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M Kiso

Publications and source records attributed to M Kiso.

At least 55 records · Page 3Linked to original sources
PubMed1998

Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain.

A membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100,000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4,851.3 micromol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.

Animals↗
PubMed1998

Relationship of structure and biological activity of monosaccharide lipid A analogues to induction of nitric oxide production by murine macrophage RAW264.7 cells.

Lipid A is the active center of bacterial lipopolysaccharide (LPS), which exhibits diverse biological activities via the production of various mediators. We investigated the production of nitric oxide (NO), one of the mediators, by a murine macrophage cell line, RAW264. 7, upon stimulation with a series of monosaccharide lipid A analogues to elucidate the relationship of structure and activity in NO production. The production of other representative mediators, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), was also investigated to compare the structural requirements for the production of these cytokines with those for the production of NO. Structure-activity relationships in NO production correlated well with those in the production of TNF-alpha and IL-6. Among the lipid A analogues possessing different numbers of acyl groups on a 4-O-phosphono-D-glucosamine backbone, compounds like GLA-60 that possess three tetradecanoyl (C14) groups exhibited stronger activities in the production of the mediators than compounds possessing four or two C14 groups. Time course study of the production of these mediators showed that production of NO started and peaked later than those of TNF-alpha and IL-6. Neither neutralization of TNF-alpha activity by antibody nor suppression of TNF-alpha production by pentoxifylline showed a significant suppressive effect on production of NO and IL-6 upon stimulation with LPS or lipid A analogues. Neutralization of IL-6 activity by antibody showed no significant suppressive effect on production of NO and TNF-alpha. A monosaccharide lipid A analogue (GLA-58) which exhibited no detectable agonistic activity showed a suppressive effect on the production of all three mediators upon stimulation with LPS or lipid A analogues. These results indicate that signals for NO production by LPS agonists in murine macrophages are transduced in good correlation with those for production of TNF-alpha and IL-6, although they are not transduced via production of those cytokines.

Acylation↗
PubMed1998

Synthetic studies on selectin ligands/inhibitors. Synthesis and biological evaluation of sulfated and phosphorylated beta-D-galacto- and lactopyranosides containing fatty-alkyl residues of different carbon chain lengths.

To investigate the biological selectin-ligand interactions, fourteen sulfated and eight phosphorylated beta-D-galacto- and lactopyranosides containing branched fatty-alkyl residues in place of the ceramide have been synthesized. Regioselective sulfation of the parent glycolipids through the dibutylstannylene acetal with a certain amount of sulfur trioxide-trimethylamine complex produced the target sulfated glycolipids, while stepwise phosphorylation by treatment of the properly protected diol with dibenzyloxy(diisopropylamino)phosphine gave the phosphorylated glycolipids. The synthetic glycolipids showed an interesting mode of inhibition of the binding of HL-60 cells to immobilized P-, L- and E-selectins during in vitro experiments. In addition, using computer modeling techniques, we examined the molecular basis for the ligand-selectin complex formation. These glycolipids may be useful as therapeutic agents against selectin-dependent inflammation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

A convenient and efficient synthesis of sialyl Lewis X.

A convenient synthesis of the sialyl Lewis X (sLe(x)) tetrasaccharide, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc (8), as a carbohydrate ligand for selectins is described. The key step is the reaction between NeuAc alpha 2-3GalSMe (5) as a glycosyl donor and the suitably protected Fuc alpha 1-3GlcNAc derivative (4) as the glycosyl acceptor by using dimethyl(methylthio)sulfonium triflate (DMTST) as the promoter.

Carbohydrate Sequence↗
PubMed1998

First total synthesis of ganglioside GT1a alpha.

A first total synthesis of ganglioside GT1a alpha (IV3III6II3Neu5AcGg4Cer) is described. The suitably protected sialyl alpha-(2-->6) ganglioside was glycosylated with the phenylthio glycoside of sialic acid in the presence of N-iodosuccinimide-(NIS)-trimethylsily trifluoromethanesulfonate (TMSOTf), followed by further glycosylation with the methyl thioglycoside promoted by dimethyl(methylthio)sulfonium triflate (DMTST), to give the heptasaccharide. The oligosaccharide obtained was converted into the title ganglioside by the introduction of ceramide and then complete deprotection.

Antigens, Surface↗
PubMed1997

First total synthesis of ganglioside GT1a alpha.

