Characterization of molecular interactions between eosinophil cationic protein and heparin.
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.
Fan
Tan-Chi
TC
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013.
Fang
Shun-Lung
SL
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013.
Hwang
Chi-Shin
CS
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013; Department of Neurology, Taipei City Hospital, Zhong Xiao Branch, Taipei 10341.
Hsu
Chih-Yen
CY
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013.
Lu
Xin-An
XA
Department of Chemistry, National Tsing Hua University, Hsinchu 30013.
Hung
Shang-Cheng
SC
Department of Chemistry, National Tsing Hua University, Hsinchu 30013; Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Lin
Shu-Chuan
SC
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013.
Chang
Margaret Dah-Tsyr
MD
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013. Electronic address: dtchang@life.nthu.edu.tw.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D004705:Endocytosis; D047150:Eosinophil Cationic Protein / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002241:Carbohydrates; D008055:Lipids; D010455:Peptides; D006493:Heparin; D047150:Eosinophil Cationic Protein