The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications

• Published in: Clinical and experimental allergy Vol. 37; no. 2; pp. 208 - 218
• Main Authors: Trulson, A., Byström, J., Engström, Å., Larsson, R., Venge, P.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.02.2007; Blackwell
• Subjects: Allergic diseases; Animals; Biological and medical sciences; Blood Proteins - genetics; cytotoxic activity; cytotoxic protein; eosinophil; Eosinophil Cationic Protein - genetics; Eosinophil Cationic Protein - metabolism; Eosinophil Cationic Protein/genetics/metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; gene polymorphism; granule protein; Guinea Pigs; Humans; Hypersensitivity - genetics; Immunoassay - methods; Immunopathology; Medical sciences; MEDICIN; MEDICINE; Polymorphism, Genetic - genetics; Protein Processing, Post-Translational - genetics; purification; Rabbits; signal peptide cleavage; Sweden - epidemiology; Sweden - ethnology; Sweden/epidemiology/ethnology; Sweden; Immunopathology; Allergy; Purification; Genetic variability; Peptides; Granulocyte; Cytotoxicity; Genotype; gene polymorphism; cytotoxic protein; Biological activity; Eosinophil; Posttranslational modification; Heterogeneity; Signal transduction; Immunology; granule protein; Cleavage; signal peptide cleavage; cytotoxic activity; Eosinophil cationic protein; Genetic/genetics; Post-Translational/genetics; Protein Processing; Polymorphism
• ISSN: 0954-7894; 1365-2222; 1365-2222
• DOI: 10.1111/j.1365-2222.2007.02644.x

Summary Background The eosinophil is a cytotoxic cell and takes part in parasite killing and tissue‐destructive processes by secretion of proteins such as eosinophil cationic protein (ECP). A polymorphism was demonstrated in the ECP gene, giving rise to a substitution of arginine at position 97 with threonine. This polymorphism is related to disease development. Objective To investigate the functional and molecular heterogeneity of native ECP and the functional consequences of the replacement of arginine with a threonine. Methods ECP was purified from healthy blood donors by gel filtration, ion‐exchange chromatography and reversed‐phase chromatography. Recombinant ECPs i.e. rECP 97arg and rECP 97thr were produced by the pFASTBAC baculovirus expression system. The cytotoxic activity was determined against an erythroleukaemia or a small cell lung cancer cell line. Results Native ECP was purified to apparent homogeneity and showed a considerable molecular heterogeneity and a corresponding functional heterogeneity with respect to cytotoxic activity. After reduction, the native cytotoxic ECP showed three bands on sodium dodecylsulphate polyacrylamide gel electrophoresis : one major band at 18–20 kDa and two minor bands at about 10 and 5 kDa, respectively. The 5 kDa contained two masses differing with 56.2 Da, which corresponds to the difference in molecular masses of arginine and threonine. rECP 97arg was cytotoxic in contrast to rECP97thr. Deglycosylation with N‐glycosidase F did not affect the cytotoxic activity of native ECP to any measurable extent nor the activity of rECP 97arg, whereas rECP 97thr achieved cytotoxic activity. The RNase activities of the recombinant and native ECPs were similar. Conclusion We conclude that ECP is present in several molecular forms with varying biological activities. Some of this functional heterogeneity is based on the genetic polymorphism of the ECP gene and some on post‐translational modifications. In subjects carrying the ECP 97thr variant, the cytotoxic activity may be disguised by N‐linked glycosylation of the active site.