A first total synthesis of ganglioside GT1a alpha (IV3III6II3)Neu5AcGg4Cer) is described. The suitably protected sialyl alpha-(2-->6) ganglioside was glycosylated with the phenylthioglycoside of sialic acid in the presence of N-iodosuccinimide (NIS)-trimethylsilyl trifluoromethanesulfonate (TMSOTf), followed by further glycosylation with the methyl thioglycoside promoted by dimethyl(methylthio)sulfonium triflate (DMTST), to give the heptasaccharide. The oligosaccharide obtained was converted into the title ganglioside by the introduction of ceramide and then complete deprotection.

Antigens, Surface↗
PubMed1997

Sialyl-Lewis(x) sequence 6-O-sulfated at N-acetylglucosamine rather than at galactose is the preferred ligand for L-selectin and de-N-acetylation of the sialic acid enhances the binding strength.

Oligosaccharide sequences based on sialyl-Lewis(x) with 6-O-sulfation at galactose (6'-sulfo) or at N-acetylglucosamine (6-sulfo) and expressed on high endothelial venules are considered likely endogenous ligands for the leukocyte adhesion molecule, L-selectin. In the course of high performance TLC of three hexaglycosylceramides 6'-sulfo sialyl Lewis(x), 6-sulfo sialyl Lewis(x), and 6',6-bis-sulfo sialyl Lewis(x), synthesized chemically for selectin recognition studies, two minor byproducts were detected and isolated from each parent compound. By liquid secondary ion mass spectrometry these were identified as isomers containing a de-N-acetylated sialic acid or having a modified carboxyl group. Binding experiments with the parent compounds and the non-sulfated sialyl Lewis(x) glycolipid show that 6-sulfation potentiates, whereas 6'-sulfation virtually abolishes L-selectin binding. Thus the hierarchy of binding strengths were 6-sulfo sialyl > sialyl = 6',6-bis-sulfo sialyl >> 6'-sulfo sialyl Lewis(x). Whereas modification of the sialic acid carboxyl group markedly impaired L-selectin binding, de-N-acetylation resulted in enhanced binding. The natural occurrence on high endothelial venules of this 'super-active' de-N-acetylated form of 6-sulfo sialyl Lewis(x), and related structures, now deserves investigation.

Acetylation↗
PubMed1997

Binding specificities of the sialoadhesin family of I-type lectins. Sialic acid linkage and substructure requirements for binding of myelin-associated glycoprotein, Schwann cell myelin protein, and sialoadhesin.

The carbohydrate binding specificities of three sialoadhesins, a subgroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1balpha >> GD1a = GT1b >> GM3 = GM4 >> GM1, GD1b, GD3, GQ1b (nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal alpha2,3- or alpha2,8-linked sialic acids with the following rank-order potency of binding: GQ1balpha > GD1a = GD1b = GT1b = GM3 = GM4 > GD3 = GQ1b >> GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid acetamido group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.

Animals↗
PubMed1997

A synthetic approach to the c-series gangliosides containing sialyl-alpha(2-->8)sialyl-alpha(2-->8)sialic acid: synthesis of ganglioside GT4, alpha(2-->6) GT4 and GT3.

Trimeric sialic acid [Neu5Ac alpha(2-->8)Neu5Ac alpha(2-->8)Neu5Ac, 1] residue-containing gangliosides, GT4, alpha(2-->6)GT4 and GT3, have been synthesized for the first time. Methyl [phenyl] 5-acetamido-8-O-[5-acetamido-8-O-(5-acetamido-4, 7, 8, 9-tetra-O-acetyl-3, 5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylono-1", 9'-lactone)-4,7-di-O-acetyl-3,5-dideoxy-D- glycero-alpha-D-galacto-2-nonulopyranosylono-1',9-lactone]-4,7-di- O-acetyl -3,5-dideoxy-2-thio-D- glycero-D-galacto-2-nonulopyranosid]onate (3) was prepared from 1, via lactonization, methyl esterification of the carboxyl group at the reducting end, O-acetylation and conversion of the anomeric acetoxy group into a phenylthio group. Iodonium-promoted glycosylation of 3 with 2-(trimethylsilyl)ethyl 2,6-di-O-benzyl-beta-D-galactopyranoside (5), 2-(trimethylsilyl)ethyl 3-O-benzyl-beta-D-galactopyranoside (6), 2-(trimethylsilyl)ethyl 2-O-benzoyl-3-O-benzyl-beta-D-galactopyranoside (9), and 2-(trimethylsilyl)ethyl 2, 3-di-O-benzyl-beta-D-galactopyranoside (11) gave the corresponding tetrasaccharides (13-15, 17) having the (Neu5Ac)3-Gal structure. The peracylated oligosaccharides 18 and 24 derived from 13 and 17, and the previously reported lactose derivative 29 were converted into the alpha-trichloroacetimidates 20, 26 and 31, and coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (21) to afford the corresponding beta-glycosides 22, 27 and 32. These protected azidosphingosine derivatives were each transformed into the target gangliosides GT4, alpha(2-->6)GT4 and GT3 via selective reduction of the azido group, subsequent coupling with octadecanoic acid, O-deacylation and saponification of the methyl ester and lactone groups.

