| 1. |
- Heiskanen, Annamari, et al.
(author)
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N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.
- 2007
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In: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 25:1, s. 197-202
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Journal article (peer-reviewed)abstract
- Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
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| 2. |
- Johansson, Petra, 1974, et al.
(author)
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Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc.
- 2005
-
In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 15:6, s. 625-36
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Journal article (peer-reviewed)abstract
- We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.
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| 3. |
- Karlsson, Anna, 1967, et al.
(author)
-
Different glycosphingolipid composition in human neutrophil subcellular compartments.
- 2001
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In: Glycoconjugate journal. - 0282-0080. ; 18:3, s. 231-43
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Journal article (peer-reviewed)abstract
- The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay. Several organelle-related differences in glycosphingolipid content were observed. The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils. Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules. Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions. A non-acid triglycosylceramide, recognized by Gal(alpha)4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules. Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane. Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.
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| 4. |
- Karlsson, Karl-Anders, 1935, et al.
(author)
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Unexpected carbohydrate cross-binding by Escherichia coli heat-labile enterotoxin. Recognition of human and rabbit target cell glycoconjugates in comparison with cholera toxin.
- 1996
-
In: Bioorganic & medicinal chemistry. - 0968-0896. ; 4:11, s. 1919-28
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Journal article (peer-reviewed)abstract
- The bacterial protein enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile toxin (LT) of Escherichia coli, induce diarrhea by enhancing the secretory activity of the small intestine of man and rabbit (animal model). This physiological effect is mediated by toxin binding to a glycolipid receptor, the ganglioside GM1, Gal beta 3GalNAc beta 4(NeuAc alpha 3)GAl beta 4Glc beta 1Cer. However, LT, but not CT, was recently shown by us to bind also to paragloboside, Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, identified in the target cells. By molecular modeling of this tetrasaccharide in the known binding site of LT, the saccharide-peptide interaction was shown to be limited to the terminal disaccharide (N-acetyllactosamine). This sequence is expressed in many glycoconjugates, and we have therefore assayed glycolipids and glycoproteins prepared from the target tissues. In addition to paragloboside, receptor activity for LT was detected in glycoproteins of human origin and in polyglycosylceramides of rabbit. However, CT bound only to GM1. Two variants of LT with slightly different sequences, human (hLT) and porcine (pLT), were identical in their binding to target glycoproteins and polyglycosylceramides, but different regarding paragloboside, which was positive for pLT but negative for hLT. This difference is discussed on basis of modeling, taking in view the difference at position 13, with Arg in pLT and His in hLT. Although N-acetyllactosamine is differently recognized in form of paragloboside by the two toxin variants, we speculate that this sequence in human glycoproteins and rabbit polyglycosylceramides is the basis for the common binding. Much work remains, however, to clear up up this unexpected sophistication in target recognition.
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| 5. |
- Leonardsson, Irene, 1953, et al.
(author)
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Isolation and partial characterization of Gal alpha-containing polyglycosylceramides from porcine tissues.
- 2004
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In: Xenotransplantation. - : Wiley. - 0908-665X .- 1399-3089. ; 11:1, s. 97-100
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Journal article (peer-reviewed)abstract
- Mammalian cell surface carbohydrate antigens are present both as glycoproteins and glycolipids. Of the glycolipids, polyglycosylceramides (PGC) have very long carbohydrate chains extending out from the cell surface. Hereto, Gal alpha-terminating xenoantigens in pig tissues have been identified in glycoproteins and short chain glycolipids but no studies of the complex PGC have been performed. In this communication, we describe the isolation and partial characterization of PGC from pig erythrocytes, small intestinal mucosa, kidney and liver. The mucosa, kidney and liver PGC fractions contained a complex pattern of Gal alpha antigens as shown by immunostaining using the Griffonia Simplicifolia isolectin B(4) while no reactivity was found with the erythrocyte PGC fractions. The mucosa PGC fractions stained strongly for blood group A antigens while the erythrocyte PGC fractions were negative. The presence of Gal alpha-terminating PGC compounds in porcine tissue adds further complexity to the distribution of this xenoantigen. Due to the long carbohydrate chains, PGC will be important targets for the Gal alpha xenoantibodies in pig to human xenotransplantation.
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| 6. |
- Miller-Podraza, Halina, 1948, et al.
(author)
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Helicobacter pylori and neutrophils: sialic acid-dependent binding to various isolated glycoconjugates.
