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Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

ABSTRACT: NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric G? proteins and/or mediating the exocytosis of the secretory granules.

SUBMITTER: Kalnina Z

PROVIDER: S-EPMC3167273 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

Kalnina Z Z Silina K K Bruvere R R Gabruseva N N Stengrevics A A Barnikol-Watanabe S S Leja M M Line A A

European journal of histochemistry : EJH 20090331 1

NUCB2 is an EF-hand Ca<sup>2+</sup> binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the ...[more]

PMID: 30256860

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Project description:The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.

Dataset Information

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

Publications

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

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