title: Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.
abstract: OBJECTIVE
  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.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Srinivasagan
authors.initials: S
authors.lastname: Ramkumar
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Fujii
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka, Japan ; Teikyo University, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Fujii
authors.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Bency
authors.initials: B
authors.lastname: Thankappan
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sakaue
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka, Japan.
authors.forename: Kim
authors.initials: K
authors.lastname: Ingu
authors.affiliation: Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Kalimuthusamy
authors.initials: K
authors.lastname: Natarajaseenivasan
authors.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Kumarasamy
authors.initials: K
authors.lastname: Anbarasu
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003720:Densitometry; D005720:Gamma Rays; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011675:Pulse Radiolysis; D015203:Reproducibility of Results; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects*
chemical_list: D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D038202:alpha-Crystallin A Chain