Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGc alpha-3Gal beta 4Glc beta Cer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well as thus explain the resistance of adult pigs to infection with E. coli K99.     

Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach

Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.     

Characterization of a reverse gyrase from the extremely thermophilic hydrogen-oxidizing eubacterium Calderobacterium hydrogenophilum

Abstract Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae. The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae. Gangliosides capable of binding K99 fimbriated E. coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG. No binding was detected to NeuAc-GM3 and NeuGc-GM1.     

Glycosphingolipid patterns of the epithelial and non-epithelial compartments of rat large intestine

The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by thin-layer chromatography, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subfraction. As for the small intestine, the two separated compartments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Galα being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major glycolipid with the structure Galα1 → 3Gal(2 ← 1αFuc)β1 → 4Glcβ1 → 1Cer, as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectroscopy to have the probable structure Galα1 → 3Ga1(2 → αFuc)β1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass spectrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The four and six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-based blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains. Quantitatively the major glycolipids of the epithelial cells of large intestine were monoglycosylceramides (glucosylceramides, and smaller amounts of galactosylceramides which were absent from small intestinal epithelium) and tetraglycosylceramides (including the A and B active species and a tetrahexosylceramide). The major lipophilic components of the epithelial cell glycolipids were phytosphingosine and long-chain hydroxy fatty acids.     

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Characterization of the binding of propionibacterium granulosum to glycosphingolipids adsorbed on surfaces. An apparent recognition of lactose which is dependent on the ceramide structure

The binding properties of a strain of Propionibacterium granulosum derived from human skin was investigated with reference to glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells using externally (125I) and metabolically [( 35S]methionine) labeled bacteria. Binding was found to lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-Cer) but not to glycolipids lacking the lactose sequence (i.e. Glc beta 1-Cer, Gal beta 1-Cer or Gal alpha 1-4Gal beta 1-Cer). In microtiter wells, binding occurred at 50 ng and became half-maximal at the theoretical value for a monomolecular layer of LacCer (i.e. 100-200 ng/well). The bacteria also bound to glycolipids with various substitutions (e.g. GalNAc beta 1-4, Gal beta 1-3GalNAc beta 1-4, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4, Gal alpha 1-3, GlcNAc beta 1-3, Gal beta 1-3GlcNAc beta 1-3, Gal beta 1-4GlcNAc beta 1-3, and Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3) at the Gal of LacCer, although only those species with GalNAc beta 1-4 or Gal beta 1-3GalNAc beta 1-4 were as active as LacCer itself. Glycolipids with other additions (e.g. Gal alpha 1-4 and NeuAc alpha 2-3) were negative. For unsubstituted LacCer, the binding required either a trihydroxy base or 2-hydroxy fatty acid, specifying the epithelial type of ceramide; LacCer composed of a dihydroxy base and nonhydroxy fatty acid was negative. This is interpreted as indicating that the proper presentation of the binding epitope depends on the ceramide structure. The relevance of this to biological membranes has not yet been established. Neither free lactose (up to 20 mg/ml) nor lactose-bovine serum albumin (5 mg/ml) prevented the binding of bacteria to LacCer, two facts that support the solid-phase binding data demonstrating a low affinity binding and the crucial importance of a particular lactose epitope.     

Animal glycolipids as attachment sites for microbes

The abundance of carbohydrate at the animal cell surface may explain why microbes have selected primarily carbohydrates as essential attachment sites for colonization or infection. Of the various surface glycoconjugates of interest, primary attention has been given to glycolipids, due in part to an efficient binding assay based on a thin-layer chromatogram with separated glycolipids. In this way the general character of carbohydrate recognition by microbes is being mapped. Mainly two examples are briefly described to illustrate some generalizations: lactosylceramide-recognition by several bacteria, and Gal alpha l----4Gal-binding by Escherichia coli and the Shiga toxin. The unique recognition of internally placed sequences, the often low-affinity binding, and the preference of certain sequences before others are interpreted to be of decisive biological value. The binding to internal parts makes it technically possible to approximate the binding epitope on a receptor glycolipid. For this the binding preferences to glycolipids carrying the binding site in different saccharide environments (isoreceptors) are compared with the computer-calculated preferred conformations (definition of steric hindrances to epitope access). Several binding epitopes dissected with this approach have a common surface character: a nonpolar area of ring hydrogens over one or two sugars, surrounded by polar oxygens or amide. This is in agreement with the recent Lemieux concept for antibody-carbohydrate interaction. This information facilitates a rational synthesis of receptor analogues for potential applications. An outline is finally given of an improved general approach for receptor analysis.     

Glycolipid binding epitopes involved in adherence of the periodontitis-associated bacterium Porphyromonas gingivalis

The ability of the periodontal pathogen Porphyromonas gingivalis to use different glycolipid structures as receptors has previously been demonstrated. The bacterium adhered to acid and nonacid glycolipids originating from human organs and to nonacid glycolipids of porcine origin. The aim of the present study was to analyze these binding epitopes by structural characterization. Glycolipid fractions with positive bacterial binding from e.g. human and porcine origin, were purified by the high performance liquid chromatography technique and thereafter used in bacterial overlay assays with (35)S-labeled P. gingivalis. Purified fractions with positive binding were structurally characterized by proton nuclear magnetic resonance spectroscopy. Complementing thin-layer chromatograms and bacterial overlay assays with pure reference glycolipid fractions and competition experiments with lactose were performed to define potential receptors. The P. gingivalis binding epitopes, including cerebrosides with nonhydroxy fatty acids, lactosylceramide with hydroxy fatty acids, sulfatides, lacto-, neolacto- and gangliotetraosylceramides, are in several instances similar to those found for other bacteria, e.g. H. pylori, H. influenzae and N. meningitidis. In addition P. gingivalis also bound to the Galalpha4Gal epitope of the globo series of glycolipids. In the future these results may be valuable for development of new treatment strategies, such as anti-adhesion therapies and vaccines specifically directed against P. gingivalis infection.     

