Eosinophils and basophils are now recognized as critical cells in the pathophysiology of bronchial asthma and other hypersensitivity diseases. Because of their potent biological activities, cationic eosinophil granule proteins are implicated as mediators of eosinophil functions. However, little information is available regarding basophil granule proteins. Prior studies under the auspices of this grant characterized the structure and function of the principal granule proteins including the major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Here, we identify three new lines of investigation. First, MBP is produced as a pro-molecule (proMBP) and converted to 14 kDa MBP. Preliminary experiments indicate that cathepsin L has proMBP converting activity, and we identify experiments to test this hypothesis. Second, the three-dimensional structures of 14 kDa MBP and proMBP will be determined by X-ray crystallography. The 14 kDa MBP have already been crystallized, and a native dataset at 2.8 angstrom has been obtained. However, 14 kDa MBP crystals are often twinned, and it may be necessary to express and crystallize proMBP in order to solve the structure of 14 kDa MBP. Third, experiments to identify novel basophil granule proteins are described. Basophils from a patient with basophilic leukemia were obtained by cytapheresis and granules isolated. High performance liquid chromatography of granule lysates revealed the presence of numerous proteins and the partial sequences of eight were obtained. We propose to stimulate umbilical cord mononuclear cells to differentiate to eosinophil/basophil hybrids and to produce a cDNA library from these hybrids. The hybrids will be compared to IL-5 stimulated umbilical cord cells by differential display analyses and by sequencing of randomly selected cDNA clones. Using degenerate oligonucleotides and novel DNA sequences from differential display and from sequencing of randomly selected clones, we will identify full-length cDNA clones and express the novel basophil proteins with the goal of producing antibody specific for the basophil. Overall, these studies will provide important new information regarding the mechanism of conversion of proMBP to MBP, the three dimensional structure of eosinophil granule proteins and the existence of novel basophil-associated proteins.
