A novel pentaglycosylceramide in ostrich liver, IV4-beta-Gal-nLc4Cer, with terminal Gal(beta1-4)Gal, a xenoepitope recognized by human natural antibodies.

Thin layer chromatograms of ostrich liver neutral glycosphingolipids were immunostained with human sera. In addition to the expected staining of the Forssman pentaglycosylceramide by some sera, more polar and less abundant unknown glycolipids could be stained. Among them, the shortest carbohydrate chain glycolipid was purified and structurally characterized by mass spectrometry, proton NMR and methylation analysis. It was a novel pentaglycosylceramide of the neolactoseries terminated with the Gal(beta1-4)Gal determinant which is not expressed in mammalian species. Human antibodies affinity-purified on a synthetic Gal(beta1-4)Gal(beta1-4)Glc-Sepharose column recognized the newly characterized Gal(beta1-4)Gal-terminated pentaglycosylceramide, and, in addition, longer chain glycolipids. Occurrence of antibodies directed at the Gal(beta1-4)Gal epitope was studied by ELISA on 108 human sera. Anti-Gal(beta1-4)Gal antibodies were predominantly IgM, and their distribution was similar to that of anti-Gal(alpha1-3)Gal and anti-Forssman IgMs. It was concluded that anti-Gal(beta1-4)Gal are natural antibodies, not previously identified in man. They can be considered as xenoantibodies directed at species which express Gal(beta1-4)Gal-terminated carbohydrate chains.

Induction of anti-Forssman antibodies in the hamster-to-rat xenotransplantation model.

BACKGROUND: In the hamster-to-rat heart xenotransplantation model, the serum response of the host contributes to determine whether the xenograft is accommodated or rejected. METHODS: To further characterize the serum response in this model, we compared anti-hamster antibodies found in naive LEW-1A rats, or in LEW-1A rats rejecting or accommodating a hamster heart, using a combination of cobra venom factor (CVF) and cyclosporin A (CsA) given for 10 days, and then CsA alone. RESULTS: Hamster hearts grafted into rat recipients contained IgG and IgA deposits to the same extent whether the xenograft was rejected or accommodated. Only immunoglobulins of the IgM isotype were found to be more abundant in recipients rejecting their graft. A significant part of this IgM response was directed toward the Forssman antigen, a sphingolipid present in the hamster but not in the rat. However, although anti-Forssman antibodies bind in situ to hamster tissues, this binding was not able to induce hyperacute rejection after antibody transfer. Furthermore, depletion of anti-Forssman antibodies from a rejecting serum did not modify its rejection properties. CONCLUSION: Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.

Characterization of a polyclonal anti-Galalpha1-3Gal antibody from chicken.

A polyclonal antibody was raised against the Galalpha1-3Gal carbohydrate epitope, which is expressed by all mammals (except man and the closest primate species) by immunizing hens with rabbit erythrocyte membranes. IgY was isolated from egg yolks, and affinity-purified on a Galalpha1-3Gal-Synsorb column. Two percent of the initial IgY fraction was recovered. The specificity of the affinity-purified antibody was characterized by: absorption with human, rabbit and pig erythrocytes; by using Synsorb columns; by inhibition with different saccharides; and by immunostaining of glycolipids separated on thin layer chromatograms. A weak reactivity was found toward blood group B or blood group Pk determinant, depending on the assay system used. Such reactivities were abolished after absorption by the appropriate sorbents, yielding a polyclonal anti-Galalpha1-3Gal antibody with narrow specificity.

Two novel isoneolacto-undecaglycosylceramides carrying Galalpha1-->3Lewis(x) on the 6-linked antenna and N-acetylneuraminic acidalpha2-->3 or Galactose alpha1-->3 on the 3-linked antenna, expressed in porcine kidney.

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Galalpha1-->3Gal, anti-type 2 lactosamine and anti-Lewis(x) antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI'3(alphaGal)2-iso-nLc8Cer, and two were novel structures differing by the number of Galalpha3Lewis(x) determinants: VI3VI'3(alphaGal)2V'3alphaFuc-iso-nLc8, and VI3VI'3(alphaGal)2 V3V'3(alphaFuc)2-iso-nLc8. The single Galalpha3Lewis(x) determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI'3(NeuAc)2-iso-nLc8, and VI3NeuAcVI'3alphaGal-iso-nLc8. A novel structure was discovered presenting a Galalpha3Lewis(x) determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI'3alphaGalV'3alphaFuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney alpha3fucosyltransferase synthesizes the Gala3Lewis(x) determinant, acting on the 6-linked before the 3-linked Galalpha3neolactosamine, and appears unable to synthesize the sialosylated Lewis(x) determinant on neolactoseries glycolipids.

