Pati Roberta, Palazzo Claudia, Valente Onofrio, Abbrescia Pasqua, Messina Raffaella, Surdo Nicoletta Concetta, Lefkimmiatis Konstantinos, Signorelli Francesco, Nicchia Grazia Paola, Frigeri Antonio
Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy.
Foundation for Advanced Biomedical Research, Veneto Institute of Molecular Medicine, 35129 Padova, Italy.
Biomolecules. 2022 Apr 25;12(5):633. doi: 10.3390/biom12050633.
AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine and serine, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of -AQP4ex in human brain water homeostasis.
水通道蛋白4外显子（AQP4ex）是最近发现的一种通过翻译通读机制产生的水通道蛋白4（AQP4）异构体。它作为超分子膜复合物（通常称为颗粒正交排列，OAP）的一个组成部分，与AQP4的经典异构体M1和M23一起，在星形胶质细胞的血管周围终足中强烈表达。先前的定点诱变实验表明，位于AQP4ex延伸肽段中的丝氨酸在水通道活性的磷酸化过程中可能发挥作用。在本研究中，我们评估了人AQP4ex的有效磷酸化情况。小规模生物信息学分析表明，人、小鼠和大鼠的AQP4ex中只有丝氨酸是保守的。通过在兔体内产生磷酸特异性抗体来评估丝氨酸的磷酸化位点。首先通过酶联免疫吸附测定（ELISA）评估抗体与磷酸化肽及其未磷酸化类似物结合的特异性，定点诱变实验进一步证实了这一点。蛋白质免疫印迹和免疫荧光实验显示，磷酸化的AQP4ex（p-AQP4ex）在人脑中强烈表达，并通过蓝绿温和变性聚丙烯酰胺凝胶电泳（BN/PAGE）实验鉴定，其定位于血管周围星形胶质细胞终足的超分子聚集体中。总之，这些数据首次揭示了在AQP4ex延伸序列中丝氨酸处存在磷酸化形式的AQP4。因此，我们预计p-AQP4ex在人脑海水稳态中具有重要的生理作用。
