Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation
Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation
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DOI：
10.1016/j.cellimm.2005.08.033
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发表时间：
2005-07-01
影响因子：
4.3
通讯作者：
Haag, F
中科院分区：
文献类型：
--
作者：
Koch-Nolte, F;Glowacki, G;Haag, F
ADP-核糖基转移酶（ART）将ADP-核糖从NAD转移到靶蛋白中的精氨酸、天冬酰胺或半胱氨酸残基。这种翻译后蛋白质修饰是霍乱毒素和其他细菌毒素在人类宿主细胞中引起病理学的机制。分子克隆已经在小鼠中鉴定出五种毒素相关的GPI锚定细胞表面ART（ART 1、ART2.1、ART2.2、ART 3和ART 4），在人类中鉴定出三种毒素相关的GPI锚定细胞表面ART（ART 1、ART 3和ART 4）。ART 2由于其通过ADP-核糖基化激活细胞溶解性P2 X7嘌呤能受体的能力而引起了人们的兴趣，ART 2由小鼠基因组中的两个功能性基因拷贝编码，而人类基因组携带两个失活的ART 2假基因。我们为每个功能性人类和小鼠ART的FLAG标记版本产生了稳定的转染子。使用基因免疫，我们提出了单克隆抗体，识别活细胞表面上的天然人类ART。这些mAb中的一些识别与小鼠ART直向同源物共享的表位，但不识别与更远的ART旁系同源物共享的表位。通过FACS筛选原代细胞和已建立的细胞系，发现非细胞、中性粒细胞和髓性白血病细胞系表达ART 1，但实体瘤来源的细胞系不表达。ART 1和ART 4分别被指定为CD 296和CD 297。(c)2005年爱思唯尔公司All rights reserved.
ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2 which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by rnonocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively. (c) 2005 Elsevier Inc. All rights reserved.