Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Tan-Chi Fan,Shun-Lung Fang,Chi-Shin Hwang,Chih-Yen Hsu,Xin-An Lu,Shang-Cheng Hung,Shu-Chuan Lin,Margaret Dah-Tsyr Chang

doi:10.1074/jbc.m803516200

Abstract

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

Highlights

IntroductionTogether with other proteins secreted from eosinophils, Eosinophil cationic protein (ECP) is thought to cause damage to epithelial cells, a common feature of airway inflammation in asthma [5]
CD spectroscopy to compare the conformations of wild-type Eosinophil cationic protein (ECP) and ECP mt1 showed that the secondary structure of ECP mt1 was very similar to that of wild-type ECP. These results strongly indicated that the decrease in heparin binding affinity resulted from a loss of the specific recognition sequence motif, but not from a conformational change in ECP mt1
We provide the first direct evidence that a linear heparin binding site within ECP contributes to its cellular binding and cytotoxicity

Summary

Introduction

Together with other proteins secreted from eosinophils, ECP is thought to cause damage to epithelial cells, a common feature of airway inflammation in asthma [5]. The binding of ECP to target cells has been attributed to its high arginine content (estimated pI ϭ 10.8), which facilitates the interaction between ECP and negatively charged molecules on the cell surface [7, 8]. Interactions between Eosinophil Cationic Protein and Heparin binding domains within proteins usually contain a high proportion of positively charged residues, which bind to the acidic groups of heparin through electrostatic interactions. It has been proposed that the three-dimensional structure of the HS chain is critical for protein binding [13]. After examining a series of heparin-binding protein sequences, Cardin and Weintraub [14] proposed that the pattern XBBBXXBX or XBBXBX (where X represents hydrophobic or uncharged amino acids, and B represents basic amino acids) is responsible for HS binding to other proteins

Objectives

Results

Conclusion