Mobile Zinc-Responsive Protein Labeling Reagent Zin-Pro Capture | Funakoshi

Zin-Pro Capture
- A protein labeling reagent in the presence of mobile zinc in the cell -

Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.

So far, to prove intracellular behavior of Zinc ion, imaging probes for metal Zinc has been used.
Such probes can only detect the location of Zinc in the cell, and it was difficult to find which proteins are related to transportation of Zinc. This is because current Zinc imaging probe is compatible with live cell imaging but not optimized for immunochemistry, as such probes are not suited for fixation.

Zin-Pro Capture is a labeling reagent which overcomes such disadvantage.

This product is commercialized by the research result of Professor Hamachi, at Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University.
Miki et al., Nat. Methods 13, 931-937 (2016)
* In this paper, Zin-Pro Capture is called as AIZin-2.

Zn²⁺ is tightly bound to proteins and plays essential roles in precise protein folding, and enzymatic function of metalloproteins as cofactor. A simulation suggests about 10% of proteins are estimated as Zn²⁺-binding proteins1). Mobile Zn²⁺, which is free form or loosely bound to protein surface, is the second class of zinc population and act as a signaling molecule. Recent evidences show an abnormal increase in mobile Zn²⁺ concentration induce pathological events including neuronal degeneration etc. Although fluorescent probes for real time monitoring of mobile Zn²⁺ in live cells have been developed, these probes could not reach comprehensive characterization of Zn²⁺ distribution mechanism and how Zn²⁺ mobiles in cells. To approach zinc homeostasis in cells and to identify proteins involved in zinc distribution on the molecular level, chemical reagents which can be applied for protein experiments are desirable. Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn²⁺ ion (Figure 1). Zin-Pro Capture has high selectivity for Zn²⁺ ion, not showing labeling activity with the other abundant metal ions such as Mn²⁺, Fe²⁺/Fe³⁺, Ca²⁺ and Mg²⁺. Its Zn²⁺-responsive labeling activity enable to monitor mobile Zn²⁺ species in the cell on the molecular basis. After labeling Zn-proximal proteins with fluorescein tag in living cells by Zin-Pro Capture, cells are lysed and the labeled proteins are subsequently enriched by immunoprecipitation with anti-fluorescein antibody. The labeled and purified proteins are characterized by western blotting with antibodies against interest protein or comprehensively identified by mass analysis. Observation of intracellular localization of labeled proteins by Zin-Pro Capture is also available in the fixed cells. Miki el al. showed that mobile Zn²⁺ in C6 glioma cells was dramatically released to cytosol from metal binding proteins such as metallothionein by oxidative stress and subsequently accumulated in vesicles²⁾. This study revealed with Zin-Pro Capture (AIZin2 in the paper2)) that oxidative stress induced Zn-rich vesicles in glioma cells were identified as COPII, COPI and ER-Golgi intermediate compartment (ERGIC) between ER and Golgi appratus^2-3).

In the absence of Zn(II) ion, protein labeling activity of Zin-Pro Capture is poor. On the other hand, in the presence of Zn(II), Zin-Pro Capture is activated by mobile Zn(II) and shows protein labeling activity of fluorescent tag to proximal proteins.

Chemical labeling activity of Zin-Pro Capture is dependent on the amount of Zn(II) ion in the cell. Highly concentrated Zn(II) increases Zin-Pro Capture activity and proteins located on Zn(II) rich compartment are densely labeled.
On the other hand, in the Zn(II) poor region, Zin-Pro Capture shows low efficiency and little proteins are labeled.
The labeled proteins can be purified by immunoprecipitation with anti-fluorescein antibody and applied for western blotting and mass spectrometry etc and also observed by microscopy in the fixed cells.

* Zin-Pro Capture is not optimized for live cell imaging.

Fig.1 Localization of labeled protein in the cells
C6 glioma cells were treated by S-nitrosocysteine (SNOC ; NO producing reagent), and labeled by Zin-Pro Capture.
Cells were fixed by methanol.

Fig. 2 Analysis of labeled proteins in western blot and Mass Spectrometer
C6 glioma cells were treated by S-nitrosocysteine 200 μM and purified by anti-fluorescein antibody (anti-FL). Analysis were done in western blot (A) and Mass Spectrometer (B).
(A) Immunoprecipitated proteins were detected in Western Blot. Strong band means the protein is located in the Zinc-rich region. Citrate synthetase (mitochondrial protein) was detected at 10 minutes after treatment, and Calnexin and GRP 94 (ER proteins) were detected at 180 minutes after treatment.
This concludes that mobile zinc were released from metallothionein in cytoplasm, and transported to ER by way of Mitochondria.
(B) Assay by Mass Spectrometer 0, 10, 180 minutes samples were labeled with stable isotope by TMT method, and analyzed by LC-MS/MS.

Zin-Pro Capture reveals mobile zinc under oxidative stress
- transiently transported into mitochondria
- accumulated in ER and ER-Golgi intermediate compartment (ERGIC).

in vitro ischemia model
Cell : C6 glioma
Stimuli : Nitric oxide-based oxidative stress

1) Andrein et al., J. Proteome Res., 5, 196–201 (2006).
2) Miki et al., Nat. Methods., 13, 931-937 (2016)
3) Motiwala and Martin, Nat. Methods., 13, 917-918 (2016)