Structural identification of two ten-sugar branched chain glycosphingolipids of blood group H type present in epithelial cells of rat small intestine

Two novel blood group H-type decaglycosylceramides with a branched core saccharide have been identified in mixture in a fraction isolated from rat small intestine. They were present exclusively in the epithelial cells. The number and sequence of sugars were established by direct inlet mass spectrometry of the permethylated and LiAlH4-reduced permethylated derivatives. Gas-liquid chromatography of the products after degradation of the native and permethylated glycolipids gave the type of sugars and the binding positions. A di- and a trisaccharide were identified by mass spectrometry after degradation of the permethylated-reduced derivative. One trisaccharide had the structure (formula see text) and was therefore additional evidence for a branched structure. Treatment of the decaglycosylceramide fraction with alpha-L-fucosidase gave free fucose and an octaglycosylceramide identified by mass spectrometry. Proton NMR spectra of the permethylated and permethylated-reduced octa- and decaglycosylceramides provided evidence for the binding configurations and the localization of type 1 and type 2 sequences in the two branches. The 3-linked branch was homogeneous with a type 1 saccharide (Gal beta 1 leads to 3GlcNAc) but the 6-linked branch had both type 1 and type 2 (Gal beta 1 leads to 4GlcNAc) sequences. Two glycolipids with the following probable structures were therefore present, making up 60 and 40% of the mixture, respectively: (formula see text) The lipophilic part contained mainly trihydroxy 18:0 long chain base (phytosphingosine) and 16:0 to 24:0 nonhydroxy fatty acids.     

Developmental changes of blood group A-active glycosphingolipids with type 1 and type 2 chains in rat small intestine

Blood group A-active glycosphingolipids of the small intestine, A-6 and A-12, which have been characterized previously in the adult rat [Breimer ME, Hansson GC, Karlsson K-A, Leffler H (1982) J Biol Chem 257:906–12], were found to appear during postnatal development, using immunostaining on thin layer chromatograms with two monoclonal anti-A antibodies, A005 and A581. In this system, A005 was found to be specific for the A determinant based on the type 2 chain, while A581 reacted mainly with the A determinant based on the type 1 chain and only weakly with the A determinant based on the type 2 chain. A-6 Type 1 was detected first at 18 days after birth. Its concentration increased markedly during the fourth week. A-6 Type 2 was detected, at a very low level, in neonates. Its concentration increased between days 15 and 20 and then decreased almost to the neonate level by 28 days. Dodecaglycosylceramide A-12 followed the same pattern of reactivity as A-6 type 1 with A581, and remained strongly reactive with A005 after 20 days. Linear A-6 and branched A-12 appeared simultaneously. Antibodies directed against blood group H determinants based on the type 1 or type 2 chains did not detect any H structure which might have appeared as a precursor of either A-6 or A-12 at the early stages of postnatal development.Abbreviations A-6, A-12, H-5, H-10 etc the glycolipids are abbreviated by giving blood group activity, and number of sugars (see also Fig. 1) - G_M3 G_M3-ganglioside, H³NeuAc-LcCer - PBS phosphate-buffered saline     

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.     

Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine

Non-acid glycosphingolipids were isolated from small intestinalepithelial cells of a single blood group A pig. One very predominantblood group compound was obtained chemically pure upon HPLCfractionation. It was characterized by mass spectrometry and¹H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide.Support for the presence of minute amounts of additional A glycolipidswas obtained by mass spectrometry and immunostaining of TLCplates with anti-A antibodies specific for A type 2 chain, Atype 3 and 4 chain, and the ALe^b determinant. Among precursorchains, globoside (type 4) and lactotetraosylceramide (type1) were immunologically identified, whereas no neolactotetraosylceramide(type 2) and gangliotetraosylceramide reactivities were detected.We addressed the question whether the predominant expressionof type 1 chain based A glycolipids reflects a restricted glycolipidprecursor chain specificity of the     

N-linked glycopeptides with blood group determinants lacking neuraminic acid from the epithelial cells of rat small and large intestine.

The N-linked type of glycans were prepared as their glycopeptides after pronase digestion of the epithelial cells from the small and large intestine of two inbred strains of rat. These glycopeptides were analysed for sugar composition, for blood-group activity, by 1H-NMR spectroscopy, and after permethylation by electron-impact mass spectrometry. The glycopeptides were of the triantennary and tetraantennary types with intersected GlcNAc. The terminal parts were, in contrast to most N-linked glycans, devoid of neuraminic acid residues. Instead they contained blood-group determinants. Blood-group-H types 1 (Fuc alpha 1-2Gal beta 1-3GlcNAc) and 2(Fuc alpha 1-2Gal beta 1-4GlcNAc) were found in the small and large intestines of both strains, although type-1 predominated. One rat strain (GOT-W) did not express blood-group-A glycopeptides in the small intestine, but the large intestine from the same strain did. The other strain (GOT-BW) expressed blood-group-A determinants in the small intestine. The lack of neuraminic acid residues in the small and large intestine and of blood-group-B activity in the large intestine differed from that found in glycosphingolipids obtained from the same organs.     

