Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

Abstract

PURPOSE:

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays.

METHODS:

Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay.

RESULTS:

αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant.

CONCLUSIONS:

Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

• Authors: Srinivasagan Ramkumar, Noriko Fujii, Norihiko Fujii, Bency Thankappan, Hiroaki Sakaue, Kim Ingu, Kalimuthusamy Natarajaseenivasan, Kumarasamy Anbarasu
• Journal: Molecular vision (Mol Vis) Vol. 20 Pg. 1002-16 ( 2014) ISSN: 1090-0535 [Electronic] United States
• PMID: 25018622 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Cross-Linking Reagents
• Mutant Proteins
• alpha-Crystallin A Chain

Topics

• Base Sequence
• Chromatography, Gel
• Chromatography, High Pressure Liquid
• Circular Dichroism
• Cross-Linking Reagents (metabolism)
• Densitometry
• Gamma Rays
• Humans
• Hydrophobic and Hydrophilic Interactions
• Molecular Sequence Data
• Mutant Proteins (chemistry, isolation & purification, radiation effects)
• Protein Structure, Quaternary
• Protein Structure, Secondary
• Pulse Radiolysis
• Reproducibility of Results
• alpha-Crystallin A Chain (chemistry, genetics, isolation & purification, radiation effects)