A. Structure/function of ECP and EDN. Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) are granule proteins with structural and functional homology to members of the mammalian ribonuclease gene family. We have traced the rapid molecular evolution of this gene family, and determined that the genes encoding EDN and ECP have accumulated non-silent mutations at rates exceeding those of all other functional coding sequences studied in primates, while retaining both the structural and catalytic components required for ribonuclease activity. Interestingly, the antibacterial activity of ECP was shown to be unrelated to ribonuclease activity. We have studied the function of a representative """"""""ancestral"""""""" sequence of the ECP/EDN gene pair; our results suggested that evolutionary constraints have promoted two novel functions--both cytotoxicity and enhanced ribonuclease activity--in the two human members of this gene family. B. Eosinophilopoiesis. We have identified conditions under which CD34+ peripheral blood progenitor cells (PBPCs) isolated from normal individuals can be induced to differentiate toward eosinophils; while faithfully replicating transcriptional events of normal eosinophilopoiesis, only three of the five granule proteins could be readily detected, suggesting the possibility of as yet unidentified eosinophilopoietic factors. Interestingly, EDN synthesized by these cells was found to be hyperglycosylated, as was EDN and ECP detected in an eosinophilic variant of the promyelocytic leukemia cell line, HL-60. Additionally, we have identified a functional promoter of the EDN gene, and shown that gene expression requires cooperation between the promoter and intronic enhancer elements, several of which may function only in hematopoeitic cells (Tiffany and Rosenberg (1995) In review). C. Charcot-Leyden crystal protein (CLC). Molecular cloning suggested a relationship between the eosinophil protein, CLC, and S-type animal lectins. Guided by this observation, we have shown that CLC can function
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