Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Modulation of maize roots H+-ATPase by sulfated polysaccharides

Vesicles derived from maize roots retain a membrane bound H⁺-ATPase that is able to pump H⁺ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H⁺-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent K_m for ATP from 0.28 to 0.95 mM and a decrease of the V_max from 14.5 to 7.1 mol P_i/mg · 30 min^–1. At pH 7.0 dextran 8000 promotes an increase in V_max from 6.7 to 11.7 mol Pi/mg · 30 min^–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K⁺ and Na⁺. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K⁺>Na⁺Li⁺).Abbreviations Mops 4-morpholinopropanesulfonic acid - EDTA ethylenediaminetetraacetic acid - ACMA 9-amino-6-chloro-2-methoxyacridine - FCCP carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone     

A new chemical tool for characterization and partial depolymerization of red algal galactans

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine complex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are liberated essentially in the non-reduced form; the reductive hydrolysis products may be determined quantitatively using gas-liquid chromatography (GLC). The method is recommended for preliminary characterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elucidation of structures of reduced sulfated oligosaccharides may be of great value for the structural analysis of complex red algal galactans.     

A COMPARATIVE CYTOCHEMICAL STUDY OF VOLVOCACEAN MATRIX POLYSACCHARIDES1

The colonial matrices of the volvocacean algae were examined for the presence of sulfated and carboxylated polysaccharides. These results were compared to a similar examination of the single-celled Chlamydomonas reinhardtii Dang. The colonial algae examined were Pandorina morum Bory, Eudorina elegans Ehr., Platydorina caudata Kofoid, Pleodorina californica Shaw, Pleodorina illinoisensis Kofoid and Volvox carteri var. nagariensis Iyengar. Alcian blue staining of whole colonies at pH 0.5 and 2.5 showed evidence for the presence of both sulfated and carboxylated polysaccharides in the extracellular matrix. Quantitative measurement of alcian blue bound to solubilized matrices supported the in vivo results. There was a trend toward an increase in sulfated polysaccharides in the more evolutionary advanced forms with the exception of Pleodorina. This trend was readily seen in the sulfate: carboxyl ratios: Pandorina morum—0.4, Eudorina elegans—1.0, Platydorina caudata—2.1 and Volvox carteri—2.2. The acidic nature of the Pleodorina matrix with a sulfate: carboxyl ratio of 0.2 appeared to be more like that of Pandorina rather than that of the more advanced Volvox.     

PRESENCE OF A SULFATED POLYSACCHARIDE IN THE EXTRACELLULAR MATRIX OF PLATYDORINA CAUDATA (VOLVOCALES,CHLOROPHYTA)1

Evidence for the presence of a sulfated polysaccharide component within the extracellular matrix of Platydorina caudata Kofoid is presented. In situ staining with alcian blue and toluidine blue O indicates accumulation of a sulfated polysaccharide in the matrix. The entire matrix was readily solubilized by a hot aqueous extraction and a sulfated proteoglycan complex was isolated. Thin-layer chromatography of hydrolysates and infrared analysis and chemical desulfation of the intact molecule indicate that the polysaccharide component is principally an arabinogalactan with ester-linked sulfate groups. Protease treatment of the extract revealed two distinct bands separable on cellulose acetate electrophoresis. The slower moving component was a sulfated glycoprotein while the faster moving component was a sulfated mucopolysaccharide essentially free of protein. This is the first report of specific chemical analyses and electrophoretic separation of a sulfated polysaccharide within the matrix of a member of the Volvocales. The cytochemistry and electrophoretic patterns of the P. caudata preparation are compared with the same type of extract made from Chlamydomonas reinhardtii Dang. The possible evolutionary significance of the electrophoretic patterns is presented.     

AN AUTORADIOGRAPHIC AND HISTOCHEMICAL LOCALIZATION OF SULFATED POLYSACCHARIDES IN EUCHEUMA NUDUM (RHODOPHYTA)1

The predominant sulfated polysaccharide, ?-carrageenan, was localized in the middle lamella of epidermal, cortical and medullary cells of Eucheumanudum J. Agardh. Autoradiographic studies with ³⁵SO₄= indicated that the label was first incorporated in the inner wall and ultimately deposited in the middle lamella of all cells, and in an outer wall layer of the epidermal cells. There was no evidence for cytoplasmic incorporation of the label. The middle lamella stained with alcian blue, was γ-metachromatic with toluidina blue O and bound diaminobenzidine-osmium tetroxide. This region was also positive with periodic acid-Schiff's (PAS) ragent, possibly demonstrating cellulose and/or a nonsulfated precursor of ?-carrageenan. A proposed model for extracellular sulfation includes production and secretion of a nonsulfated polygalactan and sulfotransferase enzyme(s) by the golgi apparatus and endoplasmic reticulum, respectively. Free sulfate in the wall would be bound to the precursor polysaccharide, with much of the resulting carrageenan migrating to the middle lamella facilitating mutual cell growth.     

The comparison of immune-enhancing activity of sulfated polysaccharidses from Tremella and Condonpsis pilosula

Based on our previous research, four sulfated polysaccharide (sPSs) from Tremella and Condonpsis pilosula, sTPS_tp, sTPS_70c, sCPPS_tp and sCPPS_50c, were prepared and their effects on splenic lymphocytes proliferation in vitro and the immune response of ND vaccine in chicken were compared taking the unmodified polysaccharide (uPS) TPS_tp as control. The results showed that four sPSs could significantly or numerically stimulate splenic lymphocyte proliferation singly or synergistically with LPS in vitro, sTPS_70c and sCPPS_tp demonstrated better effect; promote peripheral lymphocytes proliferation and enhance serum HI antibody titer in chickens vaccinated with ND vaccine, the actions of sPSs were stronger than that of uPS, and sTPS_70c at medium dosage presented the best efficacy. These indicated that sulfation modification could improve the immune-enhancing activity of TPS and CPPS, sTPS_70c possessed the strongest activity and would be expected as a component of new-type immunopotentiator.     

硫酸酯化银耳多糖的制备及抗氧化性活性研究

采用氯磺酸-吡啶法化学合成硫酸酯化银耳多糖,对硫酸酯化反应的吡啶与氯磺酸的体积比进行优化,氯化钡-明胶比浊法测定硫酸酯化银耳多糖的取代度,并对不同取代度的硫酸酯化银耳多糖进行体外抗氧化实验。结果表明:硫酸酯化银耳多糖取代度随吡啶与氯磺酸体积比的提高而增加;当吡啶与氯磺酸体积比为4∶1,反应温度60℃,反应时间3 h时,产物的取代度为1.32,在0.8～2.0 mg/mL浓度范围内对Fenton反应产生的羟基自由基体外清除率相对于银耳多糖具有明显优势。