Helicobacter pylori East Asian type CagA hijacks more SHIP2 by its EPIYA-D motif to potentiate the oncogenicity

Figures & data

Figure 1. Pathogenic effect of H. pylori CagA^E and CagA^W mediated by SHIP2 in AGS cells.

Note: (a) Intracellular p-CagA was quantified by western blot to indicate the consistent amounts of CagA injected into cells under different treatment. The ratio of the amount of p-CagA to β-actin from three independent repeats was calculated to compare CagA injected in the cells. (b) IL-8 secretion from H. pylori infected cells measured by ELISA. Migration (c) and invasion (d) ability of infected cells were examined by transwell assay. Left, representative images; Right, statistical data. sh-scramble in the figure refers to control sh-RNA, sh-SHIP2 refers to sh-RNA targeting SHIP2, CagA^E and CagA^W refer to H. pylori G27 mutants expressing CagA^E and CagA^W, respectively. ΔCagA indicates H. pylori CagA deleted strain. Error bars represent means ± SD, ns indicates no statistical significance, *** indicates p < 0.001. Scale bars in images represent 200 µm.

Figure 2. The effect of H. pylori infection on the sub-localization of SHIP2, production of PI(3,4)P₂ and activation of Akt signaling.

Note: (a) Immunofluorescence staining indicating the location of CagA and SHIP2 in H. pylori infected cells. (b) Immunofluorescence staining of PI(3,4)P₂. (c) Western blotting detection of phosphorylation levels of Akt. Bars in images represent 20 µm. Left, representative images; Right, statistical data. SHCH30 and 26695 represent wild strains expressing CagA^E and CagA^W. Error bars represent means ± SD, *p < 0.1, **p < 0.01, ***p < 0.001. The other notes represent the same meaning as above.

Figure 3. Detection of the interaction difference between CagA^E or CagA^W with SHIP2.

Note: (a) Co-immunoprecipitation to analyse the interaction between SHIP2 and CagA^E/CagA^W in H. pylori infected AGS and GES-1 cells. Antibody against SHIP2 was used as IP antibody to precipitate components containing SHIP2, antibody against p-CagA was used to indicate the amount of CagA interacted with SHIP2. The ratio of the amount of p-CagA to SHIP2 from three independent repeats was analysed to indicate the binding preference of SHIP2 to CagA^E and CagA^W. (b) SDS-PAGE analysis of the purified recombinant proteins. M lane, marker. (c) Pull-down assay demonstrating the binding of SHIP2-SH2 domain to H. pylori CagA^E and CagA^W. Cells infected with strains SHCH30 and 26695 were lysed to release intracellular p-CagA. When the lysate flow through the resin immobilizing GST-fused SHIP2-SH2 domain, p-CagA will be pulled down through its interaction with SHIP2. The difference in densitometric values of two CagA subtypes pulled down is analysed below. Error bars represent means ± SD, ***indicates p < 0.001. The other notes represent the same meaning as above.

Figure 4. Surface plasmon resonance (SPR) analysis of the interactions between SHIP2-SH2 domain with EPIpYA-D or EPIpYA-C peptides. Recombinant His tagged SHIP2-SH2 (Figure3(b)) was immobilized on a BIAcore CM5 sensor chip as the receptor. EPIpYA-C (a) or EPIpYA-D (b) at different concentrations flow through the chip. Sensorgrams were recorded by a BIAcore T100 instrument. For each peptide interaction, the response values at a fixed time point for different concentrations of peptides are marked and fitted into a stoichiometric reaction curve (c for EPIpYA-c and d for EPIpYA-D). The values of K_D were then calculated according to the curves with Biacore T100 Evaluation Software.

Figure 5. Comparative analysis of the interaction of SHIP2-SH2 domain with EPIpYA-C and EPIpYA-D peptides. (a) The binding energies of two peptides with SHIP2-SH2 domain were calculated with two algorithms, MMGBSA and MMPBSA. (b) Superimposition of SHIP2-SH2 domain interacting with EPIpYA-C and EPIpYA-D motifs. (c) Overall structure of the SHIP2-SH2-EPIpYA-C complex in two views. (d) Overall structure of the SHIP2-SH2-EPIpYA-D complex in two views. Cyan and light gray indicate EPIpYA-C and EPIpYA-D peptides respectively where their Tyr residues were both phosphorylated. Green and dark gray indicate SH2 domains interacting with EPIpYA-C and EPIpYA-D peptides respectively.

Figure 6. Key sites analysis in complexes of SHIP2-SH2-EPIpYA-C and SHIP2-SH2-EPIpYA-D. (a) Complex of SHIP2-SH2-EPIpYA-C. (b) Details of interaction surface of SHIP2-SH2-EPIpYA-C complex. (c) Complex of SHIP2-SH2-EPIpYA-D. (d) Details of interaction surface of SHIP2-SH2-EPIpYA-D. pY-115 indicates the phosphorylated tyrosine residue in synthesized peptides. Other residues on peptides are marked in black. The amino acids in green represent residues on SHIP2-SH2 domain those interact with EPIpYA-C motif, and in dark grey represent residues on SHIP2-SH2 domain those interact with EPIpYA-D peptide.

Figure 7. Confirmation of key role of Phe-120 in the interaction between EPIpYA-D and SHIP2-SH2. (a) Sequences of EPIpYA-D and its mutant peptides. (b–d) Stoichiometric reaction curves of SHIP2-SH2 interacting with EPIpYA-D or its mutant peptides from SPR analysis.

Figure 8. Verification of role of residue Phe in CagA^E-SHIP2 interaction by cell infection assays. (a) Immunoprecipitation analysis of CagA^E and its mutants (CagA^F/D and CagA^F/A) with SHIP2 in infected cells. Antibody against CagA was used as IP antibody, antibody against SHIP2 was used to reveal the amount of SHIP2 interacted with CagA. (b) Western blotting indicating activation of Akt pathway in infected cells. (c) Western blotting detecting the phosphorylation level of p-65 to indicate the activation of NF-κB pathway in infected cells. (d) Secretion of IL-8 by infected cells. Error bars represent means ± SD, **indicates p < 0.01, ***indicates p < 0.001. The other notes represent the same meaning as above.

Figure 9. Role of residue Phe in EPIYA-D motif in affecting the oncogenicity of CagA^E. SHP099 was used as an inhibitor to exclude the effect of interaction between SHP2 and CagA, as Phe in EPIYA-D motif was also reported to play roles in SHP2-CagA^E interaction to improve the cytotoxicity of CagA^E. (a) Examination of CagA induced specific hummingbird phenotype. Elongated cells with the longest axis exceeding the shortest axis by more than 2-fold were counted as the hummingbird phenotype. (b) Migration and (c) invasion of infected AGS and GES-1 cells examined by transwell assays. Bars in images represent 100 µm. Left, representative images; Right, statistical data. Error bars represent means ± SD, ns indicates no statistical significance, ***indicates p < 0.001.

Data Availability statement

The data generated during the study is available at figshare database at https://figshare.com doi.org/[10.6084/m9.figshare.25356829].