Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates

We prepared a series of oligosaccharides from porcine intestinalheparin after extensive digestion with a mixture of Flavobacteriumheparinase as well as heparitinases I and II. Previously, wereported the structures of the two glycoserines derived fromthe carbohydrate-protein linkage region [Sugahara et al., J.Biol. Chem., 267, 1528–1533 (1992)] and three tetrasaccharidesderived from the antithrombin III-binding site [Yamada et al.,J. Biol. Chem., 268, 4780–4787 (1993)]. In this study,we determined the structures of 10 other tetrasaccharides anda trisaccharide by enzymatic digestion, fast atom bombardmentmass spectrometry and 500-MHz ¹H NMR spectroscopy. These tetrasaccharidesshare the common disulphated structure,     

Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria

Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination, and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols, which are nonsulfated, sulfated, and/or phosphorylated, were obtained from the carbohydrate-protein linkage region. Six compounds, containing 0 or 1 sulfate and/or phosphate residue, represent approximately 40% of the isolated linkage hexasaccharide alditols. They were analyzed by chondroitinase ACII or alkaline phosphatase digestion in conjunction with high performance liquid chromatography, and by 500 MHz one- and two-dimensional 1H NMR spectroscopy. All six compounds have the conventional structure in common. Delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol One compound has no sulfate nor phosphate. Two of the monosulfated compounds have a O-sulfate on C-6 or on C-4 of the GalNAc residue. The third monosulfated compound has a novel O-sulfate on C-6 of the Gal residue attached to xylitol. The two phosphorylated compounds have O-phosphate on C-2 of Xyl-ol, and one of them has in addition sulfate on C-6 of GalNAc.     

Vascular permeability-increasing activity possessed byTreponema phagedenis (Reiter strain)

Treponema phagedenis possessed the ability to induce an increase in vascular permeability (blueing) in guinea pig skin, which was exerted not only by cell-free preparations but also directly by intact cells. Cell-free preparations induced maximum blueing 0 h after sample injection, while whole cells did so after 1 h. Log dose-response curves for cell-free preparations were linear within the range of blue spots with diameters of 10–24 mm, with slopes ( ) of 8.5–8.7. The vascular permeability-increasing activity was not ascribable to hyaluronidase and probably to -galactosidase; this organism did not produce any detectable hyaluronidase activity.     

Inhibitory effect of Japanese black vinegar on IgE-mediated degranulation of RBL-2H3 cells and a murine model of Japanese cedar pollinosis

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca²⁺ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Effect of organic solvents on enantioselectivity of protease catalysis

The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30 degrees C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN' always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. (c) 1997 John Wiley & Sons, Inc.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Simultaneous determination of urinary cystathionine, lanthionine, and their cyclic compounds using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization

A measurement system for cystathionine (Cysta) lanthionine (LT), and (AEC), and reduced products of their ketimines, perhydro-1,4-thiazapine-3,5-dicarboxylic acid (PHTZDC), 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA) and 1,4-thiomorpholine-3-carboxylic acid (TMA) in the urine samples of a patient with cystathioninuria and normal human subjects has been developed, using column liquid chromatography-mass spectrometry. The recoveries were about 90–105% for Cysta, LT and AEC, and about 77–87% for PHTZDC, TMDA and TMA after ion-exchange treatment. The concentrations of Cysta and PHTZDC in the urine of a patient with cystathioninuria were much higher compared with those in the urine of normal human subjects. The concentrations of AEC and TMDA were almost the same. LT and TMA could not be detected in the urine samples by this method. This method proved useful for the determination of sulfur-containing amino acids and their cyclic compounds in biological samples.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively