Lens Membrane in Relation to Human Cataractogenesis

Kansas State University, Manhattan KS

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Abstract

DESCRIPTION (provided by applicant): Although posttranslational modifications of lens proteins play a causative role in biogenesis of human senile cataracts, surprisingly little is known concerning the identity of specific amino acids that are modified in these lenses. Nonenzymatic deamidation of asparagine and glutamine residues is probably the most prevalent modification occurring in the human lens, and studies from other tissues have suggested that resistance to deamidation is a general property of stable proteins. The central hypothesis of this proposal is that the aged human lens contains gamma/beta crystallins with very low rates of deamidation, and that if deamidation does occur, it could result in deleterious effects to the lens, such as the formation of high molecular weight aggregates, low molecular weight cleavage products, and eventual cataract. To identify and quantitate deamidation, we have developed a novel approach involving the use of synthetic peptide standards, HPLC, mass spectral analysis and N-terminal sequencing. This methodology will be used to identify and quantitate deamidation of specific residues from the high molecular weight aggregate and low molecular weight cleavage products, as well as total proteins from dissected nuclei of human cataractous and aged-matched normal lenses. Molecular modeling will be used to correlate known structural properties of crystallins, with their observed rates of deamidation. Finally, human gamma/beta crystallins containing specific residues found to be deamidated will be expressed in a recombinant system and characterized by various biochemical and biophysical methods. Together, this project will identify and quantitate deamidation of specific residues of gamma/beta crystallins in the aging normal and cataractous human lens, followed by determination of the consequences of these modifications upon the stability and structural properties of the expressed deamidated proteins.