Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Abstract

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P_-1. Although RNase A also contains a P_-1 subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P_-1 subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P_-1 subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates.