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Kulandaiappan Varadaraj, Sindhu Kumari; Extracellular loop positive charges of lens aquaporin 0 play a significant role in cell-to-cell adhesion. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1685.
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Investigate the role of extracellular loops of Aquaporin 0 (AQP0) on cell-to-cell adhesion (CTCA) function.
Extracellular loops A and C of mouse AQP0 were substituted with those of AQP1 through polymerase chain reaction using specific oligonucleotide primers. We have also replaced the positively charged residues in loop A of AQP0 with structurally compatible neutral residue to create R33Q and H40Q mutants. Water permeability (Pw) and CTCA properties of the chimeric and mutant AQP0 were studied by expressing them in Xenopus oocytes through cRNA injection and by transfecting into adhesion-deficient mouse fibroblast L-cells, as appropriate. Pw was studied using the shrinking and swelling assay. CTCA was tested using a method devised by our laboratory. Intact (wild type) AQP0 and E-cadherin served as positive controls while AQP1 served as a negative control for CTCA studies.
AQP0-AQP1-loop A chimera trafficked to the plasma membrane like the intact AQP0 while AQP0-AQP1-loop C chimera did not. Pw of AQP0-AQP1-loop A was 45.3 ± 5.8 μm/s while that of intact AQP0 was 46.4 ± 3.9 μm/s; Pw of control oocytes injected with distilled water was 10.9 ± 2.5 μm/s. CTCA assays showed that the adhesion property of AQP0-AQP1-loop A chimera was significantly reduced (24%; P< 0.001) compared to that of intact AQP0. Mutants AQP0-R33Q and AQP0-H40Q trafficked and localized at the plasma membrane like the intact AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P>0.05) in Pw of AQP0-R33Q (45.7 ± 6.9 μm/s) and AQP0-H40Q (44.7 ± 7.2 μm/s) compared to that of intact AQP0. However, the CTCA property of AQP0-R33Q (~22%) and AQP0-H40Q (19%) was significantly reduced (P< 0.001) in comparison to that of intact AQP0.
The data suggest that extracellular loop A substitution or point mutation of AQP0 might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or Pw but extracellular loop C substitution inhibited protein trafficking suggesting alteration/s in protein folding. Reduction in the CTCA observed for AQP0-AQP1-loop A chimera as well as AQP0-R33Q and AQP0-H40Q mutants suggest that the conserved positive charges in the Loop A may be critical for the CTCA function of AQP0.
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