Deamidation in Human Lens βB2-Crystallin Destabilizes the Dimer

Abstract

Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. βB2 is the most abundant β-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. βB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among β-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type βB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes βB2 and may disrupt the function of βB2 in the lens.Cataract formation is the leading cause of blindness worldwide and affects an estimated 16 to 20 million people (1). Crystallins are highly soluble structural proteins that comprise 90% of lens proteins (2). The highly ordered, tightly packed crystallins make up the transparent structure of the lens and allow it to focus light onto the retina. These proteins undergo little turnover during the human life span, allowing for accumulation of modifications that may decrease crystallin solubility, alter lens transparency, and diminish vision.During normal aging, crystallins are modified by truncation, oxidation, deamidation, and disulfide bond formation (3-9). Most notable by two-dimensional gel electrophoresis are trains of deamidated species (3). Each of the major crystallin polypeptides in the lens is deamidated during aging, and most are deamidated at multiple residues. Deamidation introduces a negative charge at physiological pH by replacing an amide with a carboxyl group and causes some isomerization. These changes potentially disrupt protein structure.Recently, 34 sites of deamidations have been identified in a single cataractous lens (10). Due to the difficulty of detecting a single mass unit change resulting from deamidation, quantitative data associating deamidation with cataract formation is limited. The large numbers of † Funding provided by the National Institutes of Health, National Eye Institute, Grant Reference No. R01-EY012239 (K.J.L.), Oregon Lions Sight and Hearing Foundation grant (K.J.L.), Shriners Hospitals Shared Facilities Grant, a...

Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

“…Even the unfolding of the monomer, γD, was best fit by three transitions with the first and second transition unfolding of the N-terminal domain and the third transition unfolding of the C-terminal domain (54). However, fitting the data to a three state model resulted in large errors of at least one of the transitions for βB2 (35), γD (37), and for βA3 in this study. Silent intermediates have been hypothesized for small globular proteins, where the intermediates are less stable than the unfolded state (55).…”

Glutamine Deamidation Destabilizes Human γD-Crystallin and Lowers the Kinetic Barrier to Unfolding

“…In the ␤-crystallins, deamidation of domain interface glutamines has a large effect on stability where a monomeric intermediate is populated in equilibrium experiments (21,22). Human ␤B1 crystallin deamidated in the domain interface also had an increased tendency to aggregate at high temperatures and required more ␣-crystallin to suppress aggregation (24).…”

“…Deamidation of a glutamine in the connecting peptide of ␤B1-Crys caused the protein to form larger multimers and increased the tendency for thermal aggregation (20). In contrast, deamidation of glutamines in the domain interfaces of dimeric ␤B1-Crys and ␤B2-Crys decreased stabilities of the proteins and caused them to populate partially unfolded intermediates in equilibrium experiments (21,22,24). Studies by…”

“…Deamidation of a glutamine in the connecting peptide of ␤B1-Crys caused the protein to form larger multimers and increased the tendency for thermal aggregation (20). In contrast, deamidation of glutamines in the domain interfaces of dimeric ␤B1-Crys and ␤B2-Crys decreased stabilities of the proteins and caused them to populate partially unfolded intermediates in equilibrium experiments (21,22,24). Studies by Gupta and Srivastava (25,26) showed that ␣-crystallin also displays context-dependent effects of deamidation, including decreased in vitro chaperone activity, changes in secondary and tertiary structures, and altered oligomerization properties (25,26).…”

A Rapid, Comprehensive Liquid Chromatography-Mass Spectrometry (LC-MS)-based Survey of the Asp Isomers in Crystallins from Human Cataract Lenses

“…It is well known that WI proteins increase in aged and cataractous lenses (3). Furthermore, the lens crystallins undergo various posttranslational modifications such as isomerization and inversion of aspartic acid (Asp) residues, that is L␤-, D␤-, and D␣-formation (4 -6); deamidation of asparagine (Asn) or glutamine (Gln) residues (7)(8)(9)(10)(11); disulfide bonding of cysteine (12); oxidation of methionine or tryptophan (13,14); backbone cleavage (15); phosphorylation (16); and glycation (17) during the aging process. These modifications may induce a decrease in crystallin solubility, alter lens transparency, diminish vision, and lead to development of a cataract.…”