A novel glycosphingolipid expressed in pig kidney: Gal alpha 1-3Lewis(x) hexaglycosylceramide.

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Gal alpha 1-3Gal determinant revealed several glycosphingolipids reacting with different intensities. A minor glycosphingolipid was isolated by preparative high performance thin layer chromatography. It was characterized as a type 2 hexaglycosylceramide with the following structure Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with alpha-galactosidase followed by immunostaining with an anti-Lewis(x) monoclonal antibody. The proton NMR spectrum was found compatible with the proposed structure. Two other glycosphingolipids carrying the new determinant were partially characterized as an octa- and a branched-dodecaglycosylceramide. The expression of the Gal alpha 1-3 Lewis(x) determinant appeared to be developmentally regulated as it increased with age. The characterization of Gal alpha 1-3Le(x) in pig kidney indicates a new epitope capable of recognition by human natural antibodies in the context of xenotransplantation of pig organs to man. It also adds new members to the family of Le(x)-based glycolipids.

Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N-glycolylneuraminic acid.

Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.

Structure and genetic polymorphism of blood group A-active glycosphingolipids of the rat large intestine.

Study of blood group A- and B-active glycosphingolipid content of the epithelium of the large intestine of 16 strains of inbred rats led to the discovery of two related strains, SHR and WKY, devoid of A-active glycolipids, whereas all strains expressed B-active glycolipids. This finding evidenced a new A/non-A genetic polymorphism in the rat. Blood group A-active glycolipids were isolated from the large intestine of F344 rats and purified by affinity chromatography on immobilized Helix pomatia lectin. Three glycolipid fractions were separated by preparative thin-layer chromatography and characterized by electron-impact mass spectrometry of their permethylated and permethylated-LiAlH4-reduced derivatives. They were identified as a tetraglycosylceramide (A-4), a hexaglycosylceramide (A-6), and a difucosylated heptaglycosylceramide (A-7) with small amounts of monofucosylated octaglycosylceramide (A-8). Methylation analysis and fragmentation indicated that A6 and A-8 had a lacto- and A-7 a neolactotetraosylceramide core, respectively, identical to the core structures of B-6 and B-7 previously characterized in the large intestine of WF rats (Angström et al. (1987) Biochim. Biophys. Acta 926, 79-86). Upon methylation analysis, B-6 and B-7 purified from SHR (A-deficient) and F344 (A-expressing) were found identical to those of WF rats. This result indicated that precursor substrates for the synthesis of A-active glycolipids were available in SHR rats and thus the genetic deficiency of A-active glycolipid expression probably originated in a defect of the termination of the blood group A determinant by the alpha-3-N-acetylgalactosaminyltransferase.

Biochemical characterization of blood group-active glycosphingolipids.

Glycosphingolipids are quantitatively minor components of cell lipids. However, their segregation in the outer leaflet of the plasma membrane confers to these membranes specific structural and immunological properties. Current methods of extraction, purification and analysis of blood cell glycolipids are presented. Valuable structural data may be obtained by a combination of chemical and enzymatic degradations with thin-layer chromatography and immunological detection by monoclonal antibodies of known specificity. Examples of physical characterization by Mass Spectrometry and Proton Magnetic Resonance Spectroscopy are also presented.

Two novel decaglycosylceramides with a blood group A-active tetrasaccharide repeat in the epithelial cells of the small intestine of inbred rats.

A novel type of blood group A-active glycosphingo-lipid was isolated from the epithelial cells of the small intestine of one strain of inbred rats. Electron-impact mass spectrometry of the permethylated and LiAlH4-reduced glycolipid indicated that it is a decaglycosylceramide with a branched oligosaccharide chain. Methylation analysis, gas chromatography-mass spectroscopy of the partially methylated alditol acetates, sequential degradation by exoglycosidases and characterization of the reaction products by TLC immunostaining with appropriate anti-A and anti-H antibodies, and 1H NMR spectrometry resulted in the characterization of a decaglycosylceramide with two variants in a 7/3 ratio. It was termed AA-10. [formula: see text] The major variant has only type 1 chains, whereas the minor one has type 2 chain in the C6-linked branch. This is a novel type of glycolipid with a blood group A-active tetrasaccharide repeat. Genetic analysis demonstrated that AA-10 is inherited as an autosomal dominant trait.

