Slifman, Nancy R.

Abstract The human eosinophil granule contains a series of cationic proteins. Two of these, eosinophil-derived neurotoxin (EDN) and eosinophil protein X (EPX), are reported to have similar m.w. and both possess neurotoxic and helminthotoxic activities. Therefore, the properties of these molecules were analyzed to determine whether they differ. EDN was purified from eosinophils of patients with the hypereosinophilic syndrome and EPX from the buffy coat cells of normal individuals. By SDS-PAGE, both proteins showed a major band at 18.7 kDa and a minor band at 21.4 kDa. By two-dimensional non-equilibrium gel electrophoresis the proteins migrated identically. With radiolabeled proteins in reverse phase HPLC, both proteins eluted at the same concentration of acetonitrile and showed identical tryptic maps. Both proteins possessed comparable ribonuclease activity and both were comparably neurotoxic in the rabbit. By immunodiffusion the two proteins showed a reaction of identity; by RIA, with both polyclonal and monoclonal antibodies, the proteins had very similar inhibitory activities. These results indicate that EDN and EPX have virtually identical properties and are probably the same protein.

Abstract The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.

Two of the four principal cationic proteins of the eosinophil granule, major basic protein (MBP) and eosinophil peroxidase (EPO), were shown to be platelet agonists. Both MBP and EPO evoked a dose-dependent nonlytic secretion of platelet 5-hydroxytryptamine in unstirred platelet suspensions even in the presence of 10 microM indomethacin. MBP also evoked secretion of platelet alpha granule and lysosome components. Secretion by MBP and EPO was inhibited by 1 microM PGE1, but the nature of the inhibition differed from that observed with thrombin. Thus, MBP and EPO can be classified as strong platelet agonists with a distinct mechanism of activation.