The non-enzymatic glycation of proteins may alter their structure and eventually lead to the formation of cross-links. Glycation and cross-linking of proteins may be involved in the etiology of such diverse diabetic and age-related complications as eye lens cataract and kidney glomerular basement membrane thickening. Among the lens crystallins, the gamma-crystallins are maximally glycated, both in vivo and in vitro. Extended glycation in vitro leads to the formation of non-reducible, covalently bound dimers. The presence of such dimers has been reported in senile cataracts. Proposed here is a study on the glycation of lens gamma-crystallin subfractions and of the lens capsule. The long-term aims are to determine the effect of glycation on (i) the structure of gamma-crystallins, and (ii) the phase-separation properties of the glycated and cross-linked gamma-crystallin solutions, and the eventual development of preventive and corrective measures.
The specific aims are: 1. Lens gamma-crystallin studies - (a) In vitro determination of the kinetics of glycation of calf gamma-crystallin subfractions. The rate of formation of the glycated monomer and non-reducible cross-linked dimers will be monitored. (b) Determination of the structure of water-soluble dimers and water-insoluble aggregates, especially the nature of the cross-links. (c) Determination of the location of the phase boundaries of the water-soluble glycated and cross-linked protein solutions. (d) Determination of the age-related or diabetic changes in the proportions of normal human gamma-crystallins and estimation of the differences in the extent of their glycation. Analysis of the human lens water-insoluble fraction from normal, aged, and diabetic lenses as in (b). II. Lens capsule studies - Determination of the change in structure and stability of lens capsule upon glycation in vitro, and correlation of the spectral alterations observed, with the changes in human lens capsule from normal, aged, and diabetic lenses. Methods used will be cation-exchange and molecular-sieve chromatography for the fractionation of gamma-crystallins. Phenylboronate affinity chromatography will also be used for the glycated proteins. Absorption, fluorescence, circular dichroism, and vibrational spectroscopies will be used for general structural characterization. Labelled (13C and 2H) sugars will be used in I(b) above, to prepare advanced glycation products, which will be examined by high resolution and solid-state nuclear magnetic resonance spectroscopy and vibrational spectroscopy.
