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 assays ensitivity.P eptide arrays and SAMDI mass spectrometry were used to identify at etrapeptide 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')a nd nearest-neighbor positions (P2, P2')a nd their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity.S ubstituting FRRV into af ragment of LL37, an atural substrate of OmpT,l ed to ag reater than 400-fold improvement in OmpT catalytic efficiency,with ak cat /K m value of 6.1 10 6 Lmol À1 s À1 .Wild-type and mutant OmpT displayed significant differences in their substrate specificities,d emonstrating that even modest mutants mayn ot be suitable substitutes for the native enzyme.

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

“…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 6 A) 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 6 C), 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 ].…”

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry

“…In this study, we demonstrated the label-free measurement of PTP activity by SAMDI, but in other work, we have demonstrated the broad utility of the platform for measuring enzyme activities including kinases, deacetylases, glycosyltransferases, caspases, proteases, and others. [21][22][23][24][25][26] By…”

“…phosphatases, deacetylases, glycosyltransferases, caspases, proteases, and others. [21][22][23][24][25][26] SAMDI does not require significant alteration of substrates with labels, which can modify enzymatic activity and can require extensive development. [27,28] For example, fluorescent probes can alter enzymatic activity measurements by more than 50-fold.…”

Peptide Arrays: Development and Application

“…Wood et al used SAMDI peptide arrays to study and compare the substrate specificities of mutant and wild type OmpT. 159 Reproduced from A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT, Wood, S. E.; Sinsinbar, G.; Gudlur, S.; Nallani, M.; Huang, C.-F.; Liedberg, B.; Mrksich, M. Angew. Chem.…”