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A. Biswas, S. Lewis, B. Wang, M. Miyagi, R. H. Nagaraj; Revival and Augmentation of the Chaperone Function of Guanidinated Human A-Crystallin by Methylglyoxal. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1526.
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We demonstrated previously that modification of discrete arginine residues in human αA-crystallin by MGO made it a better chaperone and that replacement of MGO modifiable arginine residues (by site-directed mutagenesis) with neutral alanine mirrored the effects of MGO-modification [Nagaraj et al., Biochemistry 42, 10746, 2003; Biswas et al., Biochemistry 45, 4569, 2006]. The purpose of this study was to investigate the effects of introduction of additional guanidino groups and modification by MGO on the structure and chaperone function of human αA-crystallin.
Guanidino groups were introduced by conversion of lysine residues to homoarginine by treatment with o-methylisourea (OMIU). OMIU-treated αA-crystallin was further modified by incubating with 1 mM MGO. Homoarginine and homoarginine+MGO derived products (homoargpyrimidine and homohydroimidazolone) were measured by amino acid analysis and HPLC, and confirmed by LC-mass spectrometry. The chaperone function of unmodified and modified proteins was assessed using insulin (INS) and citrate synthase (CS) as substrate proteins. Surface hydrophobicity was determined by TNS binding.
Lysine residues were converted to homoarginine by OMIU in a concentration-dependent manner. Treatment with 0.25 M OMIU converted all lysine residues to homoarginine. Amino acid analyses and LC-MS data confirmed formation of homoarginine. LC-MS and HPLC results indicated formation of homoargpyrimidine and homohydroimidazolone adducts by the reaction of OMIU-modified αA-crystallin with MGO. Treatment of αA-crystallin with 0.25 M OMIU almost completely destroyed its chaperone function. Subsequent treatment with 1 mM MGO not only revived the chaperone function, but it improved it by ~ 40% and ~ 60% in INS and CS assays when compared to the unmodified protein. The surface hydrophobicity of the protein was significantly reduced by OMIU treatment but became ~ 39% higher than unmodified protein after MGO treatment. The secondary and tertiary structure of the protein didnot change due to these modifications.
Our data show a remarkable loss and gain in chaperone function by introduction of guanidino groups and their neutralization by MGO. These data lend support to the notion that guanidino groups of arginine in αA-crystallin play an important role in regulating the chaperone function.
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