Influence of lβ-, dα- and dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70–88 peptide

Abstract

Proteins have been considered to consist exclusively of L-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the D-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of D- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to D-isomers in a protein, we synthesized peptides corresponding to the 70-88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which Lα-Asp was replaced with Lβ-Asp, Dα-Asp, and Dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (Lβ-Asp, Dα-Asp, and Dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing Lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.

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

“…The isomeric peptides containing L␤-Asp, D␣-Asp, and D␤-Asp residues were more hydrophilic than the L␣-Asp-containing peptide, and the isomeric peptides lost ␤-sheet structure and changed to random structures (43). The normal peptide promoted the aggregation of insulin, whereas the other three isomers suppressed the aggregation of insulin (43). Recently, Santhoshkumar et al (44) showed that the ␣A-crystallin-derived peptide 66 SDRDKFVIFLDVKHF 80 , which accumulates in the aging lens, can inhibit the chaperone activity of ␣-crystallin and can cause aggregation and precipitation of lens crystallins.…”

“…Therefore, it is proposed that the isomerization of Asp-76 and Asp-84 residues also contributes to the insolubilization and aggregation of ␣A-crystallin. In our previous study, we synthesized peptides corresponding to residues 70 -88 (KFVIFLD 76 VKHFSPED 84 LTVK) of human ␣A-crystallin and its corresponding diastereoisomers in which L␣-Asp was replaced with L␤-Asp, D␣-Asp, and D␤-Asp at position 76 and compared their biochemical properties with that of the L␣-Asp-containing peptide (43). The isomeric peptides containing L␤-Asp, D␣-Asp, and D␤-Asp residues were more hydrophilic than the L␣-Asp-containing peptide, and the isomeric peptides lost ␤-sheet structure and changed to random structures (43).…”

“…In our previous study, we synthesized peptides corresponding to residues 70 -88 (KFVIFLD 76 VKHFSPED 84 LTVK) of human ␣A-crystallin and its corresponding diastereoisomers in which L␣-Asp was replaced with L␤-Asp, D␣-Asp, and D␤-Asp at position 76 and compared their biochemical properties with that of the L␣-Asp-containing peptide (43). The isomeric peptides containing L␤-Asp, D␣-Asp, and D␤-Asp residues were more hydrophilic than the L␣-Asp-containing peptide, and the isomeric peptides lost ␤-sheet structure and changed to random structures (43). The normal peptide promoted the aggregation of insulin, whereas the other three isomers suppressed the aggregation of insulin (43).…”

Isomerization of aspartyl residues in crystallins and its influence upon cataract

“…In order to clarify the effect of the inversion to D-isomers in a protein, we synthesized peptides corresponding to the 70-88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-β-Asp, D-α-Asp and D-β-Asp at position 76, and compared their biochemical properties with those of normal peptide. The results clearly showed that the substitution of a single Asp isomer in a peptide induced the loss of β-sheet structure and function, as well as decreasing the hydrophobicity of the peptide (Table 3b) [32]. The reversed orientation of the side chain induced by the D-isomer and elongation of the main chain induced by the β-isomer may affect the hydrophobicity and the secondary structure of the peptides, leading Table 2.…”

“…Although these methods are excellent, however, they require advanced instruments and expertise for analysis. In a previous study, we found that peptides containing four different Asp isomers (L-α-Asp, L-β-Asp, D-α-Asp and D-β-Asp) were separated into four peaks by RP-HPLC [32]. Based on this property, we tried to identify peptides containing Asp isomers in crystallins by an LC-MS/MS system [33] -a powerful tool used commonly to identify peptides in proteomics studies.…”