- 1999
-
In: Infection and immunity. - 0019-9567. ; 67:12, s. 6309-13
-
Journal article (peer-reviewed)abstract
- Helicobacter pylori has been shown to agglutinate erythrocytes in a sialic acid-dependent manner. However, very few studies have examined relevant target cells in the human stomach. Neutrophils are required for the onset of gastritis, and the inflammatory reaction may be induced on contact between bacteria and neutrophils. In the present work, glycolipids and glycoproteins were isolated from neutrophils and were studied for binding by overlay with radiolabeled bacteria on thin-layer chromatograms and on membrane blots. There was a complex pattern of binding bands. The only practical binding activity found was sialic acid dependent, since treatment of glycoconjugates with neuraminidase or mild periodate eliminated binding. As shown before for binding to erythrocytes and other glycoconjugates, bacterial cells grown on agar bound to many glycoconjugates, while growth in broth resulted in bacteria that would bind only to polyglycosylceramides, which are highly heterogeneous and branched poly-N-acetyllactosamine-containing glycolipids. Approximately seven positive bands were found for glycoproteins, and the traditional ganglioside fraction showed a complex, slow-moving interval with very strong sialic-acid-dependent binding, probably explained by Fuc substitutions on GlcNAc.
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| 7. |
- Miller-Podraza, Halina, 1948, et al.
(author)
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HELICOBACTER PYLORI BINDING TO NEW GLYCANS BASED ON N-ACETYLLACTOSAMINE.
- 2009
-
In: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19:4, s. 399-407
-
Journal article (peer-reviewed)abstract
- Previously we reported binding of Helicobacter pylori to various non-acid and sialylated neolacto carbohydrate structures using a wide range of natural and chemically modified sequences. A novel non-sialylated neolacto-based binding epitope, GlcNAcbeta3Galbeta4GlcNAc, and analogous structures carrying terminal GalNAcbeta3, GalNAcalpha3 or Galalpha3 showed the binding activity (Miller-Podraza, H. et.al. 2005. J. Biol. Chem. 280, 19695-19703). The present work reports two other H. pylori-binding non-sialylated neolacto-based structures, GlcAbeta3Galbeta4GlcNAcbeta3-R and Glcbeta3Galbeta4GlcNAcbeta3-R, and two amide derivatives (N-methyl and N-ethyl) of GlcAbeta3Galbeta4GlcNAcbeta3-R which were bound by H. pylori. The latter structures turned out to be more effective as H. pylori binders than the parent saccharide. New reducing end variants of the neolacto epitope including species containing N-acetyllactosamine linked beta6 to GlcNAc or Gal with similarity to branched polylactosamines and mucins were prepared and tested. The results extend our previous findings on binding specificities of H. pylori and show that this pathogen is able to interact with an array of lactosamine/neolacto structures, which may be of importance for the in vivo interaction of the bacterium with human cells. The information gained in this work may also be of value for rational design of anti-H. pylori drugs.
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| 8. |
- Miller-Podraza, Halina, 1948, et al.
(author)
-
Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains: structure and cross-binding properties.
- 2005
-
In: The Journal of biological chemistry. - 0021-9258. ; 280:20, s. 19695-703
-
Journal article (peer-reviewed)abstract
- Helicobacter pylori is a bacterium that colonizes the stomach of a majority of the global human population causing common gastric diseases like ulcers and cancer. It has an unusually complex pattern of binding to various host glycoconjugates including interaction with sialylated, sulfated, and fucosylated sequences. The present study describes an additional binding epitope comprising the neolacto internal sequence of GlcNAcbeta3-Galbeta4GlcNAcbeta. The binding was detected on TLC plates as an interaction with a seven-sugar ganglioside of rabbit thymus. The glycolipid was purified and characterized as Neu5Gcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3-Galbeta4Glcbeta1Cer with less than 10% of the fraction carrying a repeated lacto (type-1) core chain, Galbeta3Glc-NAcbeta3Galbeta3GlcNAcbeta. After stepwise chemical and enzymatic degradation and structural analysis of products the strongest binder was found to be the pentaglycosylceramide GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer, whereas the hexa- and tetraglycosylceramides were less active, and the trihexosylceramide was inactive. Further studies revealed that the terminal GlcNAcbeta of the pentaglycosylceramide may be exchanged for either GalNAcbeta3, GalNAcalpha3, or Galalpha3 without loss of the activity. Calculated minimum energy conformers of these four isoreceptors show a substantial topographical similarity suggesting that this binding is a result of a molecular mimicry. Although the glycoconjugate composition of human gastric epithelial cells is not known in detail it is proposed that repeating N-acetyllactosamine units of glycoconjugates may serve as bacterial attachment sites in the stomach.
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| 9. |
- Miller-Podraza, Halina, 1948, et al.
(author)
-
Recognition of glycoconjugates by Helicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides, a second sialic acid-based specificity.
- 1996
-
In: Glycoconjugate journal. - 0282-0080. ; 13:3, s. 453-60
-
Journal article (peer-reviewed)abstract
- Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way. The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins. The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12. Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids. Bacteria grown in F12 medium were metabolically labelled with 35S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus. There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides. In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level. The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis. Erythrocyte proteins as well as a range of reference glycoproteins did not bind except band 3, which was weakly active. However, this activity was resistant to periodate oxidation. These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity.
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| 10. |
- Miller-Podraza, Halina, 1948, et al.
(author)
-
Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures.
- 2004
-
In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 14:3, s. 205-17
-
Journal article (peer-reviewed)abstract
- Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.
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