The C-terminal domain of BBK32 specifically inhibits the CP in a dose-dependent manner in an ELISA based assay of complement function.

<p>CP was selectively activated by immobilization of human IgM followed by incubation of 1% human serum in GHB⁺⁺ buffer in the presence of varying concentrations of BBK32 proteins. Monoclonal antibodies were used to detect the deposition of the complement activation products (A) C3b or (B) C4b. While BBK32-FL and the BBK32-C potently inhibit the CP in a dose-dependent manner, BBK32-N showed no apparent inhibitory activity. In contrast, when conditions were used to select for (C) AP activation (LPS coating, 20% serum, GHB°, Mg-EGTA) or LP activation (mannan coating, 1% serum, GHB⁺⁺) no significant inhibition was detected for up to 1 μM concentrations of any BBK32 protein derivative. Wells containing serum only or where serum was replaced with buffer were treated as 100% and 0% signal, respectively. All experiments were performed a minimum of three times, errors are reported as the mean ± SEM, and calculated IC₅₀ values are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005404#ppat.1005404.t002" target="_blank">Table 2</a>.</p

BBK32 inhibits the enzymatic cleavage of C1s proenzyme by C1r enzyme.

<p>(A) The ability of BBK32 to inhibit the <i>in vitro</i> proteolytic cleavage of C1s proenzyme by 50 nM of activated C1r enzyme was assessed by monitoring for the presence of C1s proenzyme (single 86-kDa chain; asterisk) or activated C1s enzyme (58-kDa chain 1 and 28-kDa chain 2; arrows) by SDS-PAGE. Overnight reactions were incubated at 37°C in HBS-Ca²⁺ in the presence of various concentrations of BBK32-FL (gel 1), BBK32-C (gel 2), or BBK32-N (gel 3). In the absence of BBK32, C1r enzyme converts 100% of C1s proenzyme to C1s enzyme under the conditions used (see “No BBK32” lanes on gels 1–3). BBK32-FL and BBK32-C inhibited C1r activity in a dose-dependent manner, whereas, BBK32-N failed to inhibit C1r up to a final concentration of 10 μM. All C1r activity assays were conducted a minimum of three times and representative gel images are shown. (B) Densitometry was performed and the normalized peak intensity of the band corresponding to C1s proenzyme was plotted against the concentration of BBK32 present in each reaction. IC₅₀ values were calculated and are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005404#ppat.1005404.t002" target="_blank">Table 2</a> along with statistics for individual fits. (C) The ability of 1 μM BBK32 proteins to inhibit the cleavage of complement C4 by previously activated and purified C1s enzyme was evaluated <i>in vitro</i> by monitoring the conversion of the C4 α-chain to the C4 α’-chain by SDS-PAGE. C1s enzyme activity could not be detected under the conditions used for any BBK32 proteins, indicating that the BBK32 inhibitory activity is specific for C1r. C1s activity assays were performed in duplicate and a gel image from a representative experiment is shown.</p

The C-terminal domain of BBK32 specifically inhibits CP-mediated hemolysis.

<p>(A) The effