Abstract

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.

Highlights

  • The human lens is a tissue composed of onion-like layers of fiber cells

  • Protein Purification and Structural Characterization—To determine how these mutations, which have been linked to early onset cataract in mice, affect H␥D-Crys, three human proteins were made: V75D, I90F, and L5S. (Numbering of residues is based on that of the wild-type H␥D crystal structure, Protein Data Bank code 1HK0.) The residues Val75 and Ile90 are conserved and occur at identical positions in mouse and human ␥D-crystallins

  • The mutant alleles encoding V75D and I90F were constructed by site-specific mutagenesis of the human cDNA sequence cloned into the vector pQE1

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Summary

The abbreviations used are

H␥D-Crys, human ␥D-crystallin; M␥S-Crys, murine ␥S-crystallin; N-td, N-terminal domain; C-td, C-terminal domain; GdnHCl, guanidinium hydrocholoride; DTT, dithiothreitol; WT, wild type. Substitution of any of the six residues forming the hydrophobic domain interface of H␥D-Crys resulted in a sharp destabilization of the native state and increased population of a partially folded intermediate with an unfolded N-td and folded C-td [22]. The three amino acid substitutions, F9S in M␥S-Crys [40, 49, 50] and V76D [38] and I90F [39] in murine ␥D-crystallin, are associated with congenital or early onset cataract in mice. All proteins were expressed in Escherichia coli and purified to study their overall conformation and thermodynamic and kinetic stabilities Their significant destabilization highlights the importance of the tightly packed hydrophobic core of the ␥-crystallins and sheds light on the possible mechanisms of congenital cataract formation associated with these mutations

EXPERIMENTAL PROCEDURES
RESULTS
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