The purpose of this project is to elucidate the molecular events involved in the biosynthesis and activation of eosinophil and neutrophil granule proteins. A. Eosinophilopoiesis. We have shown that the disorder known as neutrophil specific granule deficiency (SGD) also includes the eosinophil lineage, as three of the four granule proteins are absent in circulating eosinophils from an affected individual. We have gone on to show that circulating CD34+ progenitor cells isolated from normal individuals can be induced to differentiate toward eosinophils that have transcriptional defects mimicking SGD. We have also identified a cell line in which transcription of these deficient granule proteins can be studied directly. B. Granule protein folding. We have described the high affinity, reversible interaction of the prokaryotic molecular chaperone, groEL, with nascent chains of two eosinophil granule proteins. We are developing a eukaryotic recombinant system in which folding of these proteins can be studied in their native locale. Also, using a panel of monoclonal antibodies, we have identified a novel protein that cross- reacts with a specific anti-chaperone antibody. We have also further characterized another molecular chaperone protein (hsp70RY) which was initially identified in EBV-transformed B cells (Dyer KD and Rosenberg HF. C. Structure and activity of ECP and EDN. Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) are both ribonucleases, and have structural homology to other members of the ribonuclease gene family. Using a unique approach, we have succeeded in expressing recombinant ECP and EDN in a prokaryotic system, and have shown that, for ECP, antibacterial activity is unrelated to ribonuclease activity. In addition, using antibodies that distinguish between storage and secreted forms of ECP, we have results suggesting that the released form is deglycosylated relative to the storage form (Rosenberg HF and Tiffany HL.
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