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.
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Abbreviations
- ASA:
-
Accessible surface area
- CD:
-
Circular dichroism
- GdmCl:
-
Guanidinium chloride
- H2O2 :
-
Hydrogen peroxide
- IFNA2a:
-
Interferon alpha-2a
- MRE:
-
Mean residue ellipticity
- MS:
-
Mass spectrometry
- NMR:
-
Nuclear magnetic resonance.
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Figure S1
Aggregation propensity predictions for IFNA2a by various computational programs. Black rectangles indicate overlapping regions that were predicted to be aggregation hot-spots by all the three programs. (PNG 270 kb)
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Shah, D.D., Singh, S.M. & Mallela, K.M.G. Effect of Chemical Oxidation on the Higher Order Structure, Stability, Aggregation, and Biological Function of Interferon Alpha-2a: Role of Local Structural Changes Detected by 2D NMR. Pharm Res 35, 232 (2018). https://doi.org/10.1007/s11095-018-2518-y
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DOI: https://doi.org/10.1007/s11095-018-2518-y