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A SELDI-TOF MS study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
2007 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 82, no 6, 1491-1500 p.Article in journal (Refereed) Published
Abstract [en]

Eosinophil cationic protein (ECP), a secretory protein of the eosinophil granulocyte, is a basic and highly heterogeneous protein. This heterogeneity is dependent on polymorphisms in the ECP gene and post-translational modifications, and it affects the functional properties of the protein in terms of cytotoxicity. The aim of this study was to further investigate the molecular heterogeneity, hence, an affinity capture assay based on an antigen-antibody interaction with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) technique was developed. Of three monoclonal antibodies tested, that is, EG2, 614, and 652, the 614 mab was chosen for the experiments. ECP heterogeneity of single individuals was studied in extracts of purified blood eosinophils, and the presence of approximately 5 major molecular species was demonstrated in each subject. ECP from subjects with different ECP 434(G>C) genotypes (arg97thr) showed mass differences corresponding to the amino acid shift from arginine to threonine. ECP purified from pooled leukocytes of large numbers of healthy blood donors demonstrated an extensive mass heterogeneity with approximately 10 major molecular species. By the use of a variety of glucosidases it was shown that this heterogeneity was mainly due to N-linked oligosaccharides on which sialic acid, galactose, and acetylglucosamine was positioned. We conclude that the SELDI-TOF MS technique using specific monoclonal antibodies is a convenient and versatile tool; by means of this technique, we could detect both genetic and post-translational causes of the molecular heterogeneity of the eosinophil cationic protein.

Place, publisher, year, edition, pages
2007. Vol. 82, no 6, 1491-1500 p.
Keyword [en]
eosinophil granulocyte, proteomics, glycosylation, gene polymorphism
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-96295DOI: 10.1189/jlb.0507272ISI: 000251243800017PubMedID: 17726151OAI: oai:DiVA.org:uu-96295DiVA: diva2:170824
Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2011-01-14Bibliographically approved
In thesis
1. Studies of Eosinophil Cationic Protein (ECP) in vivo and in vitro: Impact of Genetic and Posttranslational Modifications
Open this publication in new window or tab >>Studies of Eosinophil Cationic Protein (ECP) in vivo and in vitro: Impact of Genetic and Posttranslational Modifications
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Studier av Eosinophil Cationic Protein (ECP) in vivo och in vitro : Effekter av genetiska och posttranslationella modifieringar
Abstract [en]

Eosinophil granulocytes are tissue dwelling leukocytes that are implicated in host defence, particularly against helminthic parasites; they also participate in most inflammatory disorders. Although eosinophils have important roles in host defence mechanisms, their actions can also be harmful to the host as in the allergic inflammation where lung epithelium is destructed due to the release of toxic granule proteins.

The focus of the present thesis work has been to characterize the molecular and functional heterogeneity of the granule protein Eosinophil Cationic Protein (ECP). We investigated a coding ECP gene polymorphism (arg97thr) in an African population endemically exposed to the Schistosoma mansoni parasite and found a correlation between ECP genotype and disease manifestations; ECP97arg was more effective in terms of host defence against the parasite, but was also correlated to development of liver fibrosis in infected subjects.

We purified ECP97arg and ECP97thr from healthy blood donors and showed that they differ in their cytotoxic activities; ECP97arg was cytotoxic whereas ECP97thr was non-cytotoxic. They did not differ in terms of RNase activity or in their ability to stimulate fibroblast-mediated collagen gel contraction.

We developed a new SELDI-TOF MS assay to enable the study of the structure of ECP in more detail and showed that ECP is produced in several glycosylated forms, and that the degree of glycosylation determines the cytotoxic activity. Enzymatic deglycosylation significantly enhanced the cytotoxic activity of highly glycosylated ECP-variants.

To summarize, in this thesis we demonstrated that the cytotoxic activity of ECP is dependent on both a gene polymorphism and post-translational modifications, and that the cytotoxic activity is distinct from other functions of ECP. We speculate that ECP is synthesised in heavily glycosylated variants as a means to protect the host from its harmful effects and that ECP is activated by deglycosylation when required at the site of inflammation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 57 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 278
Biomedicine, Eosinophil granulocyte, granular protein, ECP, gene polymorphism, cytotoxicity, SELDI-TOF MS, glycosylation, molecular and functional heterogeneity, fibrosis, Biomedicin
urn:nbn:se:uu:diva-8261 (URN)978-91-554-6989-4 (ISBN)
Public defence
2007-11-09, Enghoffsalen, Ingång 50, bv, Akademiska Sjukhuset, Uppsala, 09:15
Available from: 2007-10-19 Created: 2007-10-19Bibliographically approved

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