Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Receptor binding and biological activity of IFNϵ and IFNκ preparations. A, receptor binding of IFNϵ and IFNκ, as well as IFNα2 and IFNω, measured as percent RO. RO corresponds to the SPR RU observed upon IFN injection over an IFNAR Biacore chip surface, divided by the theoretical RUmax of the surface, multiplied by 100. B, dose-response curves for IFNϵ, IFNκ, IFNα2, and IFNω-mediated activation of the IFI6 gene reporter in HL116 cells. The color code for the dose-response curves is the same as the receptor binding analysis in A.

Article Snippet: DNA sequences encoding the mature IFNε and IFNκ protein sequences were synthesized with optimized codons for expression in E. coli (ATUM).

Techniques: Binding Assay, Activity Assay, Injection, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: SPR analysis of IFNϵ, IFNκ, IFNα2, and IFNω binding to IFNAR1-FC, IFNAR2-FC, and the IFNAR1/IFNAR2-FC heterodimer. SPR sensorgrams for each IFN-IFNAR interaction are shown in black. The calculated sensorgrams, derived from fitting the data to a 1:1 binding model, are shown in black (IFNα2), red (IFNκ), blue (IFNϵ), and green (IFNω). Kinetic and equilibrium constants derived from the data are shown in Table 2.

Article Snippet: DNA sequences encoding the mature IFNε and IFNκ protein sequences were synthesized with optimized codons for expression in E. coli (ATUM).

Techniques: Binding Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: IFNϵ, IFNκ, and IFNα1 exhibit reduced binding to the viral IFN antagonist B18R. Type-I IFNs, at 50 (A) or 5 (B) nm concentrations, were injected over the Copenhagen strain of B18R (B19R-FC) captured on a Biacore chip. Binding is reported as fraction RU bound, which corresponds to the SPR RU observed upon IFN injection over the B19R-FC surface, divided by the theoretical RUmax of the surface. The type-III IFNλ proteins (IL29, IL28A, IL28B), which do not bind to B18R, were used as negative controls. Based on the IFN-B18R binding profiles in A and B, kinetic analyses were performed for selected strong B18R binders (IFNα14 and IFNα4) and weak B18R binders (IFNϵ and IFNα1). The experimental (black) and calculated (colored) sensorgrams for strong and weak binders are shown. The rate constants derived from the sensorgrams are found in Table 2.

Article Snippet: DNA sequences encoding the mature IFNε and IFNκ protein sequences were synthesized with optimized codons for expression in E. coli (ATUM).

Techniques: Binding Assay, Injection, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Molecular model of unique IFNϵ/κ and IFNα1 residues that modulate IFNAR2 and B18R binding. A and B, schematic models and structures of the (A) IFNα2/IFNAR1/IFNAR2 complex (PDB 3SE3) and the (B) IFNα2/B18R complex (IFNα2 from PDB 3S9D, and B18R derived from the C12R structure, PDB 3OQ3). C, ribbon diagram of the IFNα2 backbone with key residues that regulate IFNAR2 and B18R binding affinity, as discussed in the text, shown in yellow, orange, and magenta. D, superposition of IFNs in IFN/IFNAR2 and IFN/B18R structures with B18R D3 domain in red and the IFNAR2 D1 domain cyan. Three negatively charged residues conserved in IFNAR2 (green) and B18R (yellow) are positioned by IFNα arginines: Arg-35, Arg-56, and Arg=172 (magenta). E, amino acids within the AB loop region of IFNs that distinguish strong and weak IFNAR2 and B18R binders. Amino acid substitutions made to produce IFNα1 and IFNκ mutants are shown in bold.

Article Snippet: DNA sequences encoding the mature IFNε and IFNκ protein sequences were synthesized with optimized codons for expression in E. coli (ATUM).

Techniques: Binding Assay, Derivative Assay