Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function

Area of Science:

• Biochemistry
• Molecular Biology
• Ophthalmology

Background:

• Senile cataract is linked to lens protein insolubilization.
• Post-translational modifications, like aspartate isomerization, accumulate in aging lens fiber cells.
• The functional impact of alphaA-crystallin isomerization sites remains unclear.

Purpose of the Study:

• To investigate the structural and functional contributions of Asp 58 isomerization in alphaA-crystallin.
• To assess the impact of Asp 58 isomerization on alphaA-crystallin assembly, solubility, and chaperone function.
• To evaluate a novel LC-MS/MS method for analyzing amino acid residue isomerization.

Main Methods:

• Extraction and separation of alphaA-crystallin oligomers from aged human lenses.
• LC-MS/MS analysis of in-solution/gel tryptic digests to quantify Asp 58 isomerization.
• Site-directed mutagenesis of Asp 58 to assess functional consequences.

Main Results:

• Asp 58 isomerization in alphaA-crystallin is dependent on oligomer size and lens age.
• Substitution of Asp 58 with hydrophobic residues increased oligomer size and reduced solubility.
• All Asp 58 substitutions impaired the chaperone activity of alphaA-crystallin.

Conclusions:

• Asp 58 in alphaA-crystallin plays a critical role in intermolecular interactions within the lens.
• Asp 58 isomerization contributes to age-related changes in lens protein properties.
• The developed LC-MS/MS method is effective for analyzing amino acid isomerization in proteins.