ABSTRACT
Mucins are high molecular mass glycoproteins with oligosaccharides O-bonded to the protein core. beta-elimination is the most popular method used for releasing of O-glycans. However to such released glycoforms it is difficult to introduce a label to amplify a signal for oligosaccharide detection.In our study we used a combination of the beta-elimination and hydrazinolysis methods. Released glycoforms were labeled with para-amino benzooic acid ethyl ester (ABBE) and fractionated on HPLC column.This combined procedure seems to be a good tool for O-glycans analysis.
ABSTRACT
It has been suggested that Helicobacter pylori can interact via carbohydrate structures with gastric mucins. Particularly, the Lewis b structures of the secretory MUC 5AC mucin are considered to be putative receptors for bacterial adhesins. Also the epithelial MUC 1 mucin is implicated by some authors to have a major role in the mechanism of infection. The main objective of our study was to evaluate MUC 1 mucin levels in human gastric juice before and at the end of eradication therapy. Any possible changes could suggest the participation of MUC 1 in H. pylori infection. We assume that the amount of the soluble form of MUC 1 exfoliated to the juice correlates with MUC 1 expressed on epithelial cells. Gastric juice samples of 14 duodenal ulcer patients infected with H. pylori were assayed before and at the end of eradication. In all samples, DNA content was determined. Mucin fractions were isolated by gel exclusion chromatography. High molecular mass material containing MUC 1 was subjected to 4%-12% polyacrylamide gradient gels, electrotransfer to Immobilon P and immunodetection. In 12 infected patients (86%), the initial low level of MUC 1 mucin in gastric juice increased at the end of eradication. In comparison to the infected patients, neutral carbohydrate and DNA content in gastric juice diminished after treatment. Our results indicate that the bacterium affects the soluble form of MUC 1 mucin, thus suggesting a likely role of this mucin in the course of H. pylori infection.
Subject(s)
Antigens, Neoplasm/metabolism , Duodenal Ulcer/drug therapy , Duodenal Ulcer/microbiology , Gastric Juice/metabolism , Helicobacter Infections/metabolism , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Mucins/metabolism , Adult , Antigens, Neoplasm/genetics , Chromatography, Gel , DNA/genetics , Duodenal Ulcer/metabolism , Duodenal Ulcer/pathology , Female , Helicobacter Infections/drug therapy , Helicobacter Infections/pathology , Helicobacter pylori/isolation & purification , Humans , Male , Middle Aged , Mucin-1 , Mucins/geneticsABSTRACT
Mucin was purified by the gel filtration method on columns with high porous molecular sives in buffers with SDS and proteinase inhibitors. The addition of proteinase inhibitors distinctly inhibited proteolytic activity. It was found that the obtained mucin, after disulphide-bound reduction, is dissociated to mucin subunits and N-glycosylated glycoprotein of molecular weight about 75 kDa. This protein has carbohydrate and amino acid composition different from high molecular fraction. The 75 kDa protein is strongly associated with high molecular mass mucin subunits and can be separated either during electrophoresis or fractionation in buffers with 2-mercaptoethanol.
Subject(s)
Gastric Mucins/antagonists & inhibitors , Protease Inhibitors/pharmacology , Acrylic Resins , Amino Acids/analysis , Animals , Buffers , Carbohydrates/analysis , Chemical Fractionation , Chromatography , Chromatography, Gel , Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel , Gastric Mucins/analysis , Gastric Mucins/isolation & purification , Gastric Mucins/pharmacology , Glycoproteins/analysis , Mercaptoethanol/chemistry , Molecular Weight , Oxidation-Reduction , Porosity , Sodium Dodecyl Sulfate/chemistry , Surface-Active Agents/chemistry , SwineABSTRACT
Lectin isolated from wheat germ was purified by affinity chromatography on ovogel column, recrystallization and gel filtration on Ultrogel AcA 44 column, eluted with borate buffer to eliminate accidental lectin-ligand interaction products. In 87-times purified WGA preparation fucose, xylose, mannose, galactose and glucose were found, amounting 1.5% of lectin mass.
