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Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa C - J. Exp. Med. (2000)

Bottom Line: Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen.These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm.The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Nursing and Welfare, Fukui Prefectural University, Fukui 910-1195, Japan. asahi@fpu.ac.jp

ABSTRACT
Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

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Demonstration of the tyrosine-phosphorylated 145-kD protein derived from H. pylori. AGS cells were infected with [35S]methionine-labeled or nonlabeled H. pylori for 5 h at 37°C, and the cell lysates prepared with 1% Triton X-100 were immunoprecipitated by an antiphosphotyrosine mAb PY-20 or an anti-CagA polyclonal antibody. The precipitated proteins were separated by SDS-PAGE and analyzed using a radioanalytic imaging system. Arrowhead indicates the 145-kD protein. Lanes 1–3, AGS infected with [35S]methionine-labeled H. pylori; lanes 4 and 5, [35S]methionine-labeled H. pylori alone; lanes 6–8, [35S]methionine-labeled AGS infected with nonlabeled H. pylori; lanes 1 and 6, the 1% Triton X-100–soluble fraction precipitated by control normal IgG; lanes 2, 4, and 7, the 1% Triton X-100–soluble fraction precipitated by antiphosphotyrosine mAb PY-20; and lanes 3, 5, and 8, the 1% Triton X-100–soluble fraction precipitated by anti-CagA polyclonal antibody. Note that the [35S]methionine-labeled band being lower than that of 145-kD protein in lanes 3 and 5 may be bacterial protein(s) cross-reacted with the polyclonal anti-CagA antibody.
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Figure 4: Demonstration of the tyrosine-phosphorylated 145-kD protein derived from H. pylori. AGS cells were infected with [35S]methionine-labeled or nonlabeled H. pylori for 5 h at 37°C, and the cell lysates prepared with 1% Triton X-100 were immunoprecipitated by an antiphosphotyrosine mAb PY-20 or an anti-CagA polyclonal antibody. The precipitated proteins were separated by SDS-PAGE and analyzed using a radioanalytic imaging system. Arrowhead indicates the 145-kD protein. Lanes 1–3, AGS infected with [35S]methionine-labeled H. pylori; lanes 4 and 5, [35S]methionine-labeled H. pylori alone; lanes 6–8, [35S]methionine-labeled AGS infected with nonlabeled H. pylori; lanes 1 and 6, the 1% Triton X-100–soluble fraction precipitated by control normal IgG; lanes 2, 4, and 7, the 1% Triton X-100–soluble fraction precipitated by antiphosphotyrosine mAb PY-20; and lanes 3, 5, and 8, the 1% Triton X-100–soluble fraction precipitated by anti-CagA polyclonal antibody. Note that the [35S]methionine-labeled band being lower than that of 145-kD protein in lanes 3 and 5 may be bacterial protein(s) cross-reacted with the polyclonal anti-CagA antibody.

Mentions: To directly demonstrate that the tyrosine-phosphorylated 145-kD protein was of H. pylori origin, H. pylori strain NCTC11637 and AGS cells were labeled with [35S]methionine separately, and then the nonlabeled epithelial cells were infected with the [35S]methionine-labeled bacteria for 5 h (the [35S]methionine-labeled epithelial cells were infected with nonlabeled bacteria for 5 h). The infected AGS cells were lysed with 1% Triton X-100, and the soluble fraction was immunoprecipitated with antiphosphotyrosine mAb PY-20. Analysis of the precipitates revealed a 145-kD protein labeled by [35S]methionine (Fig. 4, lane 2). Although the radioactivity of the tyrosine-phosphorylated protein was low, the labeled 145-kD protein was reproducibly detected in three independent experiments. Under the same conditions, the 145-kD protein was not detected in antiphosphotyrosine mAb PY-20 immunoprecipitates from labeled bacteria alone (Fig. 4, lane 4) or from labeled AGS cells infected with nonlabeled H. pylori (lane 7). As the CagA protein of H. pylori ranges in size from 120 to 140 kD in different strains 1122 and our data suggested that the 145-kD protein was derived from H. pylori, we reasoned that the tyrosine-phosphorylated protein was CagA. The lysates of epithelial cells infected with the [35S]methionine-labeled H. pylori were immunoprecipitated by an anti-CagA polyclonal antibody. As shown in Fig. 4 (lane 3), a protein corresponding to 145 kD that was labeled by [35S]methionine was immunoprecipitated with anti-CagA antibody.


Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa C - J. Exp. Med. (2000)

Demonstration of the tyrosine-phosphorylated 145-kD protein derived from H. pylori. AGS cells were infected with [35S]methionine-labeled or nonlabeled H. pylori for 5 h at 37°C, and the cell lysates prepared with 1% Triton X-100 were immunoprecipitated by an antiphosphotyrosine mAb PY-20 or an anti-CagA polyclonal antibody. The precipitated proteins were separated by SDS-PAGE and analyzed using a radioanalytic imaging system. Arrowhead indicates the 145-kD protein. Lanes 1–3, AGS infected with [35S]methionine-labeled H. pylori; lanes 4 and 5, [35S]methionine-labeled H. pylori alone; lanes 6–8, [35S]methionine-labeled AGS infected with nonlabeled H. pylori; lanes 1 and 6, the 1% Triton X-100–soluble fraction precipitated by control normal IgG; lanes 2, 4, and 7, the 1% Triton X-100–soluble fraction precipitated by antiphosphotyrosine mAb PY-20; and lanes 3, 5, and 8, the 1% Triton X-100–soluble fraction precipitated by anti-CagA polyclonal antibody. Note that the [35S]methionine-labeled band being lower than that of 145-kD protein in lanes 3 and 5 may be bacterial protein(s) cross-reacted with the polyclonal anti-CagA antibody.
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Related In: Results  -  Collection

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Figure 4: Demonstration of the tyrosine-phosphorylated 145-kD protein derived from H. pylori. AGS cells were infected with [35S]methionine-labeled or nonlabeled H. pylori for 5 h at 37°C, and the cell lysates prepared with 1% Triton X-100 were immunoprecipitated by an antiphosphotyrosine mAb PY-20 or an anti-CagA polyclonal antibody. The precipitated proteins were separated by SDS-PAGE and analyzed using a radioanalytic imaging system. Arrowhead indicates the 145-kD protein. Lanes 1–3, AGS infected with [35S]methionine-labeled H. pylori; lanes 4 and 5, [35S]methionine-labeled H. pylori alone; lanes 6–8, [35S]methionine-labeled AGS infected with nonlabeled H. pylori; lanes 1 and 6, the 1% Triton X-100–soluble fraction precipitated by control normal IgG; lanes 2, 4, and 7, the 1% Triton X-100–soluble fraction precipitated by antiphosphotyrosine mAb PY-20; and lanes 3, 5, and 8, the 1% Triton X-100–soluble fraction precipitated by anti-CagA polyclonal antibody. Note that the [35S]methionine-labeled band being lower than that of 145-kD protein in lanes 3 and 5 may be bacterial protein(s) cross-reacted with the polyclonal anti-CagA antibody.
Mentions: To directly demonstrate that the tyrosine-phosphorylated 145-kD protein was of H. pylori origin, H. pylori strain NCTC11637 and AGS cells were labeled with [35S]methionine separately, and then the nonlabeled epithelial cells were infected with the [35S]methionine-labeled bacteria for 5 h (the [35S]methionine-labeled epithelial cells were infected with nonlabeled bacteria for 5 h). The infected AGS cells were lysed with 1% Triton X-100, and the soluble fraction was immunoprecipitated with antiphosphotyrosine mAb PY-20. Analysis of the precipitates revealed a 145-kD protein labeled by [35S]methionine (Fig. 4, lane 2). Although the radioactivity of the tyrosine-phosphorylated protein was low, the labeled 145-kD protein was reproducibly detected in three independent experiments. Under the same conditions, the 145-kD protein was not detected in antiphosphotyrosine mAb PY-20 immunoprecipitates from labeled bacteria alone (Fig. 4, lane 4) or from labeled AGS cells infected with nonlabeled H. pylori (lane 7). As the CagA protein of H. pylori ranges in size from 120 to 140 kD in different strains 1122 and our data suggested that the 145-kD protein was derived from H. pylori, we reasoned that the tyrosine-phosphorylated protein was CagA. The lysates of epithelial cells infected with the [35S]methionine-labeled H. pylori were immunoprecipitated by an anti-CagA polyclonal antibody. As shown in Fig. 4 (lane 3), a protein corresponding to 145 kD that was labeled by [35S]methionine was immunoprecipitated with anti-CagA antibody.

Bottom Line: Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen.These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm.The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Nursing and Welfare, Fukui Prefectural University, Fukui 910-1195, Japan. asahi@fpu.ac.jp

ABSTRACT
Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

Show MeSH
Related in: MedlinePlus