Abstract: SA-PO295
Phosphorylation Profile of Human AQP2 in Urine Exosome Identified by LC-MS/MS Phosphoproteomic Analysis
Session Information
- Fluid and Electrolytes: Basic - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid and Electrolytes
- 901 Fluid and Electrolytes: Basic
Authors
- Ishibashi, Kenichi, Meiji Pharmceutical university, Kiyose, Japan
- Tanaka, Yasuko, Meiji Pharmceutical university, Kiyose, Japan
- Sasaki, Sei, Meiji Pharmceutical university, Kiyose, Japan
- Yamamoto, Tadashi, Niigata University, Niigata, Japan
Background
AQP2 water channel is the key membrane protein which determines the water permeability of collecting ducts. Multiple phosphorylation sites at the C-terminal of AQP2 are identified including S256, S261 and S264. Interetingly, the amino acid at S269 of rodents AQP2 is Thr in human AQP2. The phosphorylation of S269 in rodents has been shown to be an important signal for the apical membrane accumulation. However, the phosphorylation of T269 in human is unknown. As AQP2 is excreted into the urine by the endocytosed exosomes, human AQP2 protein is easily obtained from the urine. The purpose of this study was to examine the phosphorylation status of human AQP2 from urine exosomes.
Methods
Human urine samples of volunteers were obtained from the morning first urine. Urine exosomes were isolated by differential centrifugations and digested with trypsin in solution. Tryptic peptides were purified by MonoSpin column (GL Sciences, Tokyo, Japan) and analyzed thrice by LC–MS/MS (Bruker Tims TOFpro). Western blots were used to detect the AQP2 phosphorylation with a usual and S256, S261, S264, and S269-phosphorylated AQP2-specific antibodies.
Results
Summation of thrice analysis identified total 185 PSMs (peptide spectrum match) of phosphorylated AQP2 at Ser and/or Thr. The most dominant form was S256 phosphorylated form (n=154), followed by S261 form (n=14). Small numbers of phosphorylation were observed at T244 (n=6), S264 (n=4), and T269 (n=2). Western blot of human urine exosomes detected dominant S256 and S261-phosphorylations and much lower S264 and T269-phosphrylations.
Conclusion
These results indicate that the all phosphorylation sites of human AQP2 including T269 are indeed phosphorylated and S256- and S261-phosphorylations play a dominant role in its urinary exosomal excretion. The newly identified T244 phosphorylation is intriguing and worth further studies.
Funding
- Government Support - Non-U.S.