Nucleolin as cell surface receptor for tumor necrosis factor-alpha inducing protein: a carcinogenic factor of Helicobacter pylori

Tumor necrosis factor-alpha inducing protein (Tip alpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tip alpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappa B activation, resulting in tumor development. To elucidate mechanism of action of Tip alpha, we studied a binding protein of Tip alpha in gastric epithelial cells. Tip alpha binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tip alpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. Nucleolin was co-precipitated with Tip alpha-FLAG, but not with del-Tip alpha-FLAG (an inactive mutant). After treatment with Tip alpha-FLAG, incorporated Tip alpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tip alpha to recombinant His-tagged nucleolin fragment (284-710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tip alpha-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tip alpha and expression of TNF-alpha gene. All the results indicate that nucleolin acts as a receptor for Tip alpha and shuttles Tip alpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tip alpha.