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Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin
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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Vision
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4087120/ https://www.ncbi.nlm.nih.gov/pubmed/25018622 |
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author | Ramkumar, Srinivasagan Fujii, Noriko Fujii, Norihiko Thankappan, Bency Sakaue, Hiroaki Ingu, Kim Natarajaseenivasan, Kalimuthusamy Anbarasu, Kumarasamy |
author_facet | Ramkumar, Srinivasagan Fujii, Noriko Fujii, Norihiko Thankappan, Bency Sakaue, Hiroaki Ingu, Kim Natarajaseenivasan, Kalimuthusamy Anbarasu, Kumarasamy |
author_sort | Ramkumar, Srinivasagan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4087120 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
spelling | pubmed-40871202014-07-11 Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin Ramkumar, Srinivasagan Fujii, Noriko Fujii, Norihiko Thankappan, Bency Sakaue, Hiroaki Ingu, Kim Natarajaseenivasan, Kalimuthusamy Anbarasu, Kumarasamy Mol Vis Research Article 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. Molecular Vision 2014-07-07 /pmc/articles/PMC4087120/ /pubmed/25018622 Text en Copyright © 2014 Molecular Vision. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, used for non-commercial purposes, and is not altered or transformed. |
spellingShingle | Research Article Ramkumar, Srinivasagan Fujii, Noriko Fujii, Norihiko Thankappan, Bency Sakaue, Hiroaki Ingu, Kim Natarajaseenivasan, Kalimuthusamy Anbarasu, Kumarasamy Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title | Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title_full | Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title_fullStr | Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title_full_unstemmed | Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title_short | Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin |
title_sort | comparison of effect of gamma ray irradiation on wild-type and n-terminal mutants of αa-crystallin |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4087120/ https://www.ncbi.nlm.nih.gov/pubmed/25018622 |
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