The Serpin-like Loop Insertion of Ovalbumin Increases the Stability and Decreases the OVA 323–339 Epitope Processing Efficiency

Abstract

Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323–339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323–339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323–339 epitope in vitro . These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

SARS-CoV-2 Beta Variant Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability

“…A summary of the parameters fit by non-linear regression are shown in Table 1. Changes in resistance to denaturant-induced unfolding did not correspond with protein melting temperatures as measured by a fluorescence melting assay that we have applied to the study of ovalbumin variants (Figure 4E)(20). We observed a reduction in the melting temperature of the beta variant RBD (Figure 4F).…”

“…Therefore, we hypothesized that these RBD substitutions alter RBD stability and resistance to unfolding. To test this we measured guanidine-induced unfolding of RBD variants using a fluorescence based unfolding assay 19,20 . Unfolding curves generated by measuring fluorescent dye binding in response to increasing guanidine-HCl concentration were fit by non-linear regression to estimate free energy of unfolding 21 .…”

“…Therefore, we hypothesized that these RBD substitutions alter RBD stability and resistance to unfolding. To test this we measured guanidine-induced unfolding of RBD variants using a fluorescence-based unfolding assay (20, 21). Bis-ANS fluorescence was measured for RBD variants in the presence of increasing guanidine-HCl concentration (Figure 4A).…”

“…Molecular dynamic simulations and results from unfolding studies presented here lead us to the conclusion that RBD variant of concern substitutions alter RBD structure and stability. Changes in protein structure and stability are associated with changes in proteolytic resistance(20, 21, 25, 26). Based on these previous observations and those we have reported here thus far, we hypothesized that RBD substitutions alter proteolytic susceptibility in accordance with changes in stability.…”

“…Reduced hydrogen bond content may explain the observed decrease in stability and suggests that the elimination of specific interactions between residue 484 and other RBD residues alone cannot explain the observed reduction in RBD stability. Finally, we tested the stability of RBD variants by limited proteolysis with cathepsin S. Protein susceptibility to limited proteolysis is a robust measure of protein stability and has also been linked to protein immunogenicity 19,20,23,24 . We observed that the K417N substitution increased proteolytic resistance at all time points while the E484K and all three substitutions together (K417N/E484K/N501Y) decreased resistance after 15 and 30 minutes of incubation.…”