Modifications to lens membrane proteins during lens development and their potential functional consequences

Abstract

Abstract: : Purpose: To identify and correlate posttranslational modifications to lens membrane proteins with fiber cell age and to examine functional consequences of these modifications. Methods: Lens membranes were isolated from bovine and human lens sections by centrifugation of homogenized tissue. After washes with urea and NaOH, membranes were digested with trypsin or pepsin either in solution or in–gel after SDS–PAGE. Proteolytic products were analyzed by a combination of HPLC–tandem mass spectrometry and MALDI tandem mass spectrometry. Aquaporin 0 function was examined by swelling assays in Xenopus oocytes after expression of wild–type and mutant forms. Protein–protein interactions of AQP0 were identified by affinity chromatography with C–terminal peptides or with AQP0 antibodies. Results: Fiber cell age dependent truncation, deamidation, phosphorylation, and racemization/isomerization were observed for Aquaporin 0. The effect of C–terminal truncation on water permeability revealed no change in intrinsic permeability. The effect of truncation on AQP0 protein–protein interactions is implied by identification of C–terminal interacting partners, filensin and CP49. A new lens modification was observed on bovine MP20, C–mannosylation of two tryptophan residues. The functional significance of this new modification remains to be elucidated. Conclusions: Sensitive methods have been developed to identify developmentally related posttranslational modifications on two major lens membrane proteins, AQP0 and MP20. These modifications are likely to play functional roles in regulation of protein activity and/or in protein–protein interactions, key aspects of lens development.

Keywords: protein modifications–post translational • protein structure/function • proteomics