A Simple Analysis Method for 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid by HPLC-ELSD with Column for Anion Analysis

4-Deoxy-l- erythro-5-hexoseulose uronic acid (DEH) is a rare deoxy sugar produced from alginate by the action of an exotype alginate lyase. A simple and rapid method for analyzing DEH using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed in this study. For chromatography, an isocratic elution of ammonium formate buffer including formic acid and a column for anion chromatography were used. In the developed method, DEH was detected at a retention time of 3.038 minutes and limits of detection (signal-noise ratio = 3) and quantification (signal-noise ratio = 10) were 37.5 and 124.9 µg/mL as a sodium DEH, respectively. In addition, separation and detection of alginate unsaturated oligosaccharides were also tested using the method. Within an analysis time of 10 minutes, it was possible to separate and detect unsaturated disaccharide, unsaturated trisaccharide, and unsaturated tetrasaccharide prepared using poly(β-d-mannuronate) lyase and sodium alginate of high mannuronate type. The HPLC-ELSD method established in this study will be applicable for quantitative analysis of DEH and measurement of exotype alginate lyase activity.

Comparison of ion mobility-mass spectrometry and pulsed-field gradient nuclear magnetic resonance spectroscopy for the differentiation of chondroitin sulfate isomers

The unsaturated disaccharide isomers of chondroitin sulfate (CS) obtained by enzymatic digestion of the polysaccharide can be differentiated in mixtures by ion mobility MS (A) as well as pulsed-field gradient NMR (B).

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

ABSTRACTAlginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacteriumFlammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.

Structural analysis ofClostridium acetobutylicumATCC 824 glycoside hydrolase from CAZy family GH105

Clostridium acetobutylicumATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR ofBacillus subtilisstrain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) fromBacillus subtilisstrain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase fromBacillus subtilisstrain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.

A glycosulphatase that removes sulphate from mucus glycoprotein

A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection.

Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor

Binding of basic fibroblast growth factor (bFGF) to the extracellular matrix of cultured bovine aorta smooth muscle cells is likely to be mediated via heparan sulphate, since not only exogenous addition of heparan sulphate to the culture medium but also pretreatment of the cells with heparitinase (but not chondroitinase ABC) resulted in loss of binding. Comparison of the affinity of bFGF to various glycosaminoglycan-conjugated gels showed a direct and specific binding of bFGF to heparan sulphate. Heparan sulphate also bound to a bFGF affinity gel. However, the proportion of heparan sulphate bound varied depending on the source of the HS (more than 90% and 45% with pig aorta heparan sulphate and mouse EHS tumour heparan sulphate respectively). The bound heparan sulphate had the ability to protect bFGF from proteolytic digestion, but the unbound heparan sulphate did not. The results suggest the presence in the bound heparan sulphate of a specific structure involved in binding. Limited digestion with heparitinase I of porcine aorta heparan sulphate yielded 13% oligosaccharides bound to the gel, of which the smallest were octasaccharides. Analysis of a hexadecasaccharide fraction which was obtained at the highest yield among the bound oligosaccharides was performed by h.p.l.c. of the deamination products obtained with nitrous acid and the unsaturated disaccharide products formed by heparitinase digestion. Comparison of the disaccharide unit compositions exhibited a marked difference in IdoA(2SO4)GlcNSO3 and IdoA(2SO4)GlcNSO3(6SO4) units between the bound and unbound hexadecasaccharides. The amounts measured were 3 mol and 1 mol per mol of the former and 0.4 mol and 0.6 mol per mol of the latter. It is likely that the binding of bFGF to heparan sulphate may require the domain structure of the heparan sulphate to be composed of clustering IdoA(2SO4)-GlcNSO3 units.

An unsulphated region of the rat chondrosarcoma chondroitin sulphate chain and its binding to monoclonal antibody 3B3

The chondroitin sulphate chains of proteoglycans are not uniformly sulphated. Commonly, regions of under- and over-sulphation are found. It is probable that variability in chondroitin sulphation has physiological significance, although such structure-function relationships largely remain unexplored. Chondroitin sulphate from rat chondrosarcoma proteoglycan has been found to possess no oversulphated residues. It is primarily chondroitin 4-sulphate, although a significant proportion of unsulphated disaccharides (14%) are also present. It appears that some unsulphated disaccharides are concentrated only at the point of attachment to the linkage region (i.e. it is the major unsaturated disaccharide remaining attached to chondrosarcoma proteoglycan core produced by chondroitinase ABC digestion). This proteoglycan core binds monoclonal antibody (MAb) 3B3. Although 3B3 principally binds to 6-sulphated ‘stubs’ of proteoglycan cores [Couchman, Caterson, Christner & Baker (1984) Nature (London) 307, 650-652], given a high concentration of unsulphated ‘stubs’, it can alternatively bind to these residues. It is also evident that caution must be exercised in using MAb 3B3 to identify chondroitin 6-sulphated proteoglycans.

Liquid-chromatographic determination of urinary glycosaminoglycans for differential diagnosis of genetic mucopolysaccharidoses.

Glycosaminoglycans in urine from patients with various mucopolysaccharidoses were digested with chondroitin ABC lyase (EC 4.2.2.4) or chondroitin AC lyase (EC 4.2.2.5), then converted into fluorescent pyridylamino derivatives and analyzed by "high-performance" liquid chromatography. Chromatograms of the chondroitin ABC lyase digests of samples from nine patients with Hunter's syndrome all showed a major peak for unsaturated disaccharide-4-sulfate, derived from dermatan sulfate, and another specific but unidentified peak (peak x). All samples from patients with Hurler's and Scheie's syndromes contained another specific component (peak y), besides the disaccharide-4-sulfate. A sample from a patient with Maroteaux-Lamy syndrome showed peaks for N-acetylgalactosamine-4-sulfate and the disaccharide-4-sulfate, but neither peak x nor peak y. A sample from a patient with Morquio's disease showed a much higher ratio of the 6-sulfate to 4-sulfate than in other diseases, and N-acetylgalactosamine-6-sulfate was detected. We tentatively identified peaks x and y as the pyridylamino derivatives of 2-O-sulfo-L-idosyluronic acid 4-O-sulfo-N-acetylgalactosamine and L-idosyluronic acid 4-O-sulfo-N-acetylgalactosamine, respectively. This method may be useful in differential diagnosis.