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The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. (Inflammation)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. (Inflammation)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. (Cancer Pharmacology and Informatics/Rolf Larsson)
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2007 (English)In: Clinical and Experimental Allergy, ISSN 0954-7894, E-ISSN 1365-2222, Vol. 37, no 2, 208-218 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2007. Vol. 37, no 2, 208-218 p.
Keyword [en]
Animals, Blood Proteins/*genetics, Eosinophil Cationic Protein/*genetics/metabolism, Guinea Pigs, Humans, Hypersensitivity/*genetics, Immunoassay/methods, Polymorphism; Genetic/*genetics, Protein Processing; Post-Translational/*genetics, Rabbits, Sweden/epidemiology/ethnology
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-13878DOI: 10.1111/j.1365-2222.2007.02644.xISI: 000243676700007PubMedID: 17250693OAI: oai:DiVA.org:uu-13878DiVA: diva2:41648
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2017-12-11Bibliographically approved

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Trulson, AgnetaEngström, ÅkeLarsson, RolfVenge, Per

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