Lipoproteins of rat small intestine epithelial cells in D-hypovitaminosis

Chemical composition of lipoproteins has been studied in intestinal epitheliocytes of rats in normalcy and under D-hypovitaminosis. It is found that D-hypovitaminosis induces changes in the lipid and protein composition of lipoproteins. It is supposed that disturbances in biosynthesis of the lipoprotein components and their transport may be possible reasons of such changes.     

Conformational analysis of blood group A-active glycosphingolipids using HSEA-calculations. The possible significance of the core oligosaccharide chain for the presentation and recognition of the A-determinant

Conformational analysis of four different A-active glycosphingolipids, A types 1-4, was carried out using HSEA-calculations with the GESA-program. In their minimum energy conformations the oligosaccharide chains are more or less curved; in particular the type 3 and 4 have a strongly bent shape. When the carbohydrate structures are linked to ceramide, using the conformational features predominantly observed in crystal structures of membrane lipids, rather drastic differences in the orientation of the oligosaccharide chains are obtained. For the type 1 glycosphingolipid the model study indicates that the A-determinant extends almost perpendicularly to the membrane plane whereas for type 2, 3 and 4 the terminal part of the oligosaccharide chains is more parallel to the membrane. The fucose branch on type 3 and type 4 thereby appears directed towards the environment whereas for type 2 it would face the membrane. Due to restrictions imposed by the membrane layer this core specific orientation is largely preserved even if the flexibility of the saccharide-ceramide linkage is taken into account. Hydrophilic and hydrophobic sites on the surface of the different oligosaccharide chains in their minimum energy conformation were located using the GRID-program. It is suggested that the core-dependent presentation of the A-determinant might explain the chain type specificity observed for different monoclonal anti-A antibodies. The results further suggest that assay systems ensuring a membrane-like presentation of the glycolipid antigen should be used in studies of glycolipid/protein interactions.     

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.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Tissue specificity of glycosphingolipids as expressed in pancreas and small intestine of blood group A and B human individuals

A chemical investigation has been done on blood group active glycosphingolipids of both small intestine and pancreas from two individuals, one blood group A and one blood group B. Total non-acid glycolipid fractions were prepared and the major blood group fucolipids present were purified and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradation methods. The glycolipid structures identified were a blood group Leb hexaglycosylceramide, a B-hexaglycosylceramide with a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, A-hexaglycosylceramides with types 1 and 2 (Gal beta 1 leads to 4GlcNAc) carbohydrate chains, a B-heptaglycosylceramide with a type 1 carbohydrate chain, and A-heptaglycosylceramides with type 1 and 2 carbohydrate chains. In addition several minor glycolipids having more than seven sugar residues were detected by thin-layer chromatography. The small intestine and pancreas had some distinct differences in their expression of the major fucolipids. The small intestine contained only glycolipids based upon type 1 carbohydrate chain while the pancreas had both type 1 and type 2 structures. The intestines contained mainly difucosyl compounds while the pancreas tissues contained both mono- and difucosyl glycolipids. Monofucosylglycolipids based on both types 1 and 2 saccharides were present in one pancreas while the other one contained only monofucosylcomponents based on type 1 chain. The ceramides of the intestinal glycolipids were found to be more hydroxylated (trihydroxy long-chain base, hydroxy fatty acids) compared to the pancreas glycolipids (dihydroxy long-chain base, non-hydroxy fatty acids).     

Structures of blood group glycosphingolipids of human small intestine. A relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor

Small intestinal epithelial cells (enterocytes) were isolated from specimens obtained at operation from four human individuals with different blood group ABO, Lewis, and secretor phenotypes. The non-acid glycolipids were isolated and characterized by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy and for reactivity with monoclonal antibodies on thin-layer chromatograms. Monohexosylceramides and blood group ABH (type 1 chain) and Lewis glycolipids with 5-7 sugar residues were the major compounds present in all cases, and the expression of the major blood group glycolipids was in agreement with the ABO, Lewis, and secretor phenotype of the individual donors. Small amounts of more complex glycolipids with up to 10 sugar residues were identified by mass spectrometry in all cases. In addition, small amounts of lactotetraosylceramide, a blood group H-active triglycosylceramide with the structure of Fuc alpha 1-2Gal-Hex-Cer (where Fuc is fucose, Hex is hexose, and Cer is ceramide), and dihexosylceramides were identified in some cases. Globotriaosyl- and globotetraosylceramides were absent from the epithelial cells. Small amounts of Leb-active glycolipids in blood group OLe(a+b-), non-secretor and OLe(a-b-), secretor individuals as well as trace amounts of type 2 carbohydrate chain compounds in all individuals were detected by specific monoclonal antibodies.     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

Microscale analysis of glycosphingolipids by TLC blotting/secondary ion mass spectrometry: a novel blood group A-active glycosphingolipid and changes in glycosphingolipid expression in rat mammary tumour cells with different metastatic potentials

