Isolation and characterization of a novel 2-aminoethylphosphonyl-glycosphingolipid from the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I' containing 1 mol of 2-aminoethylphosphonate residue was isolated from the skin of Aplysia kurodai using two silicic acid chromatography systems. Data obtained on methanolysis, permethylation, mild acid hydrolysis, and hydrogen fluoride treatment combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry showed that this glycolipid was 3-O-MeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 1----1Ceramide. Palmitic acid, octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine are its major aliphatic components. The new glycolipid has essentially the same structure as another major phosphonoglycosphingolipid in the skin of Aplysia, SGL-II, that contains 2 mol of 2-aminoethylphosphonate residue, suggesting a metabolic relationship between the two.     

The occurrence of glycosphingolipids containing mannose in the sea-water bivalve, Meretrix lusoria (Hamaguri)

Total neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal(beta 1----4)Glc-Cer and Man(beta 1----4)Glc-Cer (MlOse2Cer) for CDS, Man(alpha 1----3)Man(beta 1----4)Glc-Cer (MlOse3Cer) and Gal(alpha 1----3)Man(beta 1----4)Glc-Cer (II3 alpha Gal-MlOse2Cer) for CTS. To our knowledge II3 alpha Gal-MlOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4,8-sphingadienine. More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai

A phosphonoglycosphingolipid, designated as FGL-IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by permethylation studies to be attached as a ketal to O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb. By sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3(Fuc alpha 1----2) (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide. Its major aliphatic components were palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. This is the first report of the occurrence of pyruvylated galactose as a constituent of animal sphingolipid.     

Sialylpentaosylceramide detected with anti-GM2 monoclonal antibody. Structural characterization and complementary expression with GM2 in gastric cancer and normal gastric mucosa

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.     

Structure of a novel diphosphonoglycosphingolipid isolated from the skin of Aplysia kurodai

A new phosphonoglycosphingolipid containing two 2-aminoethylphosphonate residues was isolated from the skin of Aplysia kurodai, a marine gastropod, using two systems of silicic acid chromatography. By methanolysis, permethylation, mild acid hydrolysis and hydrogen fluoride treatment combined with thin layer chromatography and gas chromatography-mass spectrometry, the new phosphonoglycosphingolipid was shown to be 3-O-MeGal (1----3) GalNAc (1----3) [6'-O-(2-aminoethylphosphonyl) Gal (1----2)] [2-aminoethylphosphonyl (----6)] Gal (1----4) Glc (1----1) ceramide. Most of the fatty acid (90 per cent) was palmitic acid. Octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine were the major sphingosine bases of the new glycolipid.     

Immunogenic properties of mannose-containing ceramide tetrasaccharide from fresh-water bivalve, Hyriopsis schlegelii

Antiserum against GlcNAc beta 1----2Man alpha 1----3Man beta 1----4Glc beta 1----1Cer (MlOse4Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve Hyriopsis schlegelii, was elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin with Freund's adjuvant. The specificity of the affinity-purified antibody obtained from the serum was based on two forms of enzyme-immunodetection of its binding to structurally related glycolipids, either adsorbed to microtiter plates or chromatographed on thin-layer plates. The purified antibody exhibited a significant cross-reactivity with GlcNAc beta 1----2Man alpha 1----3(Xyl beta 1----2)Man beta 1----4Glc beta 1----1Cer, (MIXOse5Cer) containing a core structure closely related to MlOse4Cer, but almost unrelated to other glycolipids. Distribution of MlOse4Cer and MlXOse5Cer in various bivalve and snail glycolipid extracts were screened in thin-layer immunobinding assays by using this purified specific antibody. The presence of the glycolipid antigens was limited to certain taxonomic orders of shellfish species.     

Structure of a triphosphonopentaosylceramide containing 4-O-methyl-N-acetylglucosamine from the skin of the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I containing 3 mol of 2-aminoethylphosphonate residues was isolated from the skin of a sea gastropod, Aplysia kurodai. The saccharide moiety of the glycolipid was characterized as 4-O-methyl-GlcNAc alpha 1----4GalNAc alpha-1----3 [6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6)Gal beta 1----4(2-aminoethylphosphonyl----6) Glc beta 1----1-ceramide. The major aliphatic components of the ceramide portion were palmitic acid, stearic acid, octadeca-4-sphingenine, and anteisononadeca-4-sphingenine. This glycolipid is unique in containing 4-O-methyl-N-acetylglucosamine and 3 mol of 2-aminoethylphosphonate residues, one of which is attached to C-6 of glucose.     

Accumulation of a globo-series glycolipid having Gal alpha 1-3Gal in PC12h pheochromocytoma cells

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.     

