title: Functional characterization of ECP-heparin interaction: a novel molecular model.
abstract: Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) 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. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 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 Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Ta-Jen
authors.initials: TJ
authors.lastname: Hung
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Noboru
authors.initials: N
authors.lastname: Tomiya
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Tse-Hao
authors.initials: TH
authors.lastname: Chang
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Cheng
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Ping-Hsueh
authors.initials: PH
authors.lastname: Kuo
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Sim-Kun
authors.initials: SK
authors.lastname: Ng
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Pei-Chun
authors.initials: PC
authors.lastname: Lien
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China ; Department of Biology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Yuan-Chuan
authors.initials: YC
authors.lastname: Lee
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China ; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D047150:Eosinophil Cationic Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004804:Eosinophils / Q000201:enzymology; D015870:Gene Expression; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D006493:Heparin; D047150:Eosinophil Cationic Protein