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The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus

Oligomerization of the AQP11-C227A mutant. (A) Oligomerization state of Myc-hAQP11 or Myc-hAQP11-C227A was evaluated using a chemical cross-linker, paraformaldehyde. Typical examples of multimerization of Myc-hAQP11 (far left lane) and Myc-hAQP11-C227A (2nd and 3rd lanes from the left) are shown. (B) Corresponding data in 2nd and 3rd lanes from the left of A at 20-fold longer exposure time. (C) The results of densitometric analysis of cross-linked Myc-hAQP11 (white bar) and Myc-hAQP11-C227A (grey bar) are shown. The ratio of multimerized proteins (more than dimer) to total proteins (monomer + multimerized proteins) was taken as an index of the multimerization level. Values are presented as mean ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 vs. Myc-hAQP11 (Student’s t-test).
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f0020: Oligomerization of the AQP11-C227A mutant. (A) Oligomerization state of Myc-hAQP11 or Myc-hAQP11-C227A was evaluated using a chemical cross-linker, paraformaldehyde. Typical examples of multimerization of Myc-hAQP11 (far left lane) and Myc-hAQP11-C227A (2nd and 3rd lanes from the left) are shown. (B) Corresponding data in 2nd and 3rd lanes from the left of A at 20-fold longer exposure time. (C) The results of densitometric analysis of cross-linked Myc-hAQP11 (white bar) and Myc-hAQP11-C227A (grey bar) are shown. The ratio of multimerized proteins (more than dimer) to total proteins (monomer + multimerized proteins) was taken as an index of the multimerization level. Values are presented as mean ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 vs. Myc-hAQP11 (Student’s t-test).

Mentions: We have shown that AQP11 is able to form a multimerized structure [8]. Recently, our group also observed the reduced multimer formation of AQP11-C227S using a cross-linker, paraformaldehyde [13]. We therefore evaluated the multimeric structure of AQP11-C227A using the same method. As shown in Fig. 4A, paraformaldehyde was able to clearly produce a band at ∼75 and ∼50 kDa in addition to a band at ∼25 kDa for Myc-hAQP11, confirming that AQP11 has a multimerized structure. In contrast, when Myc-hAQP11-C227A was examined, the bands at ∼75 and ∼50 kDa were missing, but the band at ∼25 kDa was visible. Even when we employed a 20-fold longer exposure time, no bands at ∼75 kDa were evident (Fig. 4B). Fig. 4C shows the summarized data for Fig. 4A, indicating a reduced ability of Myc-hAQP11-C227A to form a multimerized protein in comparison with the wild-type protein. Taken together with the reported data, these results indicate that Cys227 of AQP11 has an important role in homo-multimerization.


The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Oligomerization of the AQP11-C227A mutant. (A) Oligomerization state of Myc-hAQP11 or Myc-hAQP11-C227A was evaluated using a chemical cross-linker, paraformaldehyde. Typical examples of multimerization of Myc-hAQP11 (far left lane) and Myc-hAQP11-C227A (2nd and 3rd lanes from the left) are shown. (B) Corresponding data in 2nd and 3rd lanes from the left of A at 20-fold longer exposure time. (C) The results of densitometric analysis of cross-linked Myc-hAQP11 (white bar) and Myc-hAQP11-C227A (grey bar) are shown. The ratio of multimerized proteins (more than dimer) to total proteins (monomer + multimerized proteins) was taken as an index of the multimerization level. Values are presented as mean ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 vs. Myc-hAQP11 (Student’s t-test).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048847&req=5

f0020: Oligomerization of the AQP11-C227A mutant. (A) Oligomerization state of Myc-hAQP11 or Myc-hAQP11-C227A was evaluated using a chemical cross-linker, paraformaldehyde. Typical examples of multimerization of Myc-hAQP11 (far left lane) and Myc-hAQP11-C227A (2nd and 3rd lanes from the left) are shown. (B) Corresponding data in 2nd and 3rd lanes from the left of A at 20-fold longer exposure time. (C) The results of densitometric analysis of cross-linked Myc-hAQP11 (white bar) and Myc-hAQP11-C227A (grey bar) are shown. The ratio of multimerized proteins (more than dimer) to total proteins (monomer + multimerized proteins) was taken as an index of the multimerization level. Values are presented as mean ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 vs. Myc-hAQP11 (Student’s t-test).
Mentions: We have shown that AQP11 is able to form a multimerized structure [8]. Recently, our group also observed the reduced multimer formation of AQP11-C227S using a cross-linker, paraformaldehyde [13]. We therefore evaluated the multimeric structure of AQP11-C227A using the same method. As shown in Fig. 4A, paraformaldehyde was able to clearly produce a band at ∼75 and ∼50 kDa in addition to a band at ∼25 kDa for Myc-hAQP11, confirming that AQP11 has a multimerized structure. In contrast, when Myc-hAQP11-C227A was examined, the bands at ∼75 and ∼50 kDa were missing, but the band at ∼25 kDa was visible. Even when we employed a 20-fold longer exposure time, no bands at ∼75 kDa were evident (Fig. 4B). Fig. 4C shows the summarized data for Fig. 4A, indicating a reduced ability of Myc-hAQP11-C227A to form a multimerized protein in comparison with the wild-type protein. Taken together with the reported data, these results indicate that Cys227 of AQP11 has an important role in homo-multimerization.

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus