June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
N- or C- Terminal Cleavage of Mature Fiber Cell Aquaporin 0 Does Not Impede the Water Channel and Cell-to-Cell Adhesion Functions
Author Affiliations & Notes
  • Kulandaiappan Varadaraj
    Physiology and Biophysics, State University of New York, Stony Brook, NY
    SUNY Eye Institute, New York, NY
  • Sindhu Kumari
    Physiology and Biophysics, State University of New York, Stony Brook, NY
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 485. doi:
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      Kulandaiappan Varadaraj, Sindhu Kumari; N- or C- Terminal Cleavage of Mature Fiber Cell Aquaporin 0 Does Not Impede the Water Channel and Cell-to-Cell Adhesion Functions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):485.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Investigate the effect of N- or C-terminal truncation of Aquaporin 0 (AQP0) - as in the case of lens mature fiber cells - on the water channel and cell-to-cell adhesion functions, with reference to the intact AQP0 (263 amino acids) which performs both functions.

Methods: The following amino acid deletions were introduced into the wild type AQP0 through specifically designed oligonucleotide primers and polymerase chain reaction, to create various truncation mutants mimicking the natural truncations: Amino acids (AA) 2-6, AA 244-263, AA 247-263, AA 250-263 and AA 260-263. The water channel and cell-to-cell adhesion properties of these truncated AQP0 were studied by expressing them in Xenopus oocytes through cRNA injection and by transfecting them into adhesion-deficient mouse fibroblast L-cells, respectively. Water permeability was studied using the shrinking and swelling assay. Cell-to-cell adhesion was tested using a traditional assay as well as a method devised in our laboratory; positive controls namely, intact AQP0 and E-cadherin, and a negative control, AQP1 were also tested to validate the cell-to-cell adhesion property of truncated AQP0.

Results: Water permeability and cell-to-cell adhesion were not affected (P< 0.001) in the 2-6 amino acids cleaved N-terminal truncation mutant, compared to that of the intact AQP0. C-terminal truncated (AA 244-263, AA 247-263, AA 250-263 and AA 260-263) AQP0 proteins trafficked to the plasma membrane (PM). However, the membrane localization of AA 244-263 truncated mutant was reduced, with a consequent decline in water permeability and cell-to-cell adhesion. Nevertheless, in all of the C-terminal mutants the water transport and cell-to-cell adhesion functions with respect to the amount AQP0 expressed at the membrane, were not impeded and remained comparable to those of intact AQP0.

Conclusions: The data suggest that the deleted amino acids may not be critical for the functions of AQP0. In human, the N- and C- terminal ends of AQP0 in the older fiber cells get cleaved off as early as 2 years of age, possibly to assist in the process of the compact packing of the cytoplasmic proteins and mature fiber cells, in order to adjust the refractive index of the growing lens.

Keywords: 445 cataract • 657 protein modifications-post translational • 446 cell adhesions/cell junctions  
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