Journal: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: Kinetic and thermodynamic parameters for ScpA S512A binding to C5a peptides.

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Binding Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: ScpA enzyme kinetics and activity against rhC5a dR . (a) SDS-PAGE analysis of recombinant rhC5a and rhC5a dR cleaved with ScpA. Lanes with ‘+’ and ‘-’ indicate samples with and without ScpA, respectively. rhC5a and rhC5a dR treated with ScpA produced fragments of similar size (indicated with an arrow marked ‘core’). Molecular weight ladders are shown on the left side of the gels in Fig. 1 a and 1b in kDa. Mass spectrometry of cleaved rhC5a and rhC5a dR show that the observed mass of the products (10886.1 Da and 10885.6 Da, respectively) are consistent with rhC5a core (calculated mass 10886.2 Da.). Observed and calculated (parenthesis) masses are reported for rhC5a and rhC5a dR . (b) The activity of ScpA was examined in human serum. The Coomassie stained polyacrylamide gel and Western blot analysis shows that ScpA (36 nM) cleaves rhC5a (39 µM) in human serum. The arrow indicates the expected position for the ‘core’ (P N ) product of ScpA cleavage of C5a. (c) Progress curves for ScpA cleavage of rhC5a C75 -BODIPY. Data are plotted with curves from global fitting of 6 progress curves to the Van Slyke-Cullen model. Values for k cat and K m are reported as the mean and standard deviation of the mean from 3 experiments. Data shown in the plot are from 3 experiments. A key to substrate concentrations is provided on the right-hand side of the graph.

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Activity Assay, SDS Page, Recombinant, Produced, Molecular Weight, Mass Spectrometry, Staining, Western Blot, Standard Deviation

Journal: Computational and Structural Biotechnology Journal

Article Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

doi: 10.1016/j.csbj.2021.04.024

Figure Lengend Snippet: Model of the ScpA-hC5a complex. (a) The stereo diagram shows the ScpA-C5a complex with hC5a (cartoon diagram) on the surface of ScpA. The coordinates for the model are from the docking analysis described in Kagawa et al. . ScpA is colored by domains, with the catalytic domain (‘CAT’) domain in salmon, the PA domain in blue, and the Fn1–Fn3 domains in green, cyan, and yellow, respectively. hC5a is shown as a cartoon model with the core portion (P N , orange) on the ScpA Fn2 domain and tail residues (P C , purple) extended through the prime side of the active site. The location of the scissile bond is indicated with a red arrow. Sidechains of hC5a arginine residues found to impact on binding are shown with space filling models and labelled ‘a’ (R37), ‘b’ (R40), ‘c’ (R46), and ‘d’ (R74). Panel (b) shows a stereo diagram the hC5a model indicating locations of all residues mutated in this study (shown as stick models). hC5a is colored as in panel A. The four hC5a helices are labelled ‘I’ to ‘IV’. The location of the C27 sidechain is indicated with an asterisk. Panels (c) to (h) show representative sensorgrams of ScpA S512A binding to rhC5a K4A,K5A , rhC5a K12A,K14A , rhC5a R37A , rhC5a R40A , rhC5a R46A , and rhC5a K49A respectively. Observed data (black lines) are plotted with curves obtained from global fitting of data with a 1:1 Langmuir model for binding (red lines). Mean K D values obtained from 3 experiments are reported. Panel (i) shows a reaction scheme where binding of full-length hC5a (‘S’) occurs with a conformational change in ScpA (‘E’ to ‘F’). The C-terminal ‘tail’ residues (‘P C ’) are released during the acylation step. Deacylation produces a complex (‘F∙P N ’) between the enzyme and core portion of hC5a (‘P N ’). Dissociation of P N is faster from ‘F’ and thus not rate limiting in the catalytic cycle. Binding of the ‘P N ’ product to the ‘E’ ScpA state is accompanied by a slow rate of dissociation as measured in SPR studies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Western blot analysis of the samples used polyclonal rabbit anti-human C5a antibody (MBS524143, MyBioSource, USA).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: The role of complement activation in rhabdomyolysis-induced acute kidney injury

doi: 10.1371/journal.pone.0192361

Figure Lengend Snippet: (A) Immunofluorescence staining of three complement proteins. (B) C5a, C5b-9, and CD59 protein levels were measured using western blot analysis, and the ratio of these proteins to that of GAPDH was determined using densitometry. ★ P<0.01 and ▲ P>0.05 vs. control; ◆ P<0.01 and ▼ P>0.05 vs. AKI.

Article Snippet: Slides were air-dried, fixed with methanol/acetone for 10 min, and treated with the following FITC-conjugated anti-rat antibodies (Abs): rabbit anti-rat C3 Ab (Abcam, Cambridge, MA, USA); rabbit anti-rat C1q Ab (Abcam); rabbit anti-rat MBL-A Ab (Abcam); rabbit anti-rat factors B Ab (Santa Cruz, USA); rabbit anti-rat C5a Ab (ABBIOTEC, USA); and rabbit anti-rat CD59 Ab (Santa Cruz).

Techniques: Immunofluorescence, Staining, Western Blot, Control