A SELDI-TOF MS study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein

Abstract

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.

Eosinophilic biomarkers for detection of acute exacerbation of chronic obstructive pulmonary disease with or without pulmonary embolism

“…In addition to chronic inflammatory disorders, including asthma and COPD, a number of studies have addressed the involvement of eosinophils and their granule proteins in host resistance to parasites, their antimicrobial activities toward bacterial, viral and protozoan pathogens, their function as mediators of hypersensitivity and their interactions with the coagulation system (22,38). Case reports have stated that eosinophilia in patients may be accompanied by thromboembolisms (40)(41)(42).…”

“…ECP has various biological activities, including antibacterial, antiviral, antiparasitic and neurotoxic functions, and it contributes to the regulation of fibroblast activity (21). ECP also induces airway mucus secretion and interacts with the coagulation and complement systems (22,23). Fiorini et al (24) concluded that serum ECP levels increased during AECOPD.…”

Eosinophil Cationic Protein (ECP) Is Processed during Secretion

“…Samples were analyzed on PS20 ProteinChip arrays in a PBS-IIC system or PCS 4000 Enterprise system (Bio-Rad Laboratories) as described previously (13). A monoclonal anti-ECP Ab, clone 614 provided by Diagnostics Development, was used.…”

“…Supernatants and cell extracts were incubated with or without 3 U of Nglycosidase F (Roche Diagnostics) at 37°C overnight as has been described before (13).…”

“…In a previous study we showed that eosinophil extracts from healthy blood donors contain several variants of ECP with molecular masses ranging from 15.9 to 17.2 kDa when analyzed by means of a sensitive and specific surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) technique (13). Preliminary in vitro studies of fractionated ECP, purified from buffy coats, demonstrated that only some of these molecular variants displayed cytotoxic activity.…”