Identification and Characterization of the Immunodominant Proteins of Helicobacter pylori Related to Gastric Cancer and Duodenal Ulcer

• Main Authors: Yu-Fen Lin, 林玉芬
• Other Authors: 周綠蘋
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/00511042060087944922

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 95 === Helicobacter pylori is a major cause of chronic active gastritis, duodenal ulcer, gastric ulcer and is strongly related to gastric cancer. Chronic inflammation induced by colonization of H. pylori is thought to contribute to pathogenesis in the gastroduodenal tract. Although many virulence factors of H. pylori have been reported, the association between the virulence factors and variant gastric diseases are unclear and not universal. As the distinct distribution and pattern of gastritis occurred, the developed duodenal ulcer and gastric cancer are considered as clinically divergent events, with much lower risk for progressing to gastric cancer among patients with duodenal ulcer than with gastric ulcer or other precancerous diseases such as atrophic gastritis and intestinal metaplasia. Owing to clinical differences between gastric cancer and duodenal ulcer, we compared 2D-immunoblot profiles of acid-glycine extracts from H. pylori by probing with sera of patients with gastric cancer (n = 15) and duodenal ulcer (n = 15). In the first part of studies, we aimed to search for immunogenic proteins related to gastric cancer and therefore extracted the proteins of the H. pylori strain isolated from a gastric cancer patient. In total, 24 protein spots were identified as antigens with better recognition in gastric cancer sera than duodenal ulcer sera. The proteins showing higher frequency of recognition in gastric cancer group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase a subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR and membrane fusion protein. Of these proteins, GroES was identified as a dominant gastric cancer-related antigen with a much higher seropositivity of gastric cancer samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for duodenal ulcer (n = 124). GroES seropositivity was more commonly associated with antral gastric cancer than the non-antral gastric cancer (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). For functional analysis of GroES, we monitored the expression levels of pro-inflammatory cytokines, and marker proteins related to cell proliferation by using reverse transcriptase-polymerase chain reaction (RT-PCR), an enzyme-linked immunosorbent assay (ELISA), Western blotting and a cell viability assay. In peripheral blood mononuclear cells, GroES induced expression of ilterleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1beta, tumor necrosis factor (TNF)-alpha, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). In gastric epithelial cells (KATO-III cells), GroES up-regulated the expression of IL-8, c-jun, c-fos, mutated p53, cyclin D1, p21WAF1/Cip1 and survivin, but down-regulated the p27Kip1 level. Therefore we concluded that GroES is a novel gastric cancer-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation. In the second part of the studies, we aimed to identify H. pylori antigens showing a high seropositivity in DU and to develop a platform for rapid and easy diagnosis for DU. Since DU and gastric cancer (GC) are considered clinical divergent gastroduodenal diseases, we compared two-dimensional immunoblots of an acid-glycine extract of an H. pylori strain from a patient with DU probed with serum samples from patients with DU (n = 10) or GC (n = 10), to identify DU-related antigens. Of the 11 proteins that were strongly recognized by serum IgG from DU patients, translation elongation factor EF-G (FusA), catalase (KatA), and urease alpha subunit (UreA) were identified as DU-related antigens, showing higher seropositivity in DU samples (n = 124) than in GC samples (n = 95) (FusA: 70.2% vs. 45.3%, KatA: 50.8% vs. 41.1%, UreA: 44.4% vs 27.4%). In addition, we found that the use of multiple antigens improved the discrimination between patients with DU and those with GC, as the odds ratios increased from 1.82 (95% CI: 0.79-4.21, P = 0.1607) for seropositivity for FusA, KatA, or UreA alone to 4.95 (95% CI: 2.05-12.0, P = 0.0004) for two of the three antigens, and to 5.71 (95% CI: 1.86-17.6, P = 0.0024) for all three antigens. Moreover, a protein chip containing the 3 DU-related antigens was developed to test the idea of using multiple biomarkers in diagnosis. We conclude that FusA, KatA, and UreA are DU-related antigens of H. pylori and the combination of these proteins on a protein array provided is a rapid and convenient method for detecting serum antibody patterns of DU patients.