Degradation of human intestinal glycosphingolipids by extracellular glycosidases from mucin-degrading bacteria of the human fecal flora

Certain normal strains of human fecal bacteria are unique in producing extracellular glycosidases that degrade the oligosaccharide chains of gut mucin glycoproteins. We have studied the action of such glycosidases partially purified from the cell-free supernates of five of these strains on intestinal glycosphingolipids isolated from human meconium. The glycolipids were sialosyl-lactosylceramide, lactosylceramide, and fucolipids with A, B, H, Lea, or Leb blood group determinants. In addition to the strain-specific high blood group A-degrading activities (Ruminococcus torques strains VIII-239 and IX-70), B-degrading activity (Ruminococcus AB strain VI-268), and H-degrading activities (all strains) corresponding to alpha 1-3-N-acetylgalactosaminidase, alpha 1-3-galactosidase and alpha 1-2-fucosidase, respectively, all strains also degraded sialosyl-lactosylceramide and Lea and Leb antigenic glycolipids, indicating the presence of alpha 2-3-neuraminidases and alpha 1-4-fucosidases. Enzyme preparations from Bifidobacterium infantis strain VIII-240 and R. torques strain VIII-239 hydrolyzed the Lea active glycolipid directly to lactosylceramide, suggesting the presence of endo-beta 1-3-N-acetylglucosaminidase activities. Similar endo-beta-N-acetylglucosaminidase activities were identified in four of the five enzyme preparations. The enzymes produced by R. AB strain VI-268 lacked this activity as well as beta 1-3-galactosidase, and thus degradation stopped at lactotetraosylceramide. With enzyme preparations from the other strains lactosylceramide was the single major degradation product from complex glycosphingolipids with less than 30% further degradation to glucosylceramide within 48 h. We conclude that glycosidases from mucin-degrading strains of human enteric bacteria degrade oligosaccharide chains of lactoseries fucolipids and gangliosides of intestinal origin primarily to lactosylceramide. Since several genera of enteric bacteria bind preferentially to lactosylceramide in vitro, mucin-degrading strains may have an important ecological role in host-microbial associations in the human gut.     

Enhancing effects of bile salts on the degradation of glycosphingolipids by glycosidases from bacteria of the human fecal flora

Different concentrations of ionic and non-ionic detergents were examined for optimization of the in vitro degradations of intestinal glycosphingolipids by alpha- and beta-glycosidases from human fecal bacteria. In 5 mM Triton X-100 the enzymes hydrolyzed glycosphingolipids with lactoseries type 1 and 2 chains essentially to lactosylceramide (LacCer). In 5 mM sodium di- and trihydroxy bile salts lactosylceramide was degraded to glycosylceramide (GlcCer) in varying extent by enzymes from all five strains. The minimal bile salt concentrations for optimal 1,4-beta-galactosidase activities varied between 1 and 20 mM, i.e., close to or above the critical micellar concentrations (cmc). Dihydroxy bile salts were the most efficient in promoting conversion of LacCer to GlcCer at concentrations below 10 mM and conjugation with a taurine residue did not markedly lower the GlcCer yield. The optimal detergent concentrations for hydrolyses of the p-nitrophenyl (pnp) glycosides Gal beta 1-pnp and GalNAc alpha 1-pnp were approximately 0.05 mM for Triton X-100 and 0.5 mM for sodium taurodeoxycholate, i.e., clearly below their reported cmc values. Galabiosylceramide, globotria- and globotetraosylceramides, not degraded in the Triton X-100 micelles, were also resistant to hydrolysis using the sodium bile salts as detergents. In contrast, lactotetraosylceramide and isoglobotriaosylceramide were significantly more degraded by enzymes from a Ruminococcus gnavus strain and gangliotetraosylceramide by enzymes from a Bifidobacterium bifidum and a Bifidobacterium infantis strain using bile salt detergents. All strains but R. gnavus released terminal GalNAc from para-Forssman but not from the globotetraosylceramide or Forssman structures using 5 mM sodium deoxycholate as detergent. GM1 desialylation by two Ruminococcus torques strains and the R. gnavus and B. bifidum strains were enhanced under identical conditions. We conclude that the observed effects on glycosphingolipid hydrolyses reflects variations in the micellar presentation of the substrates. In addition, detergents seem to have a direct stimulating effect on the glycosidases, however at concentrations 10-100-times below the ones optimal for glycolipid degradations. These results with optimized bile salt concentrations, further support our previous observations that these five fecal bacterial strains produce enzymes with selected specificities towards glycosphingolipid core chains of the lactoseries type 1 and 2.     

Carbohydrate recognition by enterohemorrhagic Escherichia coli: characterization of a novel glycosphingolipid from cat small intestine

A key virulence trait of pathogenic bacteria is the ability to bind to receptors on mucosal cells. Here the potential glycosphingolipid receptors of enterohemorrhagic Escherichia coli were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two nonacid glycosphingolipids of cat small intestinal epithelium was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, identified as Galalpha3Galbeta4Glcbeta1Cer (isoglobotriaosylceramide) and Galalpha3Galalpha3Galbeta4Glcbeta1Cer. The latter glycosphingolipid has not been described before. The interaction was not based on terminal Galalpha3 because the bacteria did not recognize the structurally related glycosphingolipids Galalpha3Galalpha4Galbeta4Glcbeta1Cer and Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer (B5 glycosphingolipid). However, further binding assays using reference glycosphingolipids showed that the enterohemorrhagic E. coli also bound to lactosylceramide with phytosphingosine and/or hydroxy fatty acids, suggesting that the minimal structural element recognized is a correctly presented lactosyl unit. Further binding of neolactotetraosylceramide, lactotetraosylceramide, the Le(a)-5 glycosphingolipid, as well as a weak binding to gangliotriaosylceramide and gangliotetraosylceramide, was found in analogy with binding patterns that previously have been described for other bacteria classified as lactosylceramide-binding.     

Binding of Haemophilus ducreyi to carbohydrate receptors is mediated by the 58.5-kDa GroEL heat shock protein

The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.     

An improved technique for separation of neutral glycosphingolipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) procedure for separation of O-acetyl-N-p-nitrobenzoyl derivatives of six neutral glycosphingolipids: glucosylceramide, lactosylceramide, globotriaosylceramide, lactotriaosylceramide, globotetraosylceramide, and neolactotetraosylceramide. The recoveries of glucosylceramide and globotetraosylceramide for the derivatization procedure and HPLC analysis were approximately 75%, and one nanomole of glycolipid could be detected. The procedure was used for analysis of human erythrocyte neutral glycolipids.     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

Characterization of novel nonacid glycosphingolipids as biomarkers of human gastric adenocarcinoma

Changes in glycosphingolipid structures have been shown to occur during the development of several types of human cancers, generating cancer-specific carbohydrate structures that could be used as biomarkers for diagnosis and therapeutic targeting. In this study, we characterized nonacid glycosphingolipids isolated from a human gastric adenocarcinoma by mass spectrometry, enzymatic hydrolysis, and by binding with a battery of carbohydrate-recognizing ligands. We show that the majority of the complex nonacid glycosphingolipids had type 2 (Galβ4GlcNAc) core chains (neolactotetraosylceramide, the Le^x, H type 2, x₂, and the P1 pentaosylceramides, and the Le^y, A type 2, and neolacto hexaosylceramides). We also found glycosphingolipids with type 1 (Galβ3GlcNAc) core (lactotetraosylceramide and the H type 1 pentaosylceramide) and globo (GalαGal) core chains (globotriaosylceramide and globotetraosylceramide). Interestingly, we characterized two complex glycosphingolipids as a P1 heptaosylceramide (Galα4Galβ4GlcNAcβ3Galβ4GlcNAcβ3Gal β4Glcβ1Cer) and a branched P1 decaosylceramide (Galα4Gal β4GlcNAcβ3(Galα4Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4Glc β1Cer). These are novel glycosphingolipid structures and the first reported cases of complex glycosphingolipids larger than pentaosylceramide carrying the P1 trisaccharide. We propose that these P1 glycosphingolipids may represent potential biomarkers for the early diagnosis of gastric cancer.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Isolation and characterization of the major glycosphingolipids from the liver of the rainbow trout (Oncorhynchus mykiss): identification of an abundant source of 9-O-acetyl GD3

The carbohydrate structures of the major glycosphingolipids from the liver of the rainbow trout Oncorhynchus mykiss have been examined. We have isolated and identified four major neutral (glucosylceramide, galactosylceramide, lactosylceramide, and globoside) and five acidic (sulfatide, GM3, GM2, GD1a, and 9-O-Acetyl GD3) glycosphingolipids from trout liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, fast atom bombardment mass spectrometry, and specific monoclonal antibodies. Significantly, the relatively scarce ganglioside 9-O-acetyl GD3 was found to comprise approximately 23% of the total ganglioside content of normal rainbow trout liver. 9-O-Acetyl GD3 is, however, abundant in human melanoma and as such, trout liver may be a suitable source of this antigen.     

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     

Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.

