Modifications of human βA1/βA3‐crystallins include S‐methylation, glutathiolation, and truncation

Abstract

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human ␥-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that ␤A1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of ␤A1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.Keywords: human lens crystallins; cataract; in vivo protein modification; S-methylation; glutathiolation A high concentration of proteins in the ocular lens provides the refractive index required for focusing light on the retina. Major structural proteins, called crystallins, which have molecular masses of 20-28 kDa, account for >90% of the lens proteins. Based on sequence homology and the size of aggregates isolated under physiological conditions, human crystallins are organized into three classes, ␣-, ␤-and ␥-crystallins. Because lens crystallins undergo little or no turnover, there is opportunity for numerous post-translational modifications as the lens ages (David et al. 1996;Lampi et al. 1998;Ma et al. 1998;Takemoto and Boyle 1998;Slingsby and Clout 1999;Hanson et al. 2000). Some modifications, identified in young clear lenses, may reflect normal development and maturation of the lens, while others, associated with aged lenses, could negatively impact crystallin conformation, aggregation state, or solubility, resulting in increased light scattering and eventual loss of lens transparency.The free sulfhydryls of cysteine residues are among the most reactive functional groups in proteins. Cysteine residues in lens crystallins are susceptible to oxidation forming mixed disulfides with low molecular weight thiols such as glutathione, cysteine, and ␥-glutamylcysteine (S-thiolation) (Dickerson and Lou 1993), and protein-protein disulfide bonds (Spector and Roy 1978;Lapko et al. 2002a). Although the physiological role of mixed disulfides is not well understood (Lou and Dickerson 1992;Kamei 1993;Feng et al. 2000), there is a correlation between these modifications and the color and opalescence of the lens nuclei . Also, the association of protein-protein disulfide bonds with crystallin aggregation and insolubilization suggests that intermolecular disulfide bonding has a role in cataractogenesis Stephan et al. 1999;Pande et al. 2000;Lapko et al. 2002a). I...

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

“…The only crystallins where methylation was not detected were βB3 and the chaperone protein, αB crystallin, which lacks Cys residues. This study extends earlier reports of Cys methylation in γS, γD and βA3-crystallins from human lenses (Lapko et al, 2002, 2003, 2005; Searle et al, 2005). In the case of β-crystallins, these authors detected substantial levels of S-methylcysteine only in βA1/A3-crystallin.…”

“…S-methylation, an unusual post-translational modification (PTM), was first reported in human lens nearly a decade ago, (Lapko et al, 2002, 2003, 2005; Searle et al, 2005) but no plausible mechanism was proposed. More recent work (Wilmarth et al, 2006) on modifications of aged human lens crystallins indicated that methylation was found at other sites and was an abundant lens PTM; again no mechanism was suggested.…”

Incorporating structural characteristics for identification of protein methylation sites

“…[3][4][5][6][7] Methylation can also occur on the oxygen atoms of glutamate and aspartate (Omethylation) 8 and the sulfur atom of cysteine (S-methylation). 9 These modifications are carried out by a protein family called methyltransferases, which use S-adenosylmethionine as a sub-strate to transfer a methyl group. 10 Most studies of protein methylation have focused on methylated arginine and lysine residues.…”

Degradation of an Old Human Protein

“…The lens contains few major polypeptides, there is no protein turnover (9), and consequently post-translational modifications (PTMs) 2 accumulate in these life-long polypeptides over time. Abundant PTMs are racemization (10,11), methylation (12,13), and deamidation (14,15). Some lens proteins, such as the ␣-crystallins (16 -20) and aquaporin 0 (21)(22), also undergo progressive and extensive truncation.…”