2017 Fiscal Year Research-status Report
A new concept of salt handling by Na-binding proteins that immobilize excess Na+ to ease salt stress of seawater teleost fish
| Project/Area Number |
16K18575
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| Research Institution | The University of Tokyo |
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Principal Investigator |
黄 國成 東京大学, 大気海洋研究所, 特任研究員 (40526901)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Na-binding protein / osmoregulation / hypertension / protein purification |
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| Outline of Annual Research Achievements |
Stepping into the second year of the project, I have expanded the project scale in depths and horizons. To study the Na-binding molecule comparatively, I started the study using eels as the major model and have extended the established method to study tilapia, which is another euryhaline model with rapid increase in plasma osmolality after freshwater to seawater transfer. Several methods including 2D molecule electrophoresis, Na-22 autoradiography, transcriptomic analysis, have been used to identify the Na-binding molecules, and eventually the use of HPLC column purification successfully purified the Na-binding molecules from the eel esophageal and intestinal mucus. The purified Na-binding molecule is unexpectedly small in size (1.3 - 1.8 kDa), and highly hydrophilic. The molecule is now being analyzed by MS/MS de novo sequencing to identify the sequence and possible molecule modification such as glycosylation.
I have published the first discovery paper on the Na-binding molecules in Zoological Letters, which is frequently read by investigators worldwide, receiving 302 access at Zoological Letters homepage and 62 reads on Researchgate in 2 months. In terms of international presentation, I have attended a Jointed Symposium between University of Hawaii and University of Tokyo, and the presented topic on Na-binding in fish has attracted many researchers. After the symposium, I have started the collaboration with researchers from University of Hawaii to study the Na-binding phenomenon in tilapia.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
There was a shift in the research methods but the research plan is being performed accordingly. Instead of using the transcriptome database to search for the Na-binding molecules, I use HPLC column purification for the identification. Even with the partially purified molecule, the analysis using MS/MS is not straightforward because the Na-binding molecule is an unknown protein with no prior information. There is neither described protein with Na-binding property, nor there is motif known to bind Na. The MS/MS fingerprint failed to match with all known protein database, and I am proceeding to identify the Na-binding protein using de novo sequencing. However, as the protein is highly likely to be glycosylated, the MS/MS algorithms have to be optimized. Currently, I am collaborating with the Medical Biochemistry Department of the Jichi University to analyze the de novo sequencing of Na-binding proteins.
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| Strategy for Future Research Activity |
I will carry on the identification of Na-binding molecules with the MS/MS collaboration with Jichi University. After knowing the sequences of the Na-binding molecules, I will raise specific antibody against the molecules and identify the genes that transcribe the protein with the existing transcriptome database. I will start studying the Na-binding using in vivo eel model including tissue culture and the hormonal regulation of Na-binding. International collaboration will be extended through presenting the topic in international conferences. One important goal of the project is to introduce the concept of Na-binding to the mainstream of osmoregulation studies.
The discovery of esophageal club cells that carry Na-binding molecules in freshwater eels was a breakthrough. Freshwater is an ion-deficient environment and Na-binding mechanism was likely evolved to conserve ions in an energy-efficient way. Instead of pumping ions against a large concentration gradient between body fluid and ambient freshwater, Na-binding could prove an alternative strategy to manipulate the osmotic potential of the ions. I am now preparing a manuscript that describes the discovery of Na-binding in club cells , which is novel to vertebrate science since the roles of club cells in human were not fully understood.
I will spend the funding and effort on producing specific antibody for Na-binding molecules after the identification with MS/MS analysis. I will develop the in vivo Na-binding models which may allow further study on the control and regulation of this novel physiological process.
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| Causes of Carryover |
We planned to use the incurring amont on the purchase of eels but there was a shortage of eel supply during the time. Therefore, we transfer the remaining amount to next fiscal year to purchase animal for the experiment.
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Research Products
(6 results)
