This grant has focused on the molecular biology and functions of the eosinophil's granule-associated cationic and other proteins to elucidate key structure-function relationships for their potent inflammatory actions in allergic responses, host-parasite interactions, tissue damage and fibrosis. This renewal focuses on the Charcot-Leyden crystal (CLC) protein, which forms the distinctive bipyramidal crystals which are hallmarks of eosinophil (or basophil) participation in allergic and related inflammatory reactions. CLC protein was originally identified as eosinophil lysophospholipase (LPLase), but we have now shown it to be a member of the galectin superfamily of animal lectins based on amino acid sequence, 3D protein structure, lack of LPLase activity and gene structure. The physiologic/effector role this highly abundant eosinophil constituent is therefore unresolved. Quantitative considerations alone (CLC is 10 percent of eosinophil protein), but also its secretion by activated eosinophils, elevated blood levels in patients with eosinophilia, and increased levels in sputum and BAL of asthmatics, all argue for an important role in eosinophil function. Our cloning of the CLC cDNA and analyses of CLC amino acid sequence identified similarities to b-galactoside-binding animal galectins, but not to any LPLases or other lipolytic enzymes. Our X-ray crystal structure showed CLC protein to be nearly identical to human galectins-1, -2, -3 and -7, and to possess a carbohydrate recognition domain capable of binding mannose, but not standard b-galactoside sugars. We demonstrated that CLC protein is not eosinophil LPLase, which is likely identical to human pancreatic LPLase. The goal of this renewal is to investigate the mechanisms by which eosinophils, through their considerable LPLase activity or the lectin activities of CLC protein, function in allergic pulmonary inflammation. Three questions are addressed with regard to the structure and functions of CLC protein and eosinophil LPLase in terms of their pathophysiologic roles in acute and chronic allergic airways inflammation: (1) What is the biologically relevant glycoconjugate ligand for CLC protein and what does it tell us about CLC's role in eosinophil (or basophil) biology and function. We will identify the physiologically relevant ligand(s) for CLC protein; (2) What are the structure-function relationships for CLC protein's carbohydrate-binding """"""""galectin-like"""""""" activities and ligand specificity? Site-specific mutagenesis will be used to characterize CLC protein's carbohydrate recognition domain and its mechanism of binding for the glycoconjugate ligands we define; (3) What is the role of eosinophil LPLase in allergic pulmonary inflammation involving eosinophils, and in asthma pathophysiology? We will determine whether eosinophil LPLase alters pulmonary surfactant function, resulting in decreased patency of distal airways in asthma, or modulates allergic pulmonary inflammation. We will analyze the effects of eosinophil LPLase on pulmonary surfactant function in vitro, and in vivo by determining the relationships between eosinophil recruitment and secretion of LPLase in the lung and its localization in distal airways in human asthma and murine allergic asthma models. We will analyze the effects of targeting overexpression of LPLase in the lung using transgenic approaches, and determine the effects of knocking out the eosinophil LPLase gene in murine models of eosinophilic airways inflammation. The proposed work should elucidate unique aspects of the structural biology and functions of CLC protein in eosinophils (and basophils), and the pathophysiologic actions of eosinophil LPLase in disease processes associated with eosinophilic inflammation in the lung and other tissues.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Lung Biology and Pathology Study Section (LBPA)
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Plaut, Marshall
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University of Illinois at Chicago
Schools of Medicine
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