Studies on the binding of bacteria to glycolipids. Two species of Propionibacterium apparently recognize separate epitopes on lactose of lactosylceramide

Two species of Propionibacterium were analysed regarding their binding to glycosphingolipids. Bacteria were labeled with 125I and selective interaction with glycolipids on thin-layer chromatograms was revealed by autoradiography. The carbohydrate site in common for active molecular species appeared to be lactose. The two bacteria differed, however, in the overall binding pattern on the chromatogram, probably due to recognition of separate epitopes on lactose. P. freudenreichii bound only to lactosylceramide while P. granulosum also recognized substituted lactosylceramide: Gal alpha 1----3Gal beta 1----4Glc beta Cer, GlcNAc beta 1----3Gal beta 1----4Glc beta Cer and Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta Cer were active, but Gal-alpha 1----4Gal beta 1----4Glc beta Cer was inactive. Also, there was an interesting dependence on ceramide structure in the case of lactosylceramide. P. freudenreichii bound to lactosylceramide with sphingosine and non-hydroxy fatty acids but not to species with sphingosine and 2-hydroxy fatty acids, phytosphingosine and non-hydroxy fatty acids or phytosphingosine and 2-hydroxy fatty acids. For P. granulosum the situation was reversed. This may be explained by an influence of ceramide structure on the presentation of the two lactose epitopes at the assay surface. These results were supported by curves from the binding of labeled bacteria to glycolipids coated in microtiter wells and in part by binding to glycolipid-coated chicken erythrocytes.     

Role of intestinal surfactant-like particles as a potential reservoir of uropathogenic Escherichia coli

The binding of uropathogenic Escherichia coli is mediated at the tips of pili by the PapG adhesin, which recognizes the Galalpha(1-4)Gal disaccharide on the uroepithelial surface. These receptors have been identified unequivocally in the human and murine urinary tracts but not in intestinal epithelium, yet uropathogenic E. coli strains are commonly found in normal colonic microflora. The gastrointestinal tract from duodenum to rectum elaborates a phospholipid-rich membrane particle with surfactant-like properties. In these studies, we report that purified murine particles contain a receptor recognized by the class I PapG adhesin because: (1) PapD-PapG complexes and class I pili bound to surfactant-like particles in a solid-phase assay, whereas binding was not detected in microvillous membranes derived from the same tissues, (2) purified PapD-PapG complex bound to a glycolipid receptor detectable in lipid extracts from the particles, and (3) soluble Galalpha(1-4)Gal inhibited the adhesin by 72% from binding to surfactant-like particles. The Galalpha(1-4)Gal receptor present in the intestinal surfactant-like particle which overlies the intestinal mucosa could provide one means to establish an intestinal habitat for uropathogenic E. coli.     

Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal alpha 1-4Gal-containing isoreceptors.

Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells. PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1-4Gal-containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding. Their hemagglutination patterns confirmed the existence of three receptor-binding specificities. While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG. These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes. The high prevalence of PrsG-like specificities observed among wild-type dog uropathogenic E. coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors. The receptor-binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E. coli isolates varied considerably among the three receptor-binding groups of adhesins, but only little within each group.     

Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1

The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.     

Carbohydrates act as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis: a study of bacterial binding to glycolipids

In this study we show for the first time the use of carbohydrate chains on glycolipids as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis. Previous studies have shown that this bacterium has the ability to adhere to and invade the epithelial lining of the dental pocket. Which receptor(s) the adhesin of P. gingivalis exploit in the adhesion to epithelial cells has not been shown. Therefore, the binding preferences of this specific bacterium to structures of carbohydrate origin from more than 120 different acid and nonacid glycolipid fractions were studied. The bacteria were labeled externally with (35)S and used in a chromatogram binding assay. To enable detection of carbohydrate receptor structures for P. gingivalis, the bacterium was exposed to a large number of purified total glycolipid fractions from a variety of organs from different species and different histo-blood groups. P. gingivalis showed a preference for fractions of human and pig origin for adhesion. Both nonacid and acid glycolipids were used by the bacterium, and a preference for shorter sugar chains was noticed. Bacterial binding to human acid glycolipid fractions was mainly obtained in the region of the chromatograms where sulfated carbohydrate chains usually are found. However, the binding pattern to nonacid glycolipid fractions suggests a core chain of lactose bound to the ceramide part as a tentative receptor structure. The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the dental pocket epithelium, subsequently leading to damage to periodontal tissue and tooth loss.     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2.

The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis.

A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1---- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc-containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1----2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc-containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed.     

Globo-A--a new receptor specificity for attaching Escherichia coli

Uropathogenic Escherichia coli strains designated as ONAP, based on their O negative A positive agglutination of human P1 erythrocytes, were shown to prefer the globo-A glycolipid as a receptor structure. The dependence on both the A terminal and the globoseries chain was confirmed by agglutination of human AP1, but not Ap or OP1 erythrocytes and by binding to the globo-A glycolipid on TLC plates. Neither Gal alpha 1----4Gal beta nor the A trisaccharide GalNAc alpha 1----3(Fuc alpha 1----2)Gal beta alone functioned as receptors. The bacteria thus appeared to recognize an epitope resulting from the combination of the terminal and internal structures.