Considerable evidence indicates that the eosinophil is important in resistance to parasites and in hypersensitivity diseases, such as bronchial asthma. The eosinophil is equipped with weapons to damage helminths and host tissues, including cationic proteins, reactive oxygen species, leukotrienes, and the ability to elaborate cytokines. In human diseases, eosinophils accumulate in massive numbers and discharge their granule proteins onto affected tissues, often in association with damage or frank necrosis. Under the auspices of this grant, we have investigated the structure and function of eosinophil granule proteins, including major basic protein (MBP1), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO). These proteins are toxins and damage cells and tissues; MBP1 and EPO are able to stimulate cells. Further, MBP1 causes bronchial hyperreactivity in monkeys and guinea pigs. We have discovered a novel granule protein, the MBP homologue (MBP2). Like MBP1, MBP2 damages and activates target cells. However, whether MBP2 possesses distinctive properties not shared by MBP1 is unknown. MBP1 is synthesized as a 32 kDa promolecule (proMBP1) and is processed to its 14 kDa mature form; but little is known of the proMBP convertase. Also, the crystal structure of proMBP1 is unknown. Preliminary analyses of the eosinophil granule proteome indicate the presence of numerous novel peptides. Here we identify a series of interrelated goals to investigate these issues. First, we will compare the properties of MBP1 and MBP2 and determine whether unique properties exist for MBP2. Second, we will characterize the enzyme(s) converting 32 kDa proMBP to 14 kDa MBP (proMBP convertase). Third, in collaboration, we will crystallize recombinant proMBP and solve its structure. Fourth, we will characterize the novel acidic eosinophil granule proteins discovered using two-dimensional electrophoresis and mass spectroscopy. Finally, we will determine whether two novel markers of eosinophil activation are detectable in patients with eosinophilia. Overall, these studies will expand our knowledge of the molecules composing the eosinophil granule and provide new tools to dissect eosinophil function in disease.
