Figure 1
A novel FRET peptide assay to detect H. pylori HtrA activity. (A) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x-axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca++-binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. (B) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO2) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. (C) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM HEPES buffer (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (****p < 0.0001). (D) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.