Carbohydrate Sequence↗
PubMed1997

Valency dependent patterns of binding of human L-selectin toward sialyl and sulfated oligosaccharides of Le(a) and Le(x) types: relevance to anti-adhesion therapeutics.

The human L-selectin is known to bind to immobilized 3'-sialyl-Le(x) and -Le(a) oligosaccharides both under static and physiological flow conditions. Here the reactivities toward 3'-sulfated and 3'-sialyl-Le(a) and -Le(x) pentasaccharides are compared by in-vitro binding and inhibition assays using preparations of human L-selectin-IgG-Fc chimera in which the selectin is predominantly in di- and tetrameric form (paucivalent) or in the form of a complex with anti-IgG (multivalent). Affinity for the sulfated ligands is marginally greater than for the sialyl ligands, as judged by concentrations required to give 50% inhibition of the multivalent selectin binding to the immobilized sulfated and sialyl ligands. There is a striking difference, however, in the avidities of binding of the two L-selectin forms toward the sulfated and sialyl ligands when these are immobilized in the clustered state: the paucivalent selectin gives detectable binding only to the sulfated ligands when these are immobilized as neoglycolipids on plastic microwells (up to 100 pmol immobilized per well) whereas the multivalent L-selectin binds well to both classes of ligand. Moreover, binding of the paucivalent selectin form is effectively inhibited only by the sulfated ligand, although binding of the multivalent selectin is inhibitable by both the sulfated and sialyl ligands. Such striking valency-dependent differences in ligand binding avidity and inhibitability may be manifest in vivo with the membrane-bound L-selectin, as marked variations occur in its density of expression on leukocytes. Thus, for the purpose of selecting inhibitors for development of therapeutic anti-inflammatory compounds, experimental designs based on the paucivalent L-selectin would more clearly single out compounds with broad spectrum anti-adhesive activities toward the both the high- and low-avidity interactions of the cell adhesion protein.

Animals↗
PubMed1997

Selectin-ligand interactions revealed by molecular dynamics simulation in solution.

Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.

Carbohydrate Sequence↗
PubMed1997

Sulfated sialyl Lewis X, the putative L-selectin ligand, detected on endothelial cells of high endothelial venules by a distinct set of anti-sialyl Lewis X antibodies.

Endothelial cells of high endothelial venules (HEV) in human peripheral lymph nodes expressed a distinct type of sialyl Lewis X antigen, which was detected preferentially with a set of anti-sialyl Lewis X antibodies, 2F3, 2H5 and HECA-452 in immunohistochemistry, while another set of anti-sialyl Lewis X antibodies, FH-6 and CSLEX-1, failed to detect it. The adhesion of cells expressing L-selectin to HEV was inhibited by members of the former set of antibodies in Stamper-Woodruff assays performed on frozen sections of human peripheral lymph nodes. Transfection of a cultured endothelial cell line with a human alpha1-->3 fucosyltransferase, Fuc-T VII, resulted in the expression of a distinct type of sialyl Lewis X antigen having the reactivity similar to that of HEV; i.e., the antigen appearing on the transfectant clone was detectable only with the set of 2F3, 2H5 and HECA-452, but not with the set of FH-6 and CSLEX-1. Treatment of transfectant cells with sodium chlorate, a metabolic inhibitor of sulfation, resulted in reactivity to the members of the latter set of antibodies, suggesting that sulfation of sialyl Lewis X moiety was the cause of the discrepancy in the reactivity of the anti-sialyl Lewis X antibodies. When tested against various authentic sulfated sialyl Lewis X determinants, 6-sulfo sialyl Lewis X and 6,6'-bis-sulfo sialyl Lewis X were found to be reactive to the antibodies, 2F3, 2H5 and HECA-452, but not with antibodies FH-6 and CSLEX-1, suggesting that the distinct type of sialyl Lewis X determinant on the HEV endothelial cells and Fuc-T VII-transfected endothelial cell clone are mainly 6-sulfo and/or 6,6'-bis-sulfo sialyl Lewis X determinants.