In the course of work on a systematic structural mapping of nonacid glycosphingolipids of human meconia, special attention was given to a major component preliminarily identified as an isomer of neolactotetraosylceramide (paragloboside). This component was isolated in its pure form from meconium of a blood group O individual and subjected to detailed structural analyses, using mass spectrometry and proton NMR spectroscopy on intact permethylated and permethylated-reduced (LiAlH4) derivatives, and gas liquid chromatography on degradational products of native, permethylated, and permethylated-reduced derivatives. The isolated compound was conclusively shown to have the structure Galp beta 1 yields 3GlcNAcp beta 1 yields 3Galp beta 1 yields 4Glcp beta 1 yields 1Cer, and is thus identified as lactotetraosylceramide. The major fatty acids were 2-hydroxy fatty acids with 16 and 20 to 24 carbon atoms, and the bases were sphingosine and phytosphingosine. This glycolipid, although not isolated and structurally characterized before, has long been thought of as a precursor substance of the Lewis active glycolipids and of ABH-active glycolipids with a type 1 saccharide chain.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Lactotetraosylceramide, a novel glycosphingolipid receptor for Helicobacter pylori, present in human gastric epithelium

The binding of Helicobacter pylori to glycosphingolipids was examined by binding of (35)S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. In addition to previously reported binding specificities, a selective binding to a non-acid tetraglycosylceramide of human meconium was found. This H. pylori binding glycosphingolipid was isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, were identified as Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer (lactotetraosylceramide). When using non-acid glycosphingolipid preparations from human gastric epithelial cells, an identical binding of H. pylori to the tetraglycosylceramide interval was obtained in one of seven samples. Evidence for the presence of lactotetraosylceramide in the binding-active interval was obtained by proton NMR spectroscopy of intact glycosphingolipids and by gas chromatography-electron ionization mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis. The lactotetraosylceramide binding property was detected in 65 of 74 H. pylori isolates (88%). Binding of H. pylori to lactotetraosylceramide on thin-layer chromatograms was inhibited by preincubation with lactotetraose but not with lactose. Removal of the terminal galactose of lactotetraosylceramide by galactosidase hydrolysis abolished the binding as did hydrazinolysis of the acetamido group of the N-acetylglucosamine. Therefore, Galbeta3GlcNAc is an essential part of the binding epitope.     

Characterization of ISRgn1, a novel insertion sequence of the IS3 family isolated from a bacteriocin-negative mutant of Ruminococcus gnavus E1

ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1. The copy number of ISRgn1 in R. gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn1 is not indigenous to the R. gnavus phylogenetic group but that it can transpose in this bacterium.     

Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids

The adherence of the human respiratory pathogen, Bordetella pertussis, to purified glycosphingolipids was investigated using thin layer chromatography overlay assays. Both virulent and avirulent strains of B. pertussis bound to asialo GM1. The bacterium did not bind to the gangliosides GM1, GD1a, GD1b, and GT1b, nor to lactosylceramide, trihexosylceramide, globoside, or Forssman antigen. However, after treatment of the chromatography plates with sialidase, B. pertussis bound to the gangliosides GM1, GM2, GD1a, GD1b, and GT1b but not to GM3. Comparison of the oligosaccharide structures of these gangliosides suggests that the minimum sugar structure needed for avid bacterial binding is GalNAc beta 4Gal. This structure has been previously implicated as a receptor for other human respiratory pathogens (Krivan, H. C., Roberts, D. D., Ginsburg, V. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 6157-6161). Virulent strains of B. pertussis also bound specifically to sulfatide. This response was dose-dependent and inhibited by the anionic polysaccharide dextran sulfate. The sulfated-sugars dextran sulfate, fucoidan, and heparin inhibited the attachment of virulent strains of B. pertussis to human WiDr cells and to hamster trachea cells indicating that sulfatides on the surface of mammalian cells may function as a receptor for B. pertussis. The occurrence of both sulfatides and asialo GM1 in human lung and trachea suggests that these glycolipids may serve as specific receptors for B. pertussis.     

Clostridium glycyrrhizinilyticum sp. nov., a glycyrrhizin-hydrolysing bacterium isolated from human faeces

Screening of faecal bacteria for glycyrrhetic acid (GA) production by hydrolysing of glycyrrhizin (GL) resulted in the isolation of two strains, designated ZM35T and ZM38. Strains ZM35T and ZM38 were Gram-positive, obligate anaerobic, non-spore-forming and rod-shaped bacteria. Analysis of the 16S rRNA gene sequences indicated that strains ZM35T and ZM38 belonged to cluster XIVa of the genus Clostridium. The 16S rRNA gene sequences of strains ZM35T and ZM38 were identical. Strain ZM35T exhibited approximately 94% to 95% identity with the validly described species, Clostridium oroticum(94.5%), Eubacterium contortum(93.8%), Ruminococcus gnavus(94.5%) and R. torques(95.1%). In an experiment of DNA-DNA hybridization, it was confirmed that strains ZM35T and ZM38 were the same species. The guanine-plus-cytosine (G+C) content of strain ZM35T is 45.7 mol%. Based on the phylogenetic and phenotypic findings, we propose that strains ZM35T and ZM38 be assigned to a novel species named Clostridium glycyrrhizinilyticum. The type strain is ZM35T (=JCM 13368T=DSM 17593T).     

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.