Isolation and characterization of Clostridium perfringens mutants altered in both hemagglutinin and sialidase production.
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The relationship between the production of hemagglutinin and sialidase activities by Clostridium perfringens was investigated by screening for mutants producing reduced levels of hemagglutinin activity. Twelve mutants were isolated; all produced reduced levels of sialidase activity and several had other altered phenotypic markers. Revertants that regained the ability to produce active hemagglutinin were isolated. All of these revertants produced increased sialidase activity. These results show that the production of hemagglutinin activity is directly related to the production of sialidase activity. Evidence is also presented that the processes of sporulation and the production of extracellular proteins are interrelated. (+info)
N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.
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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. (+info)
The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O-linked mucin carbohydrate containing N-glycolylneuraminic acid.
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The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins. (+info)
Binding properties of Clostridium botulinum type C progenitor toxin to mucins.
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It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures. (+info)
In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria.
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Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does. (+info)
Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation.
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Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells. (+info)
N-linked glycopeptides with blood group determinants lacking neuraminic acid from the epithelial cells of rat small and large intestine.
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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. (+info)
Synthesis of nor-C-linked neuraminic acid disaccharide: a versatile precursor of C-analogs of oligosialic acids and gangliosides.
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The Neu5Acalpha(2,8)Neu5Ac disaccharide is an important constituent of tumor related antigen, however, the O-linkage is catabolically unstable. Vaccination with a catabolically stable sialic acid C-glycoside analog might enhance immunogenicity. The synthesis of Neu5Ac nor-C-disaccharide 20R/S, corresponding to versatile precursors of C-analogs of oligosialic acid and gangliosides, is reported. The synthesis of the protected acceptor was not straightforward, as ester, silyl ether, and isopropylidene protection failed to afford desired C-linked disaccharide. Allyl ether protection of hydroxyl groups and acetyl protection of the acetamido facilitated the successful synthesis of the 8-aldehyde neuraminyl acceptor. Samarium mediated C-glycosylation afforded the desired nor-C-disaccharide as a mixture of two separable diastereomers. (+info)