Biochemical analysis of the calcium activated chloride channelhCLCA1 and functional analysis of its mouse homologue Gob-5.

摘要

hCLCA1 and its murine homologue, Gob-5, are members of the calcium activated chloride channel family of proteins and have been associated with human asthma and preclinical models of allergic inflammation. They have also been used as biomarkers for goblet cell hyperplasia and mucus production. In this thesis, the biochemical characteristics of hCLCA1 were examined. Analysis of conditioned media from heterologous overexpression of hCLCA1 in COS and HEK293 cells revealed that hCLCA1 exists as a secreted macromolecule and that hCLCA1 secretion was inhibited by the secretory pathway inhibitor brefeldin A. PNGase F digestion of secreted hCLCA1 demonstrated that hCLCA1 is highly N-glycosylated and analysis by non-reducing SIDS-PAGE revealed that hCLCA1 exists as a dimer in solution. Confocal microscopic analysis showed an association of hCLCA1 with defined markers of the secretory pathway in heterologously overexpressing as well as untransfected cells. Subcellular fractionation studies of cells overexpressing hCLCA1 revealed that there was an association with fractions containing extracellular matrix proteins. Soluble CLCA1 and Gob-5 can be detected in the bronchoalveolar lavage (BAL) fluid from animals with experimentally induced allergic inflammation. CLCA1 expression in a monkey model of allergic inflammation can be inhibited by the pretreatment of the animals with 1 mg/kg dexamethasone.; To address the pathophysiologic significance of hCLCA1 and Gob-5 in asthma, the response of Gob-5 deficient mice was measured in multiple preclinical models of allergic inflammation. Sensitized Gob-5 -/- mice, when challenged with aerosolized ovalbumin, exhibited a 50% increase in total inflammation compared with wild type controls that was the result of an increase in infiltrating neutrophils into the BAL fluid. Increased neutrophils in the BAL were also seen when Gob-5 -/- mice and wild type mice were challenged with LPS. Antigenic challenge of primed Gob-5 -/- mice also resulted in a decrease in pulmonary goblet cell metaplasia, a decrease in mucus production in the lung, and a decrease in airway hyperresponsiveness compared to controls. The results presented in this thesis reinforce the association of Gob-5 with goblet cell hyperplasia and implicate Gob-5 in the regulation of the inflammatory response, more specifically, the neutrophilic component of airways inflammation.