Abstract
Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. Here, a novel molecular model for ECP/EDN binding of heparin hexasaccharide, [GlcNS(6S)-IdoA(2S)]3, was predicted. Interestingly, Gln40 and His64 on ECP formed a clamp-like structure to stabilize heparin hexasaccharide in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln40-His64 clamp contributed to ECP-heparin interaction significantly. Besides, binding affinities of a multi-functional peptide (NYRWRCKNQN, CPPecp), containing major heparin binding region of ECP, to heparin derivatives and glycosaminoglycans (GAGs) were determined by quartz crystal microbalance (QCM) and magnetic reduction assay (MRA). Moreover, a peptide containing minimum length to interact with heparin (YRWRCK, HBPecp) was also used. In conclusion, our in silico and in vitro data demonstrate that ECP uses not only major heparin binding region but also other surrounding residues to interact with heparin. Discovery of such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN, as well as differential binding mode, binding affinity, and cellular interaction between ECP and EDN facilitate further understanding of molecular mechanisms of immune diseases.