Fig 1.
Structure of the BTLA/HVEM(23–39) peptide complex.
HVEM(23–39) peptide is shown in dark blue ribbon, BTLA in beige and the side chains of residues involved in the inter-molecular hydrogen bonds in stick representation with nitrogen atoms colored in blue, oxygen colored in red and hydrogen atoms in white. Hydrogen bonds are represented by light blue lines.
Fig 2.
Microcolumn affinity test results for binding of HVEM(23–39) fragment to BTLA: (A) supernatant, (B) last wash and (C) elution fractions. The MS measurements were done with the use of MALDI TOF/TOF 5800 (ABSciex, Germany). As a matrix α-cyano-4-hydroxycinnamic acid (CHCA, 10 mg/ml, Sigma-Aldrich) was used. The measurements were done in reflector positive mass mode with previous mass calibration with commercial standard peptide mixture (The Peptide Mass Standards Kit for Calibration of AB SCIEX MALDI-TOF™ Instruments).
Table 1.
Microcolumn affinity test.
Fig 3.
HVEM(23–39) peptide partially blocks the BTLA/HVEM binding.
Inhibition of the BTLA/HVEM interaction was assessed by ELISA (at least two experiments in triplicate). The graph shows percentages of inhibition of the BTLA/HVEM ligation, relative to the negative control (PBS), in the presence of different concentrations (5, 1 and 0.1 mg/mL) of HVEM(23–39) and corresponding scrambled peptide (Ctrl peptide). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). *: p< 0.05 following non-parametric One-way ANOVA and Dunn’s post-test.
Fig 4.
Contribution of individual HVEM(23–39) amino acid residues to the inhibition of BTLA/HVEM interaction.
The relative involvement of alanine-substituted HVEM(23–39) peptide analogs was assessed by ELISA (at least two experiments in triplicate) in the presence of graded peptide concentrations (5, 1 and 0.1 mg/mL). The percentage of inhibition of the BTLA/HVEM binding was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of modified peptide to the same concentration of wild-type peptide HVEM(23–39). **: p< 0.01 following non-parametric One-way ANOVA and Dunn’s post-test.
Table 2.
Sequences of tested peptides.
Fig 5.
Cysteines from HVEM(23–39) are the main amino acid residues involved in the inhibition of BTLA/HVEM interaction.
Increasing concentrations of HVEM(23–39) or HVEM(31–39) modified peptides, where one or both cysteines were substituted, were tested in ELISA and compared to corresponding wild-type and scrambled (Ctrl peptide) peptides (at least two experiments in triplicate). The percentage of inhibition of the BTLA/HVEM binding was calculated in relation to the negative control (PBS). Statistical analysis was performed by comparing each concentration of each peptide to the same concentration of wild-type peptide HVEM(23–39). **: p< 0.01 following non-parametric One-way ANOVA and Dunn’s post-test.
Fig 6.
Free cysteine blocks the ligation between BTLA and HVEM.
(A) The effect of increasing concentrations (5, 1 and 0.1 mg/mL) of free amino acids were tested in BTLA/HVEM ELISA (three experiments in triplicate). The percentage of inhibition was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of residue or peptide to the same concentration of Cys. (B) Assessment of free thiol groups over time in rhBTLA-Fc protein and HVEM(23–39) peptide alone or mixed together. **: p< 0.01 and ***: p< 0.001 following One-way ANOVA and Dunn’s (A) or Dunnett’s (B) post-test.
Fig 7.
Blocking capacity of HVEM(23–39) fragment and free amino acids in a cellular assay.
293T cells expressing human BTLA were incubated with peptides or free amino acids (5mg/mL) prior labeling with rhHVEM-Fc and AF647-conjugated anti-human IgG antibody (at least two experiments in triplicate). The graph shows the Geometric Fluorescence Intensity (GMFI). *: p< 0.05 and ***: p< 0.001 following non-parametric One-way ANOVA and Dunn’s post-test.