Fragmented CagA Protein is Highly Immunoreactive in Japanese Patients

Abstract

Background: High‐molecular‐weight cell‐associated proteins (HM‐CAP) assay is the most popular serological immunoassay worldwide and has been developed from US isolates as the antigens. The accuracy is reduced when the sera are from adults and children in East Asia including Japan. To overcome the reduced accuracy, an enzyme immunoassay using Japanese strain–derived HM‐CAP (JHM‐CAP) was developed, in which the antigens were prepared by exactly the same procedure as HM‐CAP. The performance of JHM‐CAP was better than that of HM‐CAP in Japanese adults as well as in children. The higher sensitivity was because of the presence of 100‐kDa protein that was absent in the preparation of HM‐CAP antigen. Materials and Methods: Immunoblot analysis and peptide mass fingerprinting methods were used to identify the distinctive 100‐kDa protein present in JHM‐CAP antigens. The peptide sequence and identification were analyzed by Mascot Search on the database of Helicobacter pylori. The identified protein was confirmed by immunoblot with a specific antibody and inhibition assay by the sera. Results: The distinctive 100‐kDa protein was a fragment of CagA derived from Japanese clinical isolates, and the sera of Japanese patients had strongly reacted to the protein, probably to the exposed epitope on the fragmented CagA. The fragmentation of CagA had occurred in the process of antigen preparation in Japanese isolates, not in US isolates even under the same preparation. Conclusion: The distinctive 100‐kDa protein was a fragment of CagA protein of H. pylori derived from Japanese clinical isolates, and Japanese patients including children are likely to react strongly to the exposed epitopes on fragmented CagA.

Helicobacter Pylori Infection: Diagnostic Strategies in Primary Diagnosis and After Therapy

“…As the H. pylori strains differ worldwide, the performance of serological kits show high variation in populations from different geographical areas. Antibody tests evaluated in the East may not be appropriate for clinical diagnosis in the West [1,14,15]. Use of pooled antigen preparations might overcome this limitation.…”

Polymorphism in the Helicobacter pylori CagA and VacA toxins and disease

“…104 Additionally, sera from Japanese patients was highly reactive to a 100 kDa fragment of CagA that was isolated from Japanese clinical isolates using a high-molecular weight cell-associated proteins (HM-CAP) assay. 118 In contrast, when this assay was performed using isolates from the United States, a larger CagA fragment that was not as reactive as the Japanese HM-CAP fragment was generated. 118 These different sized cleavage products suggest that there may also be structural differences in the various CagA proteins.…”

“…118 In contrast, when this assay was performed using isolates from the United States, a larger CagA fragment that was not as reactive as the Japanese HM-CAP fragment was generated. 118 These different sized cleavage products suggest that there may also be structural differences in the various CagA proteins. 118 Of note, these proposed structural differences do not appear to alter the stability of CagA within the host cell since CagA EPIYA-ABD and CagA EPIYA-ABCCC do not exhibit a statistically significant difference in their half-lives.…”

“…118 These different sized cleavage products suggest that there may also be structural differences in the various CagA proteins. 118 Of note, these proposed structural differences do not appear to alter the stability of CagA within the host cell since CagA EPIYA-ABD and CagA EPIYA-ABCCC do not exhibit a statistically significant difference in their half-lives. 83 Finally, the first definitive evidence for the role of the EPIYA-ABD motif in cancer development was obtained from a large scale molecular epidemiological study of South Korean H. pylori isolates; this work was the first to show that the presence of an EPIYA-ABD motif was statistically associated with the development of gastric cancer.…”

Proteomic Characterization of Helicobacter pylori CagA Antigen Recognized by Child Serum Antibodies and Its Epitope Mapping by Peptide Array

“…Serological diagnosis of H. pylori for children, however, has been problematic for Japanese children; we found that the diagnostic performance of an enzyme immune assay (EIA) kit based on Japanese strain-derived high-molecular-weight cell-associated protein (JHM-CAP kit) was much better than that based on U.S. strain-derived high-molecular-weight cell-associated protein (HM-CAP kit) [17], although the performances of the two kits for adult populations were not much different [18]. Such a clear difference between the two kits for diagnosis of H. pylori infection in Japanese children was shown to be attributed to the 100-kDa antigen protein contained only in JHM-CAP, which was later identified as a fragmented CagA protein [19]. Intriguingly, 100-kDa CagA fragments were also identified in gastric cells and phagocytic cells after infection by western H. pylori strain [20], [21].…”