The glycosphingolipid compositions of rat mammary tumour cell lines with different metastatic potentials for the lung [a parental tumour cell line (MTC) and its subclones MTLn2 (a non metastatic subclone) and MTLn3 (a subclone with high metastatic potential to the lung)] were studied using a newly developed TLC blotting/secondary ion mass spectrometry system and crude glycosphingolipids obtained from 0.5–1×10⁷ cells of each cell line. G_M3 and G_M2 were the major components of the MTC cell line, but they were very minor components in the MTLn2 and MTLn3 cell lines, G_Dla being the major ganglioside. HexNAc-fucosyl-G_Mla was found in the MTLn2 cells by the TLC blotting/SIMS method, and the terminal sugar linkage was shown to be a blood group A-type structure by immunostaining. These findings suggest that the ganglioside is a novel type of blood group A-active ganglioside, GalNAc1-3(Fuc1-2)G_Mla. No blood group A-active lipid was present in MTLn3 cells, whereas Hex-G_Mla and neutral glycosphingolipids with more than 5 sugar residues were. Abbreviations: TLC, thin-layer chromatography; HPTLC plate, high performance thin-layer chromatography-plate; PVDF, polyvinylidene difluoride; SIMS, secondary ion mass spectrometry; GC-MS, gas chromatography-mass spectrometry; C16:0, hexadecanoic acid; C18:0, octadecanoic acid; C22:0, docosanoic acid; C24:0, tetracosanoic acid; d18:1, 2-amino-4-octadecene-1,3-diol; Hex, hexose; HexNAc,N-acetylhexosamine; Gal, galactose; Glc, glucose; GalNAc,N-acetylgalactosamine; Lac, lactose; NeuAc,N-acetylneuraminic acid; Cer, ceramide; Glob, globoside; iGlob, isogloboside; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb₃Cer, Gal1-4Gal1-4Glc1-1Cer; Gb₄Cer (Glob), GalNAc1-3Gal1-4Glc1-1Cer; iGb₃Cer, Gal1-3Gal1-4Glc1-1Cer; iGb₄Cer (iBlob), GalNAc1-3Gal1-4Glc1-1Cer; Ganglio-series gangliosides are named according to Svennerholm [1].     

The presence of blood group A-active glycolipids in cancer tissues from blood group O patients

Glycolipids were isolated from human gastric cancer tissues and normal mucosae. Sulfogalactosylceramide, ganglioside and neutral glycolipid fractions were separated by DEAE-Sephadex and silica gel column chromatography. Sulfogalactosylceramide contents were higher in the cancer tissues than in the normal mucosae. Ganglioside contents showed considerable variations but in the cancer tissues in mole percentage of ganglioside GM3 was higher than in the normal mucosae. The cancer tissues contained more neutral glycolipids than normal tissues. Glycolipids of lacto-series, including fucolipids, were markedly increased in the cancer tissues. Blood group A-active glycolipids were found in the cancer tissues from two patients with blood group O but not found in the uninvolved tissue associated with the cancer tissue.     

Ontogeny of two vitamin A-metabolizing enzymes and two retinol-binding proteins present in the small intestine of the rat.

The patterns of expression of cellular retinol-binding protein (CRBP), cellular retinol-binding protein, type two [CRBP(II)], lecithin: retinol acyltransferase (LRAT), and microsomal retinal reductase were examined for rat small intestine during the perinatal period. CRBP was present (15 pmole per mg soluble protein) at the earliest time examined, the 16th day of gestation, declining by 70% by birth, maintained to adulthood. In contrast, CRBP(II) appeared 2-3 days before birth, rising to its highest level (500 pmole per mg soluble protein) by day 3 after birth, then declining by 50% during the late suckling period to the adult level. Immunohistochemistry revealed that CRBP(II) initially appeared in the epithelial cell layer in a patchy manner, resolving by birth into an even staining of all villus-associated enterocytes. In contrast, CRBP was evenly expressed in the epithelial cell layer at day 17/18 but was absent by birth. Intestinal LRAT activity increased rapidly in the 2 days prior to birth, then declined at weaning to the adult level. Microsomal retinal reductase was measurable in the intestine at birth, but not detected during the early suckling period, reappearing at day 21. Considerable increase was then observed coincident with weaning, when carotenes, from which retinal is derived, became an important source of vitamin A. The pattern of appearance of these elements appears to prepare the intestine for the necessary processing of vitamin A required after birth.     

Close relationships between the cells of the immune system and the epithelial cells in the rat small intestine

Summary A possible contribution of the intestinal epithelium to the immune defence system was studied by electron microscopy in the rat small intestine. The cells of the immune system (CIS) such as lymphocytes, eosinophils and macrophages penetrate the basal lamina into the epithelium and make close relationships with the absorptive cells. At the points of close apposition, the two cell membranes run parallel at a regular distance of about 20 nm. On the other hand, about 5% of the intestinal absorptive cells also penetrate the basal lamina into the lamina propria via their basal protrusions and show a similar close association with CIS. The basal protrusions contain many microfilaments; this indicates that they are structures with a definite function rather than a simple hernia. These findings are discussed with respect to the transport of antigenic molecules and of intercellular communication between CIS and the intestinal epithelium.