Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

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 sitedirected mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with >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 ␣2 and strand ␤1, 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.Eosinophil cationic protein (ECP), 2 a member of the ribonuclease A (RNase A) superfamily, is found in the specific granules of eosinophilic leukocytes. It is a single polypeptide with a molecular mass ranging from 16 to 21.4 kDa due to varying degrees of glycosylation. It shows a 67% amino acid sequence identity with eosinophil-derived neurotoxin (EDN), another eosinophil-secreted RNase. Although ECP shares the overall three-dimensional structure of RNase A, it has relatively lower RNase activity (1). ECP released by activated eosinophils contributes to the toxicity against helminth parasites, bacteria, and single strand RNA viruses (2-4). 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 mechanism underlying the cytotoxic property of ECP is unclear. It has been hypothesized that ECP cytotoxicity is due to destabilization of lipid membranes of target cells (6), and the degree of cytotoxicity is dependent on the cellular concentration (7). 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). Recently, we found that binding and endocytosis of ECP into bronchial epithelial cells were greatly dependent on the cell surface glycosaminoglycan, specifically heparan sulfate proteoglycans (HSPG) (9). The cytotoxicity of ECP was severely reduced toward cell lines with heparan sulfate (HS) deficiency.Heparin and HS are complex polysaccharides composed of alternating units of hexuronic acid and glucosamine. The uronic acid residues of heparin typically consist of 90% L-idopyranosyluronic acid and 10% D-glucopyranosyluronic acid (10). The N position of glucosamine may be substituted with an acetyl or sulfate grou...

The1H,13C,15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

“…This interaction is neither observed in crystal structures of ECP with a bound substrate mimics (1H1H) 24 nor without (1QMT nor 1DYT) nor in the solution structures of the ECP homologs human RNase 1 (2k11) 35 nor bovine pancreatic RNase A (2AAS). 36 Interestingly, the Tyr33 side chain, which is located in the region involved in ECP binding to heparin 25 has been recently reported to be nitrated during eosinophil maturation. 37 We propose that the nitration of Tyr33, which would endow its aromatic ring with a negative charge, could be expected to enhance its interaction with the positively charged ammonium group of Lys38.…”

Structural determinants of the eosinophil cationic protein antimicrobial activity

“…ECP shows a high binding affi nity to heparin and inhibits the enzyme RNase activity (Torrent et al , 2011a ). Critical residues involved in the interaction were identifi ed by molecular docking (Torrent et al , 2011a ) and NMR spectroscopy (Garc í a-Mayoral et al, 2010 ) and corroborated by mutagenesis studies (Fan et al , 2008 ). In particular, the 33 -38 stretch is involved in ECP binding to GAGs ( Figure 2) (Fan et al , 2008 ;Torrent et al , 2011a ).…”

“…Critical residues involved in the interaction were identifi ed by molecular docking (Torrent et al , 2011a ) and NMR spectroscopy (Garc í a-Mayoral et al, 2010 ) and corroborated by mutagenesis studies (Fan et al , 2008 ). In particular, the 33 -38 stretch is involved in ECP binding to GAGs ( Figure 2) (Fan et al , 2008 ;Torrent et al , 2011a ). The protein interaction with heparan sulphate at the matrix of epithelial cells would facilitate the protein endocytosis (Fan et al , 2007 ).…”

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

“…Residues Arg 1, Arg 7 and Arg 34 were predicted by molecular docking to provide the main driving force for heparin probe binding (Torrent et al, 2011a), and NMR spectroscopy data confirmed interactions between the a1 and b1 N-terminus secondary structure elements and the IdoA(2S)-GlcNS(6S) disaccharide (García-Mayoral et al, 2010). Residues 33-36 in L3 loop were also identified to be involved in heparin binding by site-directed mutagenesis (Fan et al, 2008), and a recent study on ECP antiparasitic activity identified Arg 34 as the key basic surface exposed residue (Singh and Batra, 2011). Interestingly, additional contribution of Arg 1 and Arg 7 is observed by NMR for an extended heparin probe (García-Mayoral et al, 2010, unpublished results).…”