Studies on by-products from the industrial extraction of alginate

The chemical composition of fucans isolated from leach-water, an industrial alginate extraction by-product, was investigated. Several fractions were obtained by anion exchange and gel permeation chromatography. They all contained fucose, but differed in the uronic acid, sulfate, xylose and galactose contents. They distributed as a continuum between uronic acid rich and sulfate poor to sulfate rich and uronic acid poor molecules. Two highly sulfated fractions were studied in particularly by chemical means (methylation, carboxy reduction, desulfation, controlled acid hydrolysis) and by¹³C nuclear magnetic resonance spectroscopy. One fraction consisted of a highly branched fucan (43.8% fucose) composed mostly of 1,2,3,4- and 1,2,4-linked fucose with some 1,4-,1,3,4- and 1,3-linkages and sulfate (23.9%) occurring on O₂ and/or O₃ and/or O₄. The other was composed mainly of fucose (31.6%), galactose (24.7%) and sulfate (23.7%). It consisted primarily of 1,6-, 1,4,6-, 1,3- and 1,3,6-linked galactose 6-and/or 4- and/or 3-sulfate on which are linked essentially terminal fucose or 1,4-linked with sulfate on O₂ and/or O₃ and/or O₄. None of these highly branched fractions contained sufficiently regular segments to yield series of homologous oligosaccharides on partial acid hydrolysis or interpretable¹³C NMR spectra.author for correspondence     

Glycosaminoglycans and glycoproteins associated with rat brain nuclei

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 μmol hexosamine/100 mg protein, comprising 57% chrondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 μg/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.     

Sialoglycopeptides from bovine milk fat globule membrane

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCI (Group A) and 0.5 M NaCI (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose.The major sialoglycopeptide of Group B released 35 % of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol galactose and sialic acid in a molar ratio of 1 : 1 : 2 was partially characterised from the cleavage mixture.The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride.The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

Glycosaminoglycans from earthworms (<Emphasis Type="Italic">Eisenia andrei</Emphasis>)

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC–ESI–MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.     

Biotransformation of Cinnamic Acid,p-Coumaric Acid,Caffeic Acid,and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana

Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid β-D-glucopyranosyl ester, p-coumaric acid, and 4-O-β-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-β-D-glucopyranosylcoumaric acid, p-coumaric acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcoumaric acid β-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-β-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-β-D-glucopyranosylcaffeic acid, 3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosylcaffeic acid, a new compound, 3-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcaffeic acid, 4-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, ferulic acid, and 4-O-β-D-glucopyranosylferulic acid. 4-O-β-D-Glucopyranosylferulic acid, ferulic acid β-D-glucopyranosyl ester, and 4-O-β-D-glucopyranosylferulic acid β-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.     

Genetic and metabolic control of sulfate metabolism in Neurospora crassa: A specific permease for choline-O-sulfate

Neurospora crassa can use choline-O-sulfate as its sole sulfur source; the utilization of this compound involves its entry followed by intracellular hydrolysis. Neurospora possesses a transport system for the uptake of choline-O-sulfate which is specific for the sulfate ester and does not transport, nor is it inhibited by, either choline or inorganic sulfate. Mutant strains of Neurospora that are unable to transport or grow on inorganic sulfate can, nevertheless, utilize choline-O-sulfate for growth and transport the intact organic sulfate at a normal rate. Methionine, which represses a number of enzymes of sulfur anabolism, also represses the synthesis of the specific permease for choline-O-sulfate. A regulatory gene, cys-3, which controls the synthesis of choline sulfatase, aryl sulfatase, and several other related enzymes, also regulates the synthesis of the choline sulfate permease. Evidence is presented that the activity of choline sulfate permease is also regulated by a turnover process, the transport system having a functional half-life of approximately 3 hr.This investigation was supported by Public Health Service Grant 1 RO1 GM-18642 from the National Institute of General Medical Services.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.     

Nature of the Carbohydrate Side Chains and Their Linkage to the Protein in Chicken Egg White Ovomucin

Some proteolytic digests of chicken egg white ovomucin were fractionated and characterized. It was shown that there are at least three types of carbohydrate side chains in ovomucin; a chain composed of galactose, galactosamine, sialic acid and sulfate in a molar ratio of about 1: 1: 1: 1, a chain composed of galactose and glucosamine in a molar ratio of about 1:1, and a chain composed of mannose and glucosamine in a molar ratio of about 1:1. It was also shown that the carbohydrate side chain composed of galactose, galactosamine, sialic acid and sulfate is linked O-glycosidically to serine or threonine in the protein core of ovomucin.     

Glycoproteins of the opium poppy

Two carbohydrate-protein fractions were isolated from the water-soluble biopolymer from opium poppy capsules by chromatography on SP-Sephadex. The carbohydrate chains are composed of arabinose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid and 4-O-methyl glucuronic acid. Methylation analysis indicated a high degree of branching suggesting a very complex structure. Treatment of the glycoprotein with NaOH in the presence of NaBH₄ resulted in a significant decrease in the serine and threonine content. The carbohydrate side chains released contained the sugar alcohol, galactitol. These results indicate that polysaccharide chains are linked to protein via serine-O-galactoside linkages.     

Preparation and characterization of tetrasaccharides from beef-lung heparin

Partial N-desulfation of beef-lung heparin prior to degradative deamination with butyl nitrite and reduction with sodium borotritide yielded many large fragments. From these, a tetrasaccharide tetra-O-sulfate (II-4NH; 8% yield from heparin) and a mixture of tetrasaccharide tri-O-sulfates (II-3NHh; 6% yield) were isolated by sequential chromatography on Sephadex G-25 and DEAE-Sephadex. For these and the other tetrasaccharide preparations, the radioactive disaccharides produced by deamination, with and without subsequent relabelling with sodium borotritide, have been quantitatively determined by the methodology described in the preceding paper. In most cases, the results permit a unique reconstruction of the relative proportions of monosaccharide components and of their sequences in the compounds present. Tetrasaccharide II-4NH appeared homogeneous and has the structure (IdoA-SO₄)(GN-O-SO₄)(IdoA-SO₄)(anhMan-SO₄). In tetrasaccharide preparation II-3NHh, the preponderant species (57%) lacks ester sulfate at the terminal l-iduronic residue in the structure just mentioned, and five other species are present. By treatment of the tetra-O-sulfate with mild acid, tetrasaccharide preparations with 3, 2, 1, and no ester sulfate were produced and could be isolated. The isomeric tetrasaccharide tri-O-sulfate species have been partially resolved. Composition and sequence data are given for all of the preparations. The resolution of numerous small fractions suggests minor irregularities in the fine structure of heparin. Ion-exchange electrophoresis was applied to the acidic oligosaccharides and was found to be a useful technique.     

Minor diterpenoid glycosides from the leaves of Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-β-d-xylopyranosyl-β-d-glucopyranosyl) ester (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (2), and 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data.     

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.     

Structures of the novel α-glucosyl linked diterpene glycosides from Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, two new minor diterpene glycosides having α-glucosyl linkage were isolated besides the known steviol glycosides including stevioside, steviolbioside, rebaudiosides A–F, rubusoside and dulcoside A. The structures of the two compounds were identified as 13-[(2-O-(3-α-O-d-glucopyranosyl)-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.     

Roles of galactose and sulfate residues in sulfatides for their antagonistic functions in the blood coagulation system

We previously reported that the sulfatide (galactosylceramide I³-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I⁶-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.     

Marine Bacterial Sulfated Fucoglucuronomannan (SFGM) Lyase Digests Brown Algal SFGM into Trisaccharides

Abstract Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp, Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp(6-O-sulfate), and Δ^4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)α1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAβ1-2D-Manpα1-)_n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAβ1-2(L-Fucp(3-O-sulfate)α1-3)D-Manpα1-)_n. Consequently, the glycosidase was deduced to be an endo-α-D-mannosidase that eliminatively cleaves the α-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense

Flavonolignans silybin and isosilybin are major components of silymarin complex isolated from seeds of the milk thistle (Silybum marianum) featuring strong antioxidant and hepatoprotective effects, and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin and isosilybin are mixtures of diastereoisomers: silybin/isosilybin A (1a, 1b) and silybin/isosilybin B (2a, 2b). The metabolism of these compounds is supposed to be strongly linked to Phase II of biotransformation and the respective conjugates are rapidly excreted in bile and urine. The aim of this study was to obtain optically pure sulfated metabolites of both silybins and isosilybins. Aryl-sulfate sulfotransferase (EC 2.8.2.22) from Desulfitobacterium hafniense was found to be a highly effective tool for the regiospecific enzymatic synthesis of silybin A-20-O-sulfate, silybin B-20-O-sulfate, isosilybin A-20-O-sulfate and isosilybin B-20-O-sulfate providing nearly quantitative yields and employing cheap p-nitrophenyl sulfate as sulfate donor. The isolated sulfated products will be used as authentic standards in metabolic studies of both silybins and isosilybins.     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR