Purchase this article with an account.
A. Biswas, A.G. Miller, T. Oya–Ito, P. Santhoshkumar, M. Bhat, R.H. Nagaraj; Role of Methylglyoxal–Modifiable Arginine Residues in the Structure and Chaperone Function of Human AlphaA–Crystallin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1516.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We reported previously that chemical modification of arginine residues at 21, 49 and 103 in human αA–crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO) enhances its chaperone function [Nagaraj et al. (2003), Biochemistry 42, 10746–10755]. The purpose of this study is to understand the role of positive charge on these three arginine residues on the structure and chaperone function of human αA–crystallin.
We cloned, expressed and purified the wild type (wt) and six mutant (R21A, R49A, R103A, R21A/R49A, R21A/R103A and R21A/R49A/R103A) human αA–crystallins. The structure of the wt and mutant proteins was determined by UV–CD, tryptophan fluorescence and dynamic light scattering. Surface hydrophobicity was determined by TNS binding. Binding constants were determined using carbonic anhydrase as a target protein. The chaperone function of wild–type and mutant proteins was assessed using two target proteins (insulin and citrate synthase).
R49A and R103A mutations significantly increased the oligomeric size of αA–crystallin, but R21A mutation had no effect. Intrinsic tryptophan fluorescence and near–UV CD spectra indicated that mutant proteins had altered tryptophan and aromatic amino acid microenvironments when compared to the wt αA–crystallin. The far–UV CD spectra of wt and mutant αA–crystallins indicated minimal perturbation in their secondary structure. Binding studies with a hydrophobic probe TNS showed an increase in the surface hydrophobocity in all mutant proteins except in R49A and R21A/R49A. When compared to the wt protein, the chaperone function of R21A and R103A mutants was considerably increased (18–39%), but it was decreased in R49A mutant. The other mutant proteins, except R21A/R49A also showed some improvement in the chaperone function. The increase in chaperone function correlated with the ability to refold denatured malate dehydrogenase.
These data demonstrate that the positive charge on R21, R49 and R103 plays a crucial role in the chaperone function of human αA–crystallin and suggest that chemical modification of arginine residues may have consequences in protein aggregation during lens aging and cataract formation.
This PDF is available to Subscribers Only