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_α proteins and/or mediating the exocytosis of the secretory granules.

Keywords: NUCB2; SEREX; chief cells; gastric cancer; parietal cells; pepsinogen secretion.; tumour-associated antigens.

Figure 1

The schematic representation of NUCB2 exon composition and the functional domains of the protein. Sp, signal peptide; Leu/Ile, Leu and Ile rich region; B, basic amino acid rich region; NLS, putative bipartite nuclear localisation signal; EF1, EF2, Ca²⁺ binding EF-hand domains; A, acidic amino acid rich region; L, leucine zipper; Hy, hydrophobic region.

Figure 2

The real-time RT-PCR analysis of NUCB2 mRNA expression in (A) a panel of normal tissue RNAs and (B) paired gastric cancer (T, only selected cases shown) and adjacent relatively normal tissues (N). BM, bone marrow.

Figure 3

The identification of novel NUCB2 transcript variants by 5′RLM-RACE. (A) The schematic representation of exon-intron composition at the 5′ end of NUCB2 gene (NC_000011.8), NUCB2 mRNA reference sequence (NM_005013.2) and newly identified transcript variants (b-c, GenBank accession nos. EU039831-EU039833). The proximal promoter is located upstream of exon 1, the newly identified distal promoter is located upstream of exon 1a. The horizontal arrows indicate the sites of primers used in the expression analysis. The analysis of promoter usage in various normal tissues (B) and paired gastric cancer and adjacent normal tissues (C) by RT-PCR. NUCB2-a represents proximal promoter found in the reference sequence, NUCB2-b – alternative distal promoter. The upper band detected in testis, ovary and Ga39T tissues represents the transcript variant containing exon 2a. 27 and 35 PCR cycles were preformed for amplification of transcript variants a and b, respectively. GAPDH was amplified as the internal control using 25 cycles of amplification.

Figure 4

Western blot analyses of NUCB2 expression. (A) NUCB2 was downregulated in the majority of gastric cancer specimens in comparison with adjacent normal tissue and the protein levels correlated with the mRNA levels in all cases. Two to 4 closely spaced bands that migrate in the range between 50 and 57 kDa were detected in gastric tissue specimens (molecular weight of the mature NUCB2 is ∼50 kDa). (B) The treatment of protein extracts with shrimp alkaline phosphatase (SAP) revealed that 53 and 52 kDa bands in normal tissues and 52 and 57 kDa bands in tumour tissue represented phosphorylated forms of NUCB2. 55 and 57 kDa bands were present only in the tumour tissues, including Ga3T – tumour tissue from the patient with anti-NUCB2 autoantibodies and AGS gastric cancer cells.

Figure 5

The immunohistochemical analysis of NUCB2 expression in stomach epithelium and gastric lesions. Normal stomach: cardiac glands (A), oxyntic glands (B) and pyloric glands (C). Chronic atrophic gastritis: mild (D), moderate (E) and severe (F). Gastric adenocarcinoma: grade I (G), grade II (H) and grade III (I). Anti-NUCB2 monoclonal antibody was used to stain the tissue sections. NUCB2-speciffic signal was observed in gastric parietal cells (black arrows), chief cells (blue arrows) and plasma cells (red arrows) infiltrated in the gastritis and gastric cancer tissues while mucous cells do not express NUCB2. No NUCB2 positive tumour cells were detected.