Eriksson Jenny, Woschnagg Charlotte, Fernvik Eva, Venge Per
Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden.
J Leukoc Biol. 2007 Dec;82(6):1491-500. doi: 10.1189/jlb.0507272. Epub 2007 Aug 28.
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.
嗜酸性粒细胞阳离子蛋白（ECP）是嗜酸性粒细胞的一种分泌蛋白，是一种碱性且高度异质性的蛋白质。这种异质性取决于ECP基因的多态性和翻译后修饰，并且在细胞毒性方面影响该蛋白的功能特性。本研究的目的是进一步研究分子异质性，因此，开发了一种基于抗原 - 抗体相互作用并结合表面增强激光解吸/电离飞行时间质谱（SELDI-TOF MS）技术的亲和捕获测定法。在所测试的三种单克隆抗体，即EG2、614和652中，选择614单克隆抗体进行实验。在纯化的血液嗜酸性粒细胞提取物中研究了个体的ECP异质性，并且在每个受试者中证实存在大约5种主要分子种类。来自具有不同ECP 434（G>C）基因型（arg97thr）的受试者的ECP显示出与从精氨酸到苏氨酸的氨基酸变化相对应的质量差异。从大量健康献血者的混合白细胞中纯化的ECP表现出广泛的质量异质性，有大约10种主要分子种类。通过使用多种糖苷酶表明，这种异质性主要归因于其上定位有唾液酸、半乳糖和N-乙酰葡糖胺的N-连接寡糖。我们得出结论，使用特异性单克隆抗体的SELDI-TOF MS技术是一种方便且通用的工具；借助该技术，我们能够检测嗜酸性粒细胞阳离子蛋白分子异质性的遗传和翻译后原因。