Animals↗
PubMed1997

Sialic acid specificity of myelin-associated glycoprotein binding.

Myelin-associated glycoprotein (MAG), a nervous system cell adhesion molecule, is an I-type lectin that binds to sialylated glycoconjugates, including gangliosides bearing characteristic structural determinants (Yang, L. J.-S., Zeller, C. B., Shaper, N. L., Kiso, M., Hasegawa, A., Shapiro, R. E., and Schnaar, R. L. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 814-818). Two cell adhesion systems, COS-1 monkey kidney fibroblasts transiently transfected to express MAG and Chinese hamster ovary (CHO) cells stably transfected to express MAG, were used to probe the structural specificity of MAG-ganglioside binding. Both cell types bound to the same gangliosides: GQ1b alpha (IV3NeuAc,III6NeuAc,II3(NeuAc)2Gg4Cer) > GT1b = GD1a > GM3 > GM1, GD1b, and GQ1b (the latter do not support adhesion). Binding was enhanced by pretreatment of MAG-expressing cells with neuraminidase. MAG-expressing Chinese hamster ovary cells bound directly to gangliosides resolved on thin layer chromatograms, allowing detection of MAG binding species in a mixture. The simplest ganglioside ligand for MAG was GM3 bearing N-acetylneuraminic acid, whereas GM3 bearing N-glycolylneuraminic acid did not support adhesion. Chemical modifications of N-acetylneuraminic acid residues (on GD1a) abrogated MAG binding. Mild periodate oxidation of sialic acids to their corresponding seven-carbon (or eight-carbon) sialic acid aldehydes abolished MAG binding, as did further conversion to the corresponding primary alcohols. Eliminating the anionic charge by ethyl esterification, amidation, or reduction also abolished MAG-mediated cell adhesion. These data demonstrate that MAG-ganglioside binding is highly specific and defines key carbohydrate structural determinants for MAG-mediated cell adhesion to gangliosides.

Animals↗
PubMed1996

Sulphatide binds to human and animal influenza A viruses, and inhibits the viral infection.

We found, by using a virus overlay assay, that influenza A virus isolates bind to sulphatide (HSO3-Gal beta 1-->1'Cer), which has no sialic acid residue, and that the infection of Madin-Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatide. A/Memphis/1/71 (H3N2) causes obvious haemagglutination and low-pH haemolysis of asialoerythrocytes reconstituted with sulphatide. All influenza A virus isolates from the species of animals so far tested bound to sulphatide. The sulphatide-binding specificity of the isolates was different from the viral sialyl-linkage specificity. Influenza A virus isolates also bound to galactosyl ceramide (GalCer; Gal beta 1-->1'Cer), as well as sulphatide, in the virus overlay assays. In contrast, the influenza virus did not bind to N-deacyl, a derivative of sulphatide, glucosyl ceramide or the other neutral glycolipids tested. These results indicate that the linkage of galactose, or sulphated galactose, to ceramide is important for viral binding.

Animals↗
PubMed1996

High affinity antibodies against Lex and sialyl Lex from a phage display library.

Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

Amino Acid Sequence↗
PubMed1996

Studies on selectin blockers. 2. Novel selectin blocker as potential therapeutics for inflammatory disorders.

As a part of our studies of selectin blockers, we prepared 1-(2-tetradecylhexadecyl)-3'-O-sulfo Le(X) 1 and 1-(2-tetradecylhexadecyl) sLe(X) 2 and examined their inhibitory activities against natural ligand (sLe(X)) binding to E-, P-, and L-selectins. Compounds 1 and 2 were 2 times more potent than the sLe(X) tetrasaccharide toward E-selectin binding and up to 4 times more potent than sLe(X) toward P- and L-selectin binding. Interestingly, compound 1 provided dose-dependent protective effects against an immunoglobulin E-mediated skin reaction in mouse ears. This protective effect was associated with diminished tissue accumulation of neutrophils in the ear (as assessed by myeloperoxidase). These findings indicate that the modification of sLe(X) or 3'-O-sulfo Le(X) with a "branched anchor", a 2-tetradecylhexadecyl group, is useful in the design of a more potent selectin blocker, which has broad inhibitory activities toward all selectins.

Animals↗