and specific aims): Eosinophils and their cationic granule proteins function as mediators of tissue damage and inflammation in asthma, allergic reactions, parasitic infections and other eosinophil-associated diseases. This application focuses on Charcot-Leyden crystal (CLC) protein, which forms distinctive hexagonal bipyramidal crystals observed in tissues and secretions, a hallmark of eosinophil participation in allergic and other inflammation. CLC is a lysophospholipase (LPLase); lysolecithin acylhydrolase), but the physiologic role remains a mystery. Quantitative considerations alone (CLC is approximately 10% of total eosinophil protein), but also its secretion during eosinophil activation, elevated levels in the blood of patients with eosinophilia, and increased levels of sputum and BAL fluids of patients with asthma, argue for its importance in eosinophil effector function. The overall goal of this project is to investigate the mechanisms by which eosinophils, through the LPLase or galectin activities of CLC protein, function in eosinophil-associated inflammation. Three specific questions are addressed with regard to CLC protein's structure-function relationships, mechanisms of catalytic activation, secretion and regulation, and potential pathophysiologic inflammatory activities in asthma: 1) What are the structure-function relationships for the LPLase activity of CLC protein and regulation of its expression? Based on the 3D structure of CLC protein, site-specific mutagenesis will be used to characterize the active site of the enzyme and elucidate the mechanisms regulating its activation and expressions by eosinophils; 2) What are the structure-function relationships for CLC protein's carbohydrate-binding (galectin) activities? Recombinant expression and site-specific mutagenesis will be used to characterize its carbohydrate recognition domain. We will identify physiologically relevant oligosaccharide or oligosaccharide-containing ligands and assess CLC's role in eosinophil adhesions; 3) What is the role of CLC protein in eosinophil effector function in asthma pathophysiology? Does this LPLase play a pro- or anti-inflammatory role, or alter surfactant function and airways architecture in asthma? This aim will analyze the effects of this LPLase on pulmonary surfactant function in vitro, CLC secretion and localization in the distal airways in asthmatic lung, relationships of CLC levels and LPLase activity in the lung with asthma severity, and the effects of LPLase administration in animal models of airways inflammation.
Showing the most recent 10 out of 35 publications
