EOSINOPHIL CATIONIC PROTEIN AND EOSINOPHIL-DERIVED NEUROTOXIN - EVOLUTION OF NOVEL FUNCTION IN A PRIMATE RIBONUCLEASE GENE FAMILY

Human eosinophil derived neurotoxin (EDN) and eosinophil cationic prot ein (ECP) are members of a unique subfamily of rapidly evolving primat e ribonuclease genes that emerged via a gene duplication event occurri ng after the divergence of Old World from New World monkeys (Rosenberg , H. F., Dyer, K. D., Tiffany, H. L., and Gonzalez, M. (1995) Nature G enet, 10, 219-223). In this work, we studied the activity of the prote in encoded by the EDN/ECP homolog of the New World monkey, Saguinus oe dipus (marmoset), a representative of the ''ancestral'' single sequenc es, Although the nucleotide sequence of the single marmoset gene (mEDN ) was equally homologous (82%) to both human genes, the en coded amino acid sequence, calculated isoelectric point, and immunoreactivity all suggested a closer relation ship with EDN. Furthermore, mEDN (at 0.3- 1.0 mu M concentrations) had no measurable anti-staphylococcal activit y, suggesting functional as well as structural similarity to EDN. Howe ver, with yeast tRNA as substrate, mEDN had significantly less ribonuc lease activity than EDN; Michaelis constants were nearly identical (K- m (mEDN) = 0.67 mu M; K-m (EDN) = 0.70 mu M), while turnover numbers d iffered by a factor of 100 (k(cat) (mEDN) = 0.91 s(-1); k(cat) (EDN) = 0.64 x 10(-2) s(-1)), Thus, evolutionary constraints appear to have p romoted two novel functions: increased cationicity/toxicity (ECP) and enhanced ribonuclease activity (EDN), The latter result is particularl y intriguing, as it suggests a crucial role for ribonuclease activity in the (as yet to be determined) physiologic function of EDN.