Abstract
Eosinophil-derived neurotoxin (EDN, also known as RNase2) is secreted by activated eosinophils and is a member of human RNase A superfamily. It contains 12 cationic residues and thus possesses a high pI value of 8.9. EDN is also an antiviral RNase and its level in biological fluid is used as an inflammatory bio-marker. This study focuses on identification and characterization of the essential heparin binding motifs in EDN, and the minimal EDN binding unit in heparin polysaccharides. Recombinant wild-type EDN, and mutant EDNs derived from site-directed mutagenesis were expressed by E. coli expression system. The interaction of EDN to bronchial epithelial cells and heparin has been demonstrated by cell ELISA and FACE, respectively. The results showed direct interaction between recombinant EDN and heparin as well as heparan sulfate (HS). In addition, competitive assays using heparin derivatives indicated that the sulfate groups contributed almost all negative charges for EDN binding, and the crucial role of N-sulfated and O-sulfated groups in heparin in the interaction to EDN was illustrated. The functional heparin binding motif has been identified by site-directed mutagenesis screening. Three heparin binding regions (HBRs) on EDN including HBR1 (34QRRCKN), HBR2 (65NKTRKN), and HBR3 (113NRDQRRD) are identified. HBR1 is the most important motif in governing EDN binding to heparin/HS, for the heparin/HS binding effect of HBR1 is higher than that of HBR2 and HBR3. Furthermore, the penetration of MBP-EDN to Beas-2B cells requires HBR1 and may correlate with the interaction of EDN on cell surface HS. Taken together, we have discovered and characterized functional heparin/HS binding motifs on hEDN.