Characterization of a novel A-active octaglycosylceramide with type 1 chain repeat inherited as a recessive trait in the epithelial cells of the small intestine of inbred rats.

Three strains of inbred rats, AVN, DA and LOU/M, were found to express human blood group A-active glycosphingolipids in the small intestine. Two A phenotypes were detected by immunostaining of thin layer chromatograms with monoclonal anti-blood group A antibody. One phenotype (DA and LOU/M) displayed a novel glycolipid which was characterized as an A-active octaglycosylceramide with a type 1 chain repeat by methylation analysis, electron-impact mass spectrometry of the permethylated and permethylated LiAlH4-reduced molecule, and 1H NMR spectroscopy. It was designated A-8. GalNAc alpha 1-3Gal beta 1-(3GlcNAc beta 1-3Gal beta 1)2-4Glc beta 1-Cer 2 Fuc alpha 1 It is the first description of a type 1 chain repeat in a linear glycolipid. Calculation of minimum energy conformations showed that the orientations of the oligosaccharide chain and A determinant of A-8 differ from those of the homologous structure with a type 2 chain repeat present in human erythrocytes (Hakomori, S., Stellner, K., and Watanabe, K. (1972) Biochem. Biophys. Res. Commun. 49, 1061-1068). Genetic analysis demonstrated that A-8 is inherited as an autosomal recessive trait.

Genetic polymorphism of rat liver gangliosides.

Liver ganglioside patterns of eight rat strains were classified according to two phenotypes: SHR type, characterized by predominance of b-series gangliosides (GD1b, GT1b, GQ1b), and DA type, characterized by predominance of a-series gangliosides (GM1, GD1a). Comparison of ganglioside pattern expressed in the liver of F1 hybrids and backcross F2 hybrids indicated that SHR type is controlled by a single autosomal-dominant gene which probably determines the expression of sialytransferase 2 activity for synthesis of GD3 from GM3.

Transient expression of type 2 chain in A-active hexaglycosylceramide of rat small intestine at weaning time. Demonstration by affinity chromatography and ceramide glycanase hydrolysis of A-active glycosphingolipids followed by gas chromatography and mass spectrometry of permethylated hexasaccharides.

The small intestine of 15- to 23-day-old rats was cut into four segments from the duodenum to the ileum. Neutral glycosphingolipids were purified from each segment and submitted to thin-layer chromatography and immunostaining with the A005 monoclonal anti-A antibody. This antibody detected an hexaglycosylceramide located mainly in the duodenum during the postnatal development. In order to characterize hexaglycosylceramides, blood group A-active glycolipids were purified by affinity chromatography on immobilized Helix pomatia lectin in organic solvent. Hexaglycosylceramides (A-6) were subsequently isolated by preparative thin-layer chromatography and hydrolyzed with ceramide glycanase. The free hexasaccharides were permethylated and analyzed by gas chromatography. Two peaks were detected in varying ratios during development, corresponding to type 1 and type 2 chain A hexasaccharides. Gas chromatography clearly demonstrated that type 2 A-6 occurred in the duodenum of developing rats, and that a shift from type 2 to type 1 A-6 occurred with growing age. The change from type 2 to type 1 chain was also assessed by methylation analysis, and by the variation of the characteristic fragmentations of type 1 and type 2 chain hexasaccharides upon mass spectometry of the permethylated A-6 oligosaccharides from the duodenum of 19-day-old and adult rats.

Regional differences in the appearance of adult-type glycosphingolipids in the small intestine of inbred rats at weaning time.

The small intestine of 15- to 33-day-old rats was cut into four segments: duodenum, proximal jejunum, distal jejunum, and ileum. Neutral glycosphingolipids and gangliosides were purified from each segment and analyzed by thin-layer chromatography in order to study the developmental appearance of adult-type glycolipids at each level of the small intestine. Type 1 A-6 glycolipid was first detected in the ileum at 15 days and subsequently in the jejunum and duodenum at 19 days of age. N-Glycolylneuraminic acid was expressed first in the ileum at 17 days, then in the proximal jejunum at 21 days, but only after 29 days in the duodenum. In each region, 6-8 days were required between first detection and full expression of N-glycolylneuraminic acid. The presence of 2-hydroxylated fatty acids in glucosylceramide was found first in the ileum at 19 days, 2-3 days before appearing in the duodenum and proximal jejunum. A period of 2-3 days was necessary to reach full adult-type level of 2-hydroxylated fatty acids in glucosylceramide. These results show that adult-type glycolipids appear earlier in the distal than in the proximal region of the rat small intestine, and that different glycolipids appear at different times and at different rates. The finding that the biochemical differentiation of the whole small intestine expands over a period of 3 days to 2 weeks, depending on the region and the glycolipid, before being fully completed indicates that, in addition to the time lag observed between the distal and the proximal region, the new cells arising from the crypt of Lieberkhün after 15 days of age are not at once fully differentiated.

Hydroxylation of CMP-NeuAc controls the expression of N-glycolylneuraminic acid in GM3 ganglioside of the small intestine of inbred rats.

An enzymatic activity responsible for the hydroxylation of CMP-NeuAc into CMP-N-glycolylneuraminic acid (CMP-NeuGc) was found in the cytosolic fraction after cellular fractionation of the mucosa of rat small intestine. It was maximum in the presence of NADPH or NADH, but it was reduced by 50% by addition of 1 mM EDTA. The Km value for CMP-NeuAc was 0.6 microM. The CMP-NeuAc hydroxylase activity paralleled the expression of the GM3 (NeuGc) phenotype in the epithelium of the small intestine and was not measurable in the mutant rats BN and SHR that only expressed GM3 (NeuAc). Furthermore, the only form of CMP-sialic acid present in the intestinal mucosa of the mutants was CMP-NeuAc, whereas in the other strains CMP-NeuGc accounted for 70-85% of the native CMP-sialic acids. Wild-type and CMP-NeuAc hydroxylase-deficient inbred rats were mated. Individuals of F1 and backcross generations were typed for the phenotypes GM3(NeuGc)/GM3(NeuAc) and the activity of CMP-NeuAc hydroxylase in the small intestine. It was found that the expression of NeuGc in GM3 depends on a single autosomal dominant gene and correlates with the activity of CMP-NeuAc hydroxylase. Two tissues other than small intestine, kidney and spleen, which expressed GM3(NeuGc) in BN and SHR, also expressed the CMP-NeuAc hydroxylase activity, as in the other strains. It was concluded that the key enzyme responsible for the presence of NeuGc in GM3 is a CMP-NeuAc hydroxylase and that mutant rats carry a defect that is specific to intestine. The comparative analysis of the respective contribution of NeuGc and NeuAc to the glycoprotein sialic acids of the small intestine showed that CMP-NeuAc hydroxylase is also responsible for part of the NeuGc present in the glycoproteins. However, the occurrence of 20-30% of NeuGc in the intestinal glycoproteins of the CMP-NeuAc hydroxylase-deficient rats indicated that there is another enzyme providing intestinal glycoproteins with NeuGc and operating under a different genetic control.

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats.

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.

Developmental changes of gangliosides of the rat stomach. Appearance of a blood group B-active ganglioside.

Rat stomach gangliosides were purified and their distribution in the different tissue compartments was established. Three major monosialogangliosides were found: GM3, GM1, and a ganglioheptaosylceramide carrying a blood group B determinant. This latter structure was characterized by exoglycosidase degradation, immunostaining with a monoclonal anti-blood group B antibody on thin layer chromatogram, permethylation analysis, electron-impact mass spectrometry of the permethylated-reduced and trimethylsilylated molecule, and 1H NMR spectroscopy of the native ganglioside. It was found to be (Formula: see text) i.e. a GM1 structure substituted with the blood group B determinant and was called B-GM1. A similar structure has been previously identified in precancerous rat liver and chemically induced rat hepatoma (Holmes, E. H., and Hakomori, S. (1982) J. Biol. Chem. 257, 7698-7703). Fucosyl-GM1 was also detected as a minor ganglioside in rat gastric mucosa. The ganglioside profile was modified during the postnatal development. The contribution of GM3 and GD3, which accounted for 95% of the ganglioside sialic acid at birth, decreased during the first 3 weeks of life. GM1, fucosyl-GM1, and B-GM1 were not detected at birth. The concentration of the fucogangliosides increased during the 2nd and 3rd weeks after birth, was stable during the 4th week and then decreased, whereas that of GM1 increased steadily between 6 days and 2 months of age. B-GM1, which has been defined as a tumor-associated ganglioside in the rat liver, was found to be a developmentally regulated antigen of the normal rat stomach.