Proteomic Analysis of Airway Epithelial Cell Autoantigens Associated with Bronchial Asthma: Target Protein Identification Using Cultured Cell Fractionation Method

Abstract

Background: Involvement of the autoimmune mechanism has been suggested for the pathogenesis of bronchial asthma and several autoantigens which are associated with bronchial asthma have been identified. Objective: To identify novel asthma-associated autoantigens by proteomic analysis of proteins which are extracted by the cultured cell fractionation method according to the physical affinity of airway epithelial cells (A549) to the culture matrix. Method: Autoantigens were screened with serum samples from patients with severe asthma and the target autoantigens were identified by mass spectrometry. Result: Cultured A549 cells were differentially fractionated into easily detachable floating cell fraction and firmly attached cell fraction. Easily detachable cell fraction containing senescent cell and necrotic cell debris had many detectable asthma-related autoantigens when screened by immunoblot analysis using IgG antibodies from patients with severe asthma. In mass spectrometry analysis of 95-kDa autoantigen, ailpha-actin 4 was identified as a new airway epithelial autoantigen which was recognized by IgG autoantibody from a patient with severe asthma. This was reconfirmed by using specific antibody. Conclusion: Proteomic analysis of the airway epithelial cells using the differential fractionation method according to the physical characteristics of cultured airway epithelial cells may be a novel method for the identification of additional asthma- related autoantigens.