The coding ECP 434(G>C) gene polymorphism determines the cytotoxicity of ECP but has minor effects on fibroblast-mediated gel contraction and no effect on RNase activity.
Eosinophil cationic protein (ECP) is a secretory protein of the eosinophil granulocyte, a cell involved in innate immunity. Functional studies have implicated ECP in numerous processes, such as tissue remodeling in allergic inflammation and cytotoxicity toward a variety of pathogens. Recent genetic studies have suggested that the ECP 434(G>C) polymorphism resulting in an arg97thr substitution would alter the function of ECP in vivo. Functional (in vitro) studies of ECP up until now have either been conducted with native preparations containing an unknown mixture of the ECP(97arg) and ECP(97thr) variants, or with recombinant proteins. Therefore, we have now for the first time extracted the native ECP(97arg) and ECP(97thr) variants from healthy blood donors and tested them functionally in vitro. Our results show that the arg97thr shift dramatically alters the cytotoxic capacity of ECP in vitro; the tested ECP(97arg) variants were cytotoxic toward the small-cell lung cancer cell line NCI-H69, whereas ECP(97thr) was noncytotoxic. RNase activity was unaffected by the arg97thr substitution. Both ECP(97arg) and ECP(97thr) stimulated fibroblast-mediated collagen gel contraction, an experimental model, which depicts wound healing, in a dose-dependent manner. In conclusion, our results demonstrate that the ECP 434(G>C) gene polymorphism affects the functional properties of native ECP, but also that there is a dissociation between different biological activities; the arg97thr substitution impairs the cytotoxic potential of ECP but less the gel contraction and not at all the RNase activity.
Rubin
Jenny
J
Department of Medical Sciences, Uppsala University, Sweden. jenny.eriksson@medsci.uu.se
Zagai
Ulrika
U
Blom
Kristin
K
Trulson
Agneta
A
Engström
Ake
A
Venge
Per
P
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000235:genetics*; D047150:Eosinophil Cationic Protein / Q000235:genetics* / Q000502:physiology / Q000633:toxicity*; D005347:Fibroblasts / Q000502:physiology*; D005782:Gels; D005838:Genotype; D006801:Humans; D011110:Polymorphism, Genetic; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism*; D013912:Threonine / Q000235:genetics
D024042:Collagen Type I; D005782:Gels; D013912:Threonine; D001120:Arginine; D012260:Ribonucleases; D047150:Eosinophil Cationic Protein; C479220:RNASE3 protein, human