Ineo Ishizuka

Characterization of gangliosides from fish brain.

Abstract Gangliosides of the brain of fish and warm-blooded animals show the same fundamental structures. They differ from each other in the occurrence of 8-O,N-diacetylneuraminic acid and the quantitative preponderance of tetra- and pentasialogang-liosides in fish brain. About 50% of the fish brain gangliosides consist of the ceramide tetra- and penta-N-acetylneuraminylganglio-N-tetraosides.

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing α1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst-/- mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst-/- mice irrespective of UUO treatment. Compared with wild-type mice, Cst-/- mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin-/- mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Structural analysis of mono- and bis-sulfated glycosphingolipids by negative liquid secondary ion mass spectrometry with high- and low-energy collision-induced dissociation

Several underivatized mono- and bis-sulfated glycosphingolipids having gangliotriaose or gangliotetraose core structure were analyzed by negative liquid secondary ion mass spectrometry (LSIMS) with high- and low-energy collision-induced dissociation (CID). In the normal negative LSIMS spectra, each mono-sulfated glycolipid gave abundant [M - H]- ions and each bis-sulfated glycolipid gave abundant [M + Na - 2H]- ions as well as the hydrogen sulfate anion [OSO3H]-. In high-energy CID spectra of the deprotonated molecule, only ions containing a sulfate ester were clearly observed. When a sulfate was present on the non-reducing terminal saccharide residue, a series of ions corresponding to sulfated mono- to tetra-saccharides, resulting from sequential cleavage of glycosidic bonds, were observed. If the sulfate was attached to an internal hexose of the sugar chain, the product ions corresponding to the non-sulfated, non-reducing terminal residue were absent. In contrast, the low-energy CID resulted in extremely simple spectra that contained only one or two major product ions characteristic of each sulfated glycolipid. These results provided clear information on the overall sugar and ceramide compositions, and allowed saccharide structures differing in location and number of sulfate esters to be distinguished.

Adaptive changes in sulfoglycolipids of kidney cell lines by culture in anisosmotic media

(1) The effects of osmolarity environments on renal glycolipid composition were examined using established renal cell lines. The profile of glycosphingolipids of Madin-Darby canine kidney cells (MDCK) in culture with anisosmotic media showed that a hyposomotic medium reduced the concentration of GalCer I3-sulfate and LacCer II3-sulfate. (2) The concentrations of sulfoglycolipids were increased by maintaining the culture in a hyperosmotic media prepared by the addition of various sodium salts to the control isosmotic medium, while the contents of most of the neutral glycolipids were reduced. The hyperosomotic medium supplemented with nonelectrolytes, mannitol, sucrose or urea, also increased the concentration of sulfoglycolipids. (3) Both sulfoglycolipids were increased linearly with gradual increases of sodium chloride in the medium. Hyperosmolarity produced by the addition of a nonelectrolyte, mannitol, also increased the levels of sulfoglycolipids. In both series of media, the most prominent accumulation was observed in LacCer II3-sulfate. (4) The incorporation of radioactive sulfate into sulfoglycolipids was elevated in cells adapted to high NaCl or mannitol. The increase of the label was observed not only in MDCK but also in three other established cell lines of renal tubular origin, JTC-12, LLC-PK1 and MDBK. (5) It was established, using the culture system of homogeneous cell lines, that the mechanism of increasing the amount of sulfoglycolipids is independent of the integral regulatory mechanism of animals and resides in the renal epithelial cell itself. These results suggest that by culture in hyperosmotic media, the elevated level of intracellular cations stimulated the activity of GalCer and LacCer sulfotransferase, inducing the increased expression of sulfoglycolipids.

Isolation and partial characterization of a novel sulfoglycosphingolipid and ganglioside GM4 from rat kidney.

Summary A novel sulfoglycolipid was isolated from the lipid extract of rat kidney by a procedure involving mild alkaline methanolysis and column chromatographies on DEAE-Sephadex and silicic acid. The component carbohydrates were galactose, glucose and galactosamine in equimolar amounts. Infrared spectroscopy, solvolysis and permethylation studies suggested that the glycolipid was GalNAc(1→4)Gal(1→4)GlcCer sulfated at the C3 hydroxyl of galactose. Ganglioside GM4 was separated from the above sulfoglycolipid on the column of DEAE-Sephadex A-25. The yields of sulfoglycolipid and GM4 were 18.7 and 12.9 nmol/g wet tissue, respectively.

Sulfatide is expressed in both erythrocytes and platelets of bovine origin

A novel sulfated glycosphingolipid containing a sulfated galactosyl residue was isolated from bovine erythrocyte ghosts, and purified to homogeneity by column chromatography on DEAE-Sephadex and silica beads. Structural characterization included compositional analyses, permethylation studies, proton nuclear magnetic resonance (NMR) spectroscopy, negative secondary ion mass spectrometry (SIMS), solvolysis and immunostaining on thin-layer chromatogram. As a result, the structure of this glycolipid is proposed as HSO3-Gal beta 1-1 Cer. The ceramide portion contained d18:1, d18:0 and t18:0, and the predominant fatty acid consisted of palmitate and palmitate with a hydroxy group, as deduced by both compositional analysis and negative SIMS mass spectrometry. The component of this glycosphingolipid probably originates from erythrocytes and platelets as indicated by the results of flow cytometry analysis using Sulph I monoclonal antibody. The yield of galactosyl sulfatide was about 0.37 mg/kg wet bovine erythrocyte membranes, about three times that of human kidney. Our results strongly suggest that galactosylceramide sulfate on erythroid cells may play an important biological role in cell to cell interaction and recognition.

Cationic Glycosphingolipids in Neuronal Tissues and Their Possible Biological Significance

During the course of studies on natural occurrence of sphingosine base in brain, cationic glycosphingolipids bound to carboxymethyl-Sephadex and eluted with triethylamine in organic solvents were isolated and characterized. Four classes of compounds were identified: (i) plasmalopsychosine-A and -B; (ii) glyceroplasmalopsychosine; (iii) glycosphingolipids having de-N-acetyl-hexosamine, e.g., de-N-acetyl-Lc3Cer; (iv) glycosylsphingosine, i.e., lysoglycosphingolipid. Only two kinds, galactosylsphingosine (psychosine) and lactosylsphingosine, were found to occur naturally in brain. All these compounds were isolated from extract of brain white matter. Their occurrence, quantity, and distribution pattern differ from one species to another. Their quantity is much lower than that of regular acidic and neutral glycosphingolipids. They may interact with regular glycosphingolipids in glycosphingolipid-enriched microdomains to elicit signal transduction, to modify cellular phenotype, although studies along this line are highly limited at this time.

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or womens sera containing sperm immobilizing antibodies, but not by normal womens sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in womens sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

Synthesis and absolute configuration of a novel aminoglycoglycerolipid, species-specific major immunodeterminant of Mycoplasma fermentans

Abstract In order to determine the absolute configuration of a novel aminoglycoglycerolipid isolated from Mycoplasma fermentans , the possible two diastereomers were stereoselectively synthesized by using (S)- and (R) -glycidols as the key building block. Their 1 H 1 H-cosy spectra compared with those of the natural product allowed us to determine the absolute configuration of the glycolipid.