Identification of adhesion sites of E–cadherin with CD8+Eß7+ T cells which are involved in the destruction of exocrine glands

Abstract

Abstract: : Purpose: We have previously reported that CD8⁺αEß7⁺T cells are involved in apoptosis of lacrimal gland cells of patients with Sjogren’s syndrome (SjS). E–cadherin on lacrimal epithelial cells may be a ligand for αEß7. To block the heterophilic adhesion between αEß7 and E–cadherin could be an effective therapy for SjS. In this study, we examined the adhesion sites of E–cadherin with αEß7. Methods: The extracellular regions of E–cadherin composed of five cadherin domains were amplified by polymerase chain reaction (PCR). The amplicons of five each domains are defined as EC1, EC2, EC3, EC4, EC5 (numbered from the N terminus), and the whole extracellular domain as EC1–5. The PCR products were inserted to pGEX–4T–2 vector to produce E–cadherin–glutathione–S–transferase (GST) fusion protein. Binding of integrin αEß7⁺– transfected K562 cells with fusion protein was examined by adhesion assay. E–cadherin–GST fusion protein was coated on 96–well culture plate. αEß7⁺K562 cells were labeled with BCECF–AM and added into each well. After washing, fluorescence intensity was measured. Results: αEß7⁺K562 cells were adhesive to EC1–5 fusion–GST fusion protein compared to GST control protein. The cells also showed significant binding to EC1, EC2, EC3 and EC4, but weak binding to EC5. Conclusions: These results suggest that not only EC1 but EC2, EC3, EC4 in human E–cadherin are responsible for binding with αEß7. EC1 has been known to be important for homophilic binding of E–cadherin, EC2, EC3 and EC4 could be better target for blocking the adhesion.

Keywords: lacrimal gland • cell adhesions/cell junctions • cell death/apoptosis