Date of Award

2003

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

College

College of Graduate Studies

First Advisor

Kevin J. Schey

Second Advisor

Rosalie K. Crouch

Third Advisor

John Hildebrandt

Fourth Advisor

John R. Raymond

Fifth Advisor

Steven A. Rosenzweig

Abstract

Aquaporin 0 (AQP0, MIP), a water channel protein found in the ocular lens fiber cells, has been proposed to maintain osmotic homeostasis by contributing to an internal circulatory system within the avascular lens. The formation of cataracts in patients with mutations in the AQP0 gene and in heterozygous AQP0 deficient mice suggests that AQP0 is necessary for establishing and preserving lens transparency. Previous structural characterization of AQP0 revealed that the C-terminus, a putative regulatory domain, is subject to many age-related posttranslational modifications. The goal of the present study was to map the spatial distribution of posttranslationally modified forms of AQP0 within normal human lenses and determine the effects of the post translational modifications on AQP0 membrane water permeability. Mass spectrometric analysis of AQP0 isolated from concentrically dissected human lenses ages 34, 35, and 38 revealed novel sites of phosphorylation at shrines 229 and 231, backbone cleavage at residues 249, 260, 261, and 262, and isomerization/racemization of L-Asp 243 to D-iso-Asp. As anticipated the extent of truncation and isomerization/racemization increased as a function of fiber cell age, whereas the level of phosphorylation was highest in the inner cortex and decreased in the lens nucleus. The water permeability of AQP0 was not affect by substitution of phosphorylation site 231, incorporation of a negative charge at sites of deamination 246 and 259, nor removal of the C-terminal twenty amino acid residues. These findings suggest that within the human lens modified AQP0 in aged fiber cells may retain the ability to transport water. Elucidation of the sites and extent of post translational modifications of AQP0 revealed that truncation at residues 24 and 259, the most abundant sites of backbone cleavage detected, may occur through a spontaneous truncation event. Identification of an age-related increase in racemized/isomerized D-iso-Asp 243 provides evidence for the presence of protein L-isoaspartate O-methyl transferase, a potentially active protein repair enzyme in the human lens. The results obtained and methods developed in this study provide the groundwork for understanding the structure and function of AQP0 in the normal human lens and future investigation of the role of AQP0 in the development of cataract.

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