A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

Abstract

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k /K value of 6.1×10 L mol s . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

“…Additionally, varying the levels of LPS during OmpT IB refolding did not significantly alter the above results (data not shown). MALDI-TOF experiments were undertaken to confirm that the substrate specificity of OmpT (RC-31(LPS) samples) for the dibasic sites was preserved ( Figure 6), in agreement with the established substrate specificity reported in the literature [19,20]. When a short synthetic peptide (Seq: GARKVG, MALDI-TOF experiments were undertaken to confirm that the substrate specificity of OmpT (RC-31(LPS) samples) for the dibasic sites was preserved ( Figure 6), in agreement with the established substrate specificity reported in the literature [17,19,20].…”

“…MALDI-TOF experiments were undertaken to confirm that the substrate specificity of OmpT (RC-31(LPS) samples) for the dibasic sites was preserved ( Figure 6), in agreement with the established substrate specificity reported in the literature [19,20]. When a short synthetic peptide (Seq: GARKVG, MALDI-TOF experiments were undertaken to confirm that the substrate specificity of OmpT (RC-31(LPS) samples) for the dibasic sites was preserved ( Figure 6), in agreement with the established substrate specificity reported in the literature [17,19,20]. When a short synthetic peptide (Seq: GARKVG, MW 627 Da; Figure 6A) carrying a dibasic site in its sequence was treated with OmpT, the signal for the full-length peptide was no longer visible in the MALDI spectra ( Figure 6C), indicating complete cleavage of the peptide.…”

“…However, this mutant was reported to have a lower activity (~30% lower) in comparison with the wild type protease [19]. In addition, we recently reported that this minor mutation in OmpT resulted in an altered substrate preference [20]. A second strategy, adopted by Wu et al (2012) [21] and described in detail in Wu's PhD thesis, involved the addition of LPS to refolded OmpT immediately after cation exchange purification to avoid autoproteolysis [21,22].…”

PITTCON‐Preise 2020

“…Mrksichs Gruppe hat Strategien zur Integration von lebenden Zellen und Mikroelektronik entwickelt, des weiteren Methoden für Hochdurchsatzassays in der Wirkstoff‐Forschung und die Herstellung synthetischer Proteine für therapeutische Anwendungen. Ihre Zuschrift über ein Bottom‐up‐Proteomikverfahren zur Identifizierung der Substratspezifität von OmpT‐Protease wurde auf dem Rück‐Innentitelbild der Angewandten Chemie vorgestellt …”

Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow

“…13,14 Here, substrates are first immobilised on a surface before treatment with an enzyme, thus determining activity and kinetic parameters. Interestingly, SAMDI has been shown to be not only compatible with peptide substrates for protein specificity, 15,16 but also carbohydrates for glycosyltransferase activity. 17 Whole cell analysis or cellular assays for evaluating compound efficacy affecting a cellular phenotype present an interesting challenge for MALDI TOF MS analysis as the system becomes inherently more complex.…”