Schwertner Katharina, Gelles Katharina, Leitner Judith, Steinberger Peter, Gundacker Claudia, Vrticka Ruben, Hoffmann-Sommergruber Karin, Ellinger Isabella, Geiselhart Sabine
Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
Institute of Immunology, Medical University of Vienna, Vienna, Austria.
Heliyon. 2023 Jul 18;9(8):e18247. doi: 10.1016/j.heliyon.2023.e18247. eCollection 2023 Aug.
The receptor for advanced glycation end products (RAGE) is encoded by , a gene that is subjected to tissue-specific alternative splicing. Splice variants of RAGE in intestine and placenta are unknown and contradictory data concerning RAGE protein expression in these tissues have been published. As a basis for future functional studies, we examined RAGE expression in small intestine, colon and placentas. PCR cloning revealed that full-length RAGE is the only RAGE transcript isoform expressed in placenta. In the small intestine, the major transcript isoform detected was RAGE_v1 encoding the C-terminally truncated soluble receptor. In the colon, both full-length RAGE as well as several splice variants were identified. Four antibodies were used to study protein expression by immunoblotting and were carefully validated. Appropriate controls were essential to avoid misinterpretation of bands caused by non-specific reactivity of antibodies. Only one of four antibodies tested detected full-length RAGE in placenta, whereas no RAGE-specific band was detected in intestinal tissues despite loading >30-fold more intestinal tissue than the positive control, human lung. RAGE expression levels in the placenta were 100-fold lower compared with human lung when analyzed by ELISA, and no significant differences in RAGE expression were detected between healthy placentas and placentas from women with preeclampsia, gestational diabetes mellitus, or fetal growth restriction. We conclude that healthy placental chorionic tissue expresses low levels of full-length RAGE, whereas expression of the tissue-specific intestinal isoforms is below the limit of detection. Low RAGE expression levels in combination with a lack of antibody validation may explain the conflicting published results on RAGE protein expression in intestine and placenta.
晚期糖基化终末产物受体（RAGE）由一个基因编码，该基因存在组织特异性可变剪接。肠道和胎盘中RAGE的剪接变体尚不清楚，并且关于这些组织中RAGE蛋白表达的相互矛盾的数据已经发表。作为未来功能研究的基础，我们检测了小肠、结肠和胎盘中RAGE的表达。PCR克隆显示全长RAGE是胎盘中唯一表达的RAGE转录异构体。在小肠中，检测到的主要转录异构体是编码C末端截短的可溶性受体的RAGE_v1。在结肠中，鉴定出了全长RAGE以及几种剪接变体。使用四种抗体通过免疫印迹研究蛋白表达，并进行了仔细验证。适当的对照对于避免因抗体非特异性反应导致的条带误判至关重要。所测试的四种抗体中只有一种在胎盘中检测到全长RAGE，而在肠道组织中未检测到RAGE特异性条带，尽管加载的肠道组织比阳性对照人肺多30倍以上。通过ELISA分析时，胎盘与人类肺相比，RAGE表达水平低100倍，并且在健康胎盘与患有先兆子痫、妊娠期糖尿病或胎儿生长受限的女性的胎盘之间未检测到RAGE表达的显著差异。我们得出结论，健康的胎盘绒毛组织表达低水平的全长RAGE，而组织特异性肠道异构体的表达低于检测限。RAGE表达水平低以及缺乏抗体验证可能解释了关于肠道和胎盘中RAGE蛋白表达的相互矛盾的已发表结果。
