Simultaneous Stereoinversion and Isomerization at Specific Aspartic Acid Residues in αA-Crystallin from Human Lens

Abstract

We characterized the primary structure of alpha A-crystallin from the lens of the human eye by detailed analyses of the amino acid sequence, mass, and stereoisomers, and found the biologically uncommon D- and beta-aspartic acid (Asp) residues in the protein. The stereoconfiguration of the Asp151 and Asp58 residues in alpha A-crystallin from old subjects (mean age: 80 years) was inverted to the D-isomer, and the residues were simultaneously isomerized to beta-aspartyl residues, which may occur via a succinimide intermediate. This is thought to be the first observation of stereoinversion of amino acids in protein in vivo. It is noteworthy that similar stereoinversion was observed in the same residues of alpha A-crystallin from young subjects (age: 11 months), with simultaneous isomerization, although the extent of isomerization was low compared with that in the aged. The conversion may take place in the early life of the lens and the resulting isomers may accumulate with aging. We also found that terminal serine173 was cleaved in aged alpha A-crystallin. Since the cleavage was not observed in young alpha A-crystallin, it may result from the in vivo aging process. The present findings also make it necessary to revise the amino acid sequence of human alpha A-crystallin presented previously: human alpha A-crystallin is composed of 173 (formerly reported as 172) amino acid residues, and the sequence from residues 153 to 155 is THA (formerly reported as -HT).

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

“…6 shows the relative amounts of the various Asp isomers of ␣A-and ␣B-crystallins from WI and WS fractions. The Asp-58 and Asp-151 of ␣A-crystallin and Asp-62 of ␣B-crystallin are highly inverted to the isomers in the WS fraction, which is entirely consistent with our previous results (4,5), and their isomeric ratios increased dramatically in the WI fraction. In recent work, Hooi et al (42) reported that Asp-151 of ␣A-crystallin converted to D␤, L␤, and D␣ in a ratio of 3:1:0.5 until age 15 and that the percentage of D␤-Asp-151 is 40% by age 20 with a fixed ratio to age 80.…”

“…Therefore, it is possible for the ratio of the isomeric peptides to be calculated from the areas of LC-MS peaks. The most abundant isomers of Asp-58 and Asp-151 of ␣A-crystallin in the WS fraction were D␤-isomers, which is consistent with our previous results using conventional methods (4). The ratios of the D␤-Asp isomers to the normal L␣-Asp form at both Asp-58 and Asp-151 of ␣A-crystallin in the WI fraction are ϳ1:1 or greater.…”

“…Deamidation of Asn or Gln introduces a negative charge at physiological pH by replacing an amide with a carboxyl group to form Asp or Glu, respectively, and causes some isomerization (4,19). Isomerization and deamidation are major non-enzymatic post-translational modifications of proteins under physiological conditions.…”

Biophysical chemistry of the ageing eye lens

“…Both αA-crystallin Asp151 and γS-crystallin Asn76 are reported to undergo gradual racemisation over their lifetime with γS-crystallin Asn76 showing particularly high modification in cataractous lenses (Hooi et al 2012). Of the 13 aspariginyl residues of αB-crystallin, Asp36 and Asp62 show extensive racemisation in aged lenses compared to young ones, a pattern repeated with βB2-crystallin Asp4 residue (Fujii et al 1994). In αA-crystallin, Asp151 and 58 are highly modified in older eyes, but the racemisation of these residues can first appear at a young age (Fujii et al 1997;Hooi et al 2013b).…”

Abnormal Protein Aggregation Due to the Presence of D-Aspartyl Residues in Cataractous Lenses

“…During the ageing process crystallins undergo several posttranslational modifications that lead to aggregation, crosslinking and insolubilization, culminating in the development of a cataract. Examples of posttranslational modification of α-crystallin that have been reported include racemization and isomerization of aspartyl residues [2,3], disulfide bonding [4], deamidation of asparagine or glutamine [5] residues, methionine oxidation [6] and backbone cleavage [7]. Among these modifications of amino acids, we have considered that the racemization of aspartyl residues to D-Asp isomers and the inversion from the normal α-linkage to the β-linkage of the peptide bond, are the most effective to change the higher order structure of protein.…”