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Nakazawa Y
,
Oka M
,
Furuki K
,
Mitsuishi A
,
Nakashima E
,
Takehana M
.
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To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lens-specific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated. Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOH-terminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOH-terminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin. The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lensAQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0. The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens.
Figure 1. Interaction between GST-AQP0-C and filensin in rat lens extract. A: The interaction between purified GST-AQP0-C and lens filensin. Western blotting analysis was performed with anti-filensin outer tail domain antibody. Lane 1: urea fraction of rat lens extract showing 94 kDa filensin. Purified GST-AQP0-C incubated with lens extract (lane 2). Filensin (94 kDa) was detected by anti anti-filensin outer tail domain antibody that interacts with the C-terminal domain of AQP0. Lane 3 contained purified GST-AQP0-C as a control. B: The interaction between lens filensin and purified GST-AQP0-C or its pseudophosphorylated forms (1, 3, or 5 sites mutated in the COOH-terminal peptide) was investigated by western blotting with anti-filensin rod antibody. Lane 1: urea fraction of rat lens extract showing 94, 50, and 38 kDa filensin protein bands. Purified GST-AQP0-C or GST-AQP0-C with 1, 3, or 5 pseudophosphorylated sites interacts with the 94 and 50 kDa filensin bands but not with the 38 kDa band (lanes 2, 4, 6, and 8, respectively). In the absence of lens extract, neither the purified GST-AQP0-C nor the 1, 3, or 5 site-mutated AQP0-C constructs interacted with the anti-filensin rod antibody (lanes 3, 5, 7, and 9, respectively). As a further control, when GST alone was incubated with lens extract, it did not interact with lens filensin (lane 10). Lane M in A and B shows the molecular markers.
Figure 2. Interaction between GST-filensin-tail or GST-filensin-rod and AQP0 in rat lens extract. The interaction between purified recombinant GST-filensin-tail or GST-filensin-rod with lens AQP0 was detected by western blotting with an anti-AQP0 COOH-terminal antibody. Lane 2 shows a 23 kDa AQP0 protein band present in the Triton-X100 extract fraction from rat lens. Incubation of GST-filensin-tail with the Triton-X100 extract fraction from rat lens revealed an AQP0 protein band at 23 kDa, illustrating the interaction between the filensin tail region and AQP0 (lane 3). In the absence of lens extract, the purified GST-filensin-tail construct did not react with the anti-AQP0 antibody (lane 4). There was no interaction between the purified filensin rod domain and AQP0 in the lens extract fraction (lane 5). In the absence of lens extract, GST-filensin-rod conjugated to glutathione beads did not contain a peptide that reacted with the anti-AQP0 antibody (lane 6). No AQP0 band was detected when GST alone was incubated with lens extract (lane 7). Anti-AQP0 antibody did not react with GST (lane8). Lane 1 shows molecular weight markers.
Figure 3. Effect of pH on the interaction between recombinant GST-AQP0-C and the recombinant filensin tail peptide. The filensin tail peptide was cleaved from the purified recombinant GST-fusion construct using preScission protease. Recombinant GST-AQP0-C and the recombinant filensin tail peptide were then incubated in different pH buffers and the interaction between the AQP0 COOH-terminal peptide and the filensin tail peptide was detected by western blotting with an anti-filensin tail antibody. Lanes 1, 2, 3, and 4 show the interaction between the filensin tail peptide and GST-AQP0-C in buffers with pH 7.0, 7.5, 8.0, and 8.5, respectively. Lane 5, GST alone incubated with the recombinant filensin tail peptide. Lane 6, purified filensin tail peptide cleaved from the GST-filensin tail fusion construct. Lane 7, GST-AQP0-C alone. Lane 8, GST alone.
Figure 4. Xenopus oocyte expressing AQP0 AQP0 in Xenopus oocytes was injected with cRNA AQP0 was detected by western blotting.
Figure 5. Change of diameter of Xenopus oocytes expressing AQP0 Xenopus oocytes expressing AQP0 were immersed in hypotonic solution (30% ND96 soluion). The diameter of Xenopus oocytes injected with 25 ng of AQP0 cRNA (closed square) or water (closed circle) was measured. The oocytes expressing AQP0 permeated more water than control.
Figure 6. Water permeability of AQP0 expressed in Xenopus oocytes. The osmotic water permeability (Pf) of AQP0 expressed on Xenopus oocyte membranes was determined by measuring the change in oocyte diameter. To balance the loading of ribosomes and obtain the same amount of transcribed AQP0 in each reaction, 12.5 ng cRNA of GST was co-injected as a blank with 12.5 ng cRNA of AQP0 (AQP0+GST). The water permeability was reduced when Xenopus oocytes were co-injected with 12.5 ng filensin COOH-terminal peptide cRNA and 12.5 ng of AQP0 cRNA, however, there was no significant difference between the water permeability value for AQP0+GST and the value obtained when 12.5 ng filensin rod domain cRNA was co-injected with 12.5 ng AQP0 (AQP0+fil rod) cRNA. These results suggested that the filensin tail region interacted with AQP0 to reduce the water permeability of AQP0, while the filensin rod peptide had no effect.
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