Dinen D Shah1, Surinder M Singh1, Krishna M G Mallela2. 1. Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 E. Montview Blvd, Aurora, Colorado, 80045, USA. 2. Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 E. Montview Blvd, Aurora, Colorado, 80045, USA. krishna.mallela@ucdenver.edu.
Abstract
PURPOSE: Oxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined. METHODS: IFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR. RESULTS: Oxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D 15N-1H HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence. CONCLUSIONS: Increased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.
PURPOSE: Oxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined. METHODS: IFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR. RESULTS: Oxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D 15N-1H HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence. CONCLUSIONS: Increased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.
Entities:
Keywords:
Oxidation; protein aggregation; protein function; protein stability; protein structure
Authors: Bin Pan; Jeff Abel; Margaret S Ricci; David N Brems; Daniel I C Wang; Bernhardt L Trout Journal: Biochemistry Date: 2006-12-26 Impact factor: 3.162
Authors: Renuka Thirumangalathu; Sampathkumar Krishnan; Pavel Bondarenko; Margaret Speed-Ricci; Theodore W Randolph; John F Carpenter; David N Brems Journal: Biochemistry Date: 2007-05-05 Impact factor: 3.162