Mutsumi Sugita

Ceramidase and ceramide synthesis in human kidney and cerebellum: Description of a new alkaline ceramidase

It has been shown that tissues of patients with Farbers disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.

Ceramidase Deficiency in Farber's Disease (Lipogranulomatosis)

Ceramidase activity could not be demonstrated in the kidney and cerebellum from a deceased patient with Farbers disease, whereas the activities of six control acid hydrolase enzymes appeared normal. This enzyme defect presumably accounts for the accumulation that has been described in two patients and may represent the biochemical basis of this disorder.

Sphingolipids in lower animals.

ABBREVIATIONS ]. INTRODUCTION II. PHOSPHOSPHINGOLIPIDS A. Phosphonosphingolipids I. Species distribution B. Ceramide phosphoethanolamine IlL GLYCOSPHINGOLIPIDS A. Nomenclature for GSL of arthropods and molluscs B. Preparation and purification C. Glycosphingnlipids in molluscs 1. Bivalves 2. Sea snails 3. Sea bare D. Glycosphingolipids in arthropods 1. Insects 2. Crustacea E. Glycosphingnlipids in parasites F. Glycosphingnlipids in annelids G. Glycosphingolipids in echinoderms IV. IMMUNOLOGICAL STUDIES A. Antibodies against mannose-containing lipids (mannolipids) from freshwater bivalves and insects 1. Anti-Ml2Cer antibody 2. Anti-Ml4Cer antibody 3. Anti-V3~,GalNAc3Me,V4flGIcA4Me-MIsCer antibody 4. Anti-Ar3Cer antibody B. Antibodies against PnGSL from sea hare, Aplysia kurodui I. Anti-SGL-II antibody 2. Anti-FGL-Ilb antibody C. Antibody against ganglioside from sea urchin Eggs 93 ACKNOWLEDGEMENTS REFESmNCm 25 26 26 26 26 26 28 29 29 30 30 32 32 33 35 37 37 38 40 40

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.

Synthetic studies on glycosphingolipids from the Protostomia phyla: Syntheses of arthro-series glycosphingolipids

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.

A novel fucosylated glycosphingolipid from the millipede, Parafontaria laminata armigera.

A novel fucosylated glycosphingolipid (GL-3a) was isolated and purified from whole tissues of the millipede, Parafontaria laminata armigera. Its chemical structure was characterized as Man beta 1-4(Fuc alpha 1-3)Glc beta 1-ceramide (I3 alpha Fuc,MlOse2Cer) by gas-liquid chromatography, permethylation study, partial acid hydrolysis, exoglycosidase degradation, TLC/enzyme-immunostaining, negative fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This compound was unique in containing a fucose branch linked to the glucose residue of the disaccharide, mannosylglucose. The ceramide moiety was mainly composed of d17:1 (64.3%) and d18:1 (20.0%) sphingoids, and 22:0 (41.8%), 23:0 (16.4%) and 24:0 (15.8%) fatty acids.

Structural characterization of a novel glycoinositolphospholipid from the parasitic nematode, Ascaris suum

A novel glycosphingolipid containing inositol phosphate as an acidic group has been demonstrated in whole tissues of the porcine roundworm, Ascaris suum. The thin layer chromatographic pattern of the total acidic glycolipid revealed the presence of several components, of which a major component (named AGL) with positive reactions toward both orcinol-sulfuric acid (sugar) and molybdate (phosphate) spray reagents was isolated and purified by the use of successive column chromatography on DEAE-Sephadex and silicic acid (latrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, methylation analysis, periodate oxidation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, the structure of AGL was deduced to be Gal alpha 1-2Ins(1-->)-P-Cer. Aliphatic constituents were lignoceric acid and its 2-hydroxy homologue as the principal fatty acids, and octadecasphinganine and branched heptadecasphinganine as the major sphingoids.

Fatty acid composition of free ceramides of kidney and cerebellum from a patient with Farber's disease.

The fatty acid composition of ceramides has been determined in kidney and cerebellum of a patient with Farbers disease, which is characterized by ceramidase deficiency. Farber cerebellum and kidney contained a five- and ten-fold excess, respectively, of free ceramides. The nonhydroxy fatty acid patterns of the ceramides from Farber kidney and cerebellum showed considerable similarities to those from control tissues, whereas large amounts of ceramides containing hydroxy fatty acids are found in Farbers disease tissues.

Structural elucidation of two novel amphoteric glycosphingolipids from the earthworm, Pheretima hilgendorfi.

The novel amphoteric glycosphingolipids containing a choline phosphate were purified from whole tissues of the earthworm, Pheretima hilgendorfi. Their chemical structures were completely characterized as cholinephosphoryl-->6(Man alpha 1-4)Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylmannosylneogalabiaosylceramide, named PGL3a) and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylneogalatriaosylceramide, named PGL3b) by compositional sugar, fatty acid and sphingoid analyses, hydrogen fluoride degradation, partial acid hydrolysis, methylation analysis, exoglycosidase degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide moieties of these lipids consisted of 22:0, 23:0 and 24:0 acids as major fatty acids, and branched octadeca- and nonadeca-4-sphingenines and octadeca-4-sphingenine as main sphingoids. Since the oligosaccharides and the ceramide moieties of PGL3a and PGL3b were identical with those of neutral glycosphingolipids found in this organism, the biosynthesis of the amphoteric glycolipids may occur by the addition of a choline phosphate residue to the corresponding neutral glycolipids, Man alpha 1-4Gal beta 1-6Gal beta 1-1Cer or Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer.

Synthetic studies on novel fucosylated glycosphingolipids from the millipede, Parafontaria laminata armigera

Abstract A novel glycosphingolipid, β- d -Man p -(1→4)-[(α- l -Fuc p -(1→3)]-β- d -Glc p -(1→1)-Cer, from the millipede, Parafontaria laminata armigera , was synthesized. A key reaction of this synthetic procedure is the formation of a spiro-orthoester and its reduction for β-selective mannosylation.