Di Stadio Chiara Stella, Altieri Filomena, Miselli Giuseppina, Elce Ausilia, Severino Valeria, Chambery Angela, Quagliariello Vincenzo, Villano Valentina, de Dominicis Gianfranco, Rippa Emilia, Arcari Paolo
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy.
Biochimie. 2016 Feb;121:151-60. doi: 10.1016/j.biochi.2015.12.010. Epub 2015 Dec 15.
AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity. In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues. To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl(-) absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the overexpression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level.
AMP18是一种在正常胃黏膜中表达但在胃癌中缺失的胃特异性分泌蛋白。AMP18在维持胃黏膜完整性方面发挥着重要作用，其特征是存在一个由约100个氨基酸组成的BRICHOS结构域，该结构域也存在于几种不相关的蛋白质中，可能具有类似分子伴侣的活性。在这项研究中，我们采用了一种功能蛋白质组学策略来鉴定潜在的AMP18相互作用蛋白，旨在增加对其在胃细胞系和组织中功能作用的了解。为此，通过亲和色谱策略纯化重组生物素化的AMP18，并将其与来自人正常胃黏膜的蛋白质提取物孵育。使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）通过肽质量指纹图谱鉴定相互作用蛋白。相互作用蛋白库包含SLC26A3，一种在肠上皮细胞顶端膜中表达的蛋白质，据推测在Cl(-)吸收和液体稳态中起关键作用。在AMP18下拉后，用抗SLC26A3的Western印迹法对转染的AGS细胞提取物进行检测，也证实了这种相互作用。此外，共聚焦显微镜也验证了AMP18与SLC26A3之间的相互作用，显示这两种蛋白质在质膜水平共定位。更重要的是，我们首次表明SLC26A3在胃癌中表达下调，并且在AMP转染的胃癌细胞中AMP18的过表达在转录和翻译水平上均上调了SLC26A3的表达，后者可能是通过激活丝裂原活化蛋白激酶（MAP）激酶途径。这些发现强烈表明，AMP18可能还通过调节SLC26A3水平在维持黏膜完整性方面发挥抗炎作用。
