To obtain a tool to study the structural characterization and the detection of mucin derived from the gastric gland mucous cells, we developed a monoclonal antibody, designated HIK1083, against mucin purified from rat gastric mucosa. In an ELISA, HIK1083 reacted strongly with the mucin purified from a deep layer of the corpus and antrum but only slightly reacted with that obtained from the surface mucosal layer. The reaction of mucin and HIK1083 was inhibited by the oligosaccharides obtained by the alkaline borohydride reduction of antigenic mucin. Two purified oligosaccharide alditols reacting with the monoclonal antibody obtained from the antigenic mucin had one and two peripheral alpha-linked N-acetylglucosamine residues, respectively, according to the evidence from NMR spectroscopy. Moreover, among the commercially available p-nitrophenyl derivatives of monosaccharides, only p-nitrophenyl-N-acetyl-alpha-D-glucosaminide inhibited the reaction of this monoclonal antibody and the antigenic mucin in a concentration-dependent manner. These results, as well as the immunohistochemical observations, indicate that alpha-linked N-acetylglucosamine residues are specifically attached to the peripheral region of the carbohydrate moiety of the mucin synthesized in and secreted from the gastric-gland-type cells, and indicate that the monoclonal antibody HIK 1083 recognizes this structure.
Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt -/-mice to better understand its role in vivo. A4gnt -/-mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt -/-mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.
Eight monoclonal antibodies (MAbs), designated RGM21 approximately RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.
Mucin, a major component of mucus, is a highly O‐glycosylated, high‐molecular‐mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site‐specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide‐alditols obtained from pig gastric mucin were fractionated by successive gel‐filtration, ion‐exchange, and normal‐phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide‐alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Fucα1–2Galβ1–3)GalNAc‐ol and Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Galβ1–3)GalNAc‐ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucα1–2Galβ1–4GlcNAc(6SO3H)β‐ sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.
Famotidine-induced suppression of gastric surface mucus cell function is prevented by combined treatment with MMSC, raising the possibility of a more effective cure of PUD.
Glycoproteins were isolated from human gastric mucosa, and their reactivities with concanavalin A, periodate oxidation and subsequent reduction, are described. Gastric glycoproteins corresponding to the paradoxical concanavalin A staining-class II and III mucins were proved biochemically. The analytical results suggest that N-acetylglucosamine residues in the glycoproteins mediate the interaction with concanavalin A.
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