Skubitz K M, Ducker T P, Goueli S A
Department of Medicine, University of Minnesota Medical School, Minneapolis.
J Immunol. 1992 Feb 1;148(3):852-60.
The CD66 Ag is a neutrophil-specific "activation Ag" in that it is detected in low density on resting cells but its surface expression is up-regulated by stimulation (with the chemotactic peptide FMLP, the calcium ionophore A23187, and 12-O-tetradeconoyl-phorbol-13-acetate). Phosphorylation is an important mechanism of regulation of protein function. Although most studies of protein phosphorylation have focused on intracellular reactions, recent studies have provided evidence for the existence of ectoprotein kinase activity on the surface of several types of cells including human neutrophils. The role of ectoprotein kinase activity in cell function is unknown and little is known about the endogenous substrates of this enzyme system. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed that CD66 mAb specifically recognize a approximately 180-kDa phosphoprotein on the surface of human neutrophils. This protein was one of the major endogenous substrates for human neutrophil ectoprotein kinase activity. Phosphoamino acid analysis of the 180-kDa protein revealed that it contained predominantly phosphotyrosine. Preclearing studies demonstrated that this protein was also recognized by CD15 mAb, and by polyclonal anticarcinoembryonic Ag antiserum. In addition, the CD66 mAb reacted with purified carcinoembryonic Ag, biliary glycoprotein, and "nonspecific cross-reacting Ag." Thus, the neutrophil protein recognized by CD66 mAb appears to be a approximately 180-kDa form of the classical "nonspecific cross-reacting Ag" on human neutrophils.
CD66抗原是一种中性粒细胞特异性的“激活抗原”，因为在静息细胞上它以低密度被检测到，但其表面表达可通过刺激（用趋化肽FMLP、钙离子载体A23187和12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯）而上调。磷酸化是蛋白质功能调节的重要机制。尽管大多数关于蛋白质磷酸化的研究都集中在细胞内反应，但最近的研究已为包括人类中性粒细胞在内的几种类型细胞表面存在胞外蛋白激酶活性提供了证据。胞外蛋白激酶活性在细胞功能中的作用尚不清楚，对该酶系统的内源性底物也知之甚少。鉴定和表征胞外蛋白激酶活性的生理底物应有助于理解该酶活性在细胞功能中的作用。用[γ - 32P]ATP标记中性粒细胞后进行蛋白质的免疫沉淀及随后的凝胶电泳显示，CD66单克隆抗体特异性识别人类中性粒细胞表面一种约180 kDa的磷蛋白。该蛋白是人类中性粒细胞胞外蛋白激酶活性的主要内源性底物之一。对180 kDa蛋白的磷酸氨基酸分析表明，它主要含有磷酸酪氨酸。预清除研究表明，该蛋白也被CD15单克隆抗体和多克隆抗癌胚抗原抗血清识别。此外，CD66单克隆抗体与纯化的癌胚抗原、胆汁糖蛋白和“非特异性交叉反应抗原”发生反应。因此，CD66单克隆抗体识别的中性粒细胞蛋白似乎是人类中性粒细胞上经典“非特异性交叉反应抗原”的一种约180 kDa的形式。
