incorporated with a small amount of hydrophobic residue. PGA alkylates, as well as fatty acid-containing polysaccharides, have potential use as multifunctional surfactants throughout various industries.

Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a Ki value of 35μM. The structure of BSTA was determined at a resolution of 2.3Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23–190) that is adjacent to the catalytic domain (a.a. 191–535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72 h conversion assay (CONV) with in vitro rumen microflora incubation for 72 h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r = 0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.

Influenza A Virus Neuraminidase Enhances Meningococcal Adhesion to Epithelial Cells through Interaction with Sialic Acid-Containing Meningococcal Capsules

The underlying mechanisms of the epidemiological association between influenza virus infections and Neisseria meningitidis invasive infections are not fully understood. Here we report that adhesion of N. meningitidis to human Hec-1-B epithelial cells is enhanced by influenza A virus (IAV) infection. A potential role of the viral neuraminidase (NA) in facilitating meningococcal adhesion to influenza virus-infected epithelial cells was examined. Expression of a recombinant IAV NA in Hec-1-B human epithelial cells increased the adhesion of strains of N. meningitidis belonging to the sialic acid-containing capsular serogroups B, C, and W135 but not to the mannosamine phosphate-containing capsular serogroup A. Adhesion enhancement was not observed with an inactive NA mutant or in the presence of an NA inhibitor (zanamivir). Furthermore, purified IAV NA was shown to cleave sialic acid-containing capsular polysaccharides of N. meningitidis. On the whole, our findings suggest that a direct interaction between the NA of IAV and the capsule of N. meningitidis enhances bacterial adhesion to cultured epithelial cells, most likely through cleavage of capsular sialic acid-containing polysaccharides. A better understanding of the association between IAV and invasive meningococcal infections should help to set up improved control strategies against these seasonal dual viral-bacterial infections.

Chemical synthesis of UDP-Glc-2,3-diNAcA, a key intermediate in cell surface polysaccharide biosynthesis in the human respiratory pathogens B. pertussis and P. aeruginosa

In connection with studies on lipopolysaccharide biosynthesis in respiratory pathogens we had a need to access potential biosynthetic intermediate sugar nucleotides. Herein we report the chemical synthesis of uridine 5'-diphospho 2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid (UDP-Glc-2,3-diNAcA) (1) from N-acetyl-D-glucosamine in 17 steps and approximately 9% overall yield. This compound has proved invaluable in the elucidation of biosynthetic pathways leading to the formation of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid-containing polysaccharides.

Carbohydrate analysis of plant materials with uronic acid-containing polysaccharides–A comparison between different hydrolysis and subsequent chromatographic analytical techniques

Acid hydrolysis, acid methanolysis, and enzymatic hydrolysis were compared for depolymerization of five different plant materials containing uronic acids. The analyzed plant materials were oat spelt, wheat straw, spruce thermomechanical pulp, aspen stemwood, and totally chlorine-free (TCF) bleached hardwood kraft pulp. Furthermore, GC (using both HP-1 and HP-5 capillary columns and FID and MSD detectors), HPAEC-PAD, and HPAEC-Borate techniques were compared for subsequent analysis of the released monosaccharides. It was shown that acid methanolysis combined with GC analysis is a convenient method for obtaining the sugar unit composition and amount of non-crystalline polysaccharides in different plant materials. The methanolysis method was generally superior to the hydrolysis method for xylan- and uronic acid-containing samples. However, acid and enzymatic hydrolysis showed the highest recoveries for bleached chemical pulp samples. Acid hydrolysis is also required for crystalline polysaccharides, but the strong acid conditions evidently lead to degradation of labile sugars. The plant methanolysates were not suitable as such for analysis on an HPAEC-PAD system. For analysis of the total amount of sugar units, hence including cellulose, other non-crystalline hemicelluloses, and pectins, a combination of the methanolysis and hydrolysis methods is recommended.

Variability in the carbazole assay for N-desulfonated/N-acylated heparin derivatives

The carbazole assay is commonly employed to quantify heparin and other uronic acid-containing polysaccharides. Heparin-derived standard curves are often employed to quantify solutions of various natural and unnatural heparin structures that have different levels of sulfate substitution, different levels of N-sulfo and N-acetyl groups, and other structural changes as a consequence of reducing molecular weight. Recent studies in our laboratory have focused on chemically modified heparin derivatives comprised of structurally diverse N-acyl moieties substituted into heparin in place of N-sulfo groups. We report here that although differing degrees of 2- N-sulfo-, 2- N-acetyl- or 2-amino- d-glucosamine residues within heparin do not affect signal intensity in the carbazole assay, replacing N-sulfo groups in heparin with structurally diverse N-acyl moieties affords products that display significant variation in the assay. The structure of different N-acyl groups, and to a lesser extent the degree of N-acylation by individual N-acyl groups, is shown to variably alter signal intensity in the carbazole assay even though content and structure of uronic acid residues is unaltered.

Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra.

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.

Complexation of vanadium(V) oxyanions with hexopyranose- and mannopyranoseuronic acid-containing polysaccharides: stereochemical considerations

Carbohydrates containing galactopyranosyl and mannopyranosyl units with vicinal cis-diols were treated with NaVO 3 in D 2O, and complexation was determined by 51V NMR spectroscopy. Me α-Gal p, Me β-Gal p (3,4- cis-diols), and Me α-Man p (2,3- cis-diol) complexed, but Me β-Man p barely did so. This low degree of complexation also occurred with a β-mannan containing alternate (1 → 3)- and (1 → 4)-linkages and an alginate having β-Man pA blocks. In contrast, branched α-mannans complexed readily, although the 51V resonances for one with side chains terminated with α-Man p-(1 → 3)-α-Man p-(1 → differed from another with only α-Man p-(1 → 2)-α-Man p-(1 → groups. The anomeric configuration of Me α-Gal p and Me β-Gal p, each with 3,4- cis-diols remote from C-1, gave rise to three 51V signals of complexes with similar shifts and proportions. The shifts of a galactomannan with terminal α-Gal p-(1 → 2)-α-Man p- were the same as those with α-Gal p-(1 → 6)-β-Man p- groups, but fewer complexes were formed with the former structure, probably due to greater steric crowding of the vanadate esters. Most of the complexes gave rise to a signal in the δ −515 region, consistent with the dimeric trigonal–bipyramidal structure.