Polar glycosphingolipids in insect: chemical structures of glycosphingolipid series containing 2'-aminoethylphosphoryl-(----6)-N-acetylglucosamine as a polar group from larvae of the green-bottle fly, Lucilia caesar.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

An antigen present in rat adenocarcinoma and normal colon non-epithelial stroma is a novel Forssman-like glycolipid based on isoglobotetraosylceramide

A glycolipid with blood group A activity detected in the non-epithelial stroma of normal rat colon but not in epithelial cells (Hansson, G.C., Karlsson, K.-A., and Thurin, J. (1984) Biochim. Biophys. Acta 792, 281-292), was purified to homogeneity from normal rat colon and rat colon adenocarcinoma. Mass spectrometry and 1H-NMR spectroscopy of the intact permethylated derivative and gas chromatography after degradation revealed the structure GalNAc alpha 1----3GAINAc beta 1----3Gal alpha 1----3Gal beta 1----4Glc beta 1----1Cer, with the predominant ceramide containing sphingosine and non-hydroxylated 24:0 fatty acid. This identifies this glycolipid as a novel Forssman-like glycolipid, which is a tumor-associated antigen by definition, since it is not present in the normal rat large intestinal epithelium cells but in rat adenocarcinoma derived from these cells.     

Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina

NeuGc alpha 2-6Glc beta 1-1Cer (M5 ganglioside) and HSO3-8NeuGc alpha 2-6Glc beta 1-1Cer (T1 ganglioside) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a downfield-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.     

Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3

Rainbow trout sperm contained almost exclusively monoanionic ganglioside fraction as a major acidic glycosphingolipid. Two monoacidic gangliosides were isolated and purified in this study and designated as sperm ganglioside 1 and 2 (sg-1 and sg-2). The two gangliosides, sg-1 and sg-2, contained the same neutral sugars, galactose and glucose in molar ratio of 1:1 and no GalNAc except for the presence of N-acetyl-neuraminic acid (NeuAc) in sg-1 and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in sg-2. The complete structures of these gangliosides were determined by a combination of methylation analysis, fast atom bombardment mass spectrometry, 400-MHz one- and two-dimensional 1H nuclear magnetic resonance spectroscopy, fatty acid analysis, and endoglycoceramidase digestion NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-1 [(NeuAc)GM3] KDN alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-2 [(KDN)GM3] where, for both sg-1 and sg-2, the ceramide moieties (Cer) were found to be made up of 4-sphingenine and mainly C16:0 fatty acid (palmitate; 95%) with a minor amount of C24:1 fatty acyl chain (nervonate, 5%). The structure of sg-2 is novel and represents the first example of a new class of gangliosides, i.e. KDN-gangliosides.     

A blood group B-like pentaglycosylceramide is the major complex glycosphingolipid of the Madin-Darby canine kidney epithelial cell line I (MDCK I)

Two sublines of the epithelial cell line MDCK differ in glycosphingolipid composition (Hansson, G.C. et al. (1986) EMBO J. 5, 483-489). The Forssman pentaglycosylceramide was an abundant glycolipid in the MDCK II subline, but was absent in the MDCK I subline. The MDCK I line instead contained another five-sugar glycolipid in relatively large amounts. This component has now been isolated and characterized with mass spectrometry, methylation analysis, exoglycosidase digestion, and proton NMR spectroscopy. The structure was concluded to be Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 Cer. This is a blood group B-like glycolipid lacking fucose, earlier found in rabbit and bovine erythrocytes.     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Studies on the chemical structure of neutral glycosphingolipids in eggs of the sea hare, Aplysia juliana.

Six neutral glycosphingolipids (GL-1-GL-6) were obtained from eggs of the sea hare (Aplysia juliana) and were characterized by FABMS, 1H-NMR, partial acid hydrolysis, methylation studies and GC analysis of the component sugars, fatty acids and long-chain bases. The following structures were determined to be Glc beta 1-1Cer (89%) and Gal beta 1-1Cer (11%) for GL-1, Glc beta 1-1Cer (47%) and Gal beta 1-1Cer (53%) for GL-2 having hydroxy fatty acids in the ceramide moiety, Gal beta 1-4Glc beta 1-1Cer for GL-3, Fuc alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-4, Gal alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-5 and GalNAc alpha 1-3(Gal alpha 1-2)Gal beta 1-4Glc beta 1-1Cer for GL-6. The fatty acid composition of each glycosphingolipid, except for GL-2, which contained 2-hydroxypalmitic acid, consisted of mostly saturated C16-C20 acids, especially palmitic acid and stearic acid. The long-chain bases of all glycosphingolipids consisted mainly of branched nonadeca-4-sphingenine and octadeca-4-sphingenine. GL-6, which was one of the major glycosphingolipids, may be a precursor of a series of phosphonoglycosphingolipids which have been isolated from the skin of A. kurodai.     

Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai.

Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)