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Characterization of a mutant E‐cadherin protein encoded by a mutant gene frequently seen in diffuse‐type human gastric carcinoma
Author(s) -
Fukudome Yuko,
Yanagihara Kazuyoshi,
Takeichi Masatoshi,
Ito Fumiaki,
Shibamoto Sayumi
Publication year - 2000
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/1097-0215(20001115)88:4<579::aid-ijc10>3.0.co;2-u
Subject(s) - exon , microbiology and biotechnology , biology , mutant , cadherin , complementary dna , alternative splicing , rna splicing , gene , intron , cell , genetics , rna
The cell–cell adhesion molecule E‐cadherin plays an essential role in the maintenance and function of epithelial tissues. Altered expression of E‐cadherin has been implicated in tumor invasion. Analysis of mutations of the human E‐cadherin gene in gastric carcinoma of the diffuse type has revealed that deletion of exon 8 or 9 in its cDNA appears to be predominant. In this study, we carried out structural and functional analyses of a mutant form of E‐cadherin in a cell line, HSC45‐M2, established from a human signet ring‐cell carcinoma. Although immunohistochemical analysis showed that the mutant cadherin was localized at cell–cell contact sites as usually seen with the wild type, these cells did not form compact colonies. HSC45‐M2 cells expressed aberrant E‐cadherin with an m.w. larger than that of the wild type. In these cells, we found deletion of the exon 9–intron 9 boundary including the splicing donor site in E‐cadherin genomic DNA. RT‐PCR indicated 2 transcripts, which appeared to be caused by the splicing defect. Northern blotting, however, showed that the transcript lacking exon 9 was predominantly detected in these cells. The electrophoretic mobilities on SDS‐PAGE of the mutant E‐cadherin protein in HSC45‐M2 cells and the protein expressed from cDNA lacking exon 9 appeared identical. Analysis of the amino‐terminal region of the mutant E‐cadherin protein revealed that the cadherin was capable of becoming a mature form by removal of its amino‐terminal peptide. However, the mutant E‐cadherin was susceptible to trypsinization in the presence of Ca 2+ , which is not the case for wild‐type E‐cadherin, suggesting that the mutant E‐cadherin frequently found in diffuse‐type gastric carcinoma may have lost its Ca 2+ ‐binding ability, leading to disruption of the tight cell–cell association. Int. J. Cancer 88:579–583, 2000. © 2000 Wiley‐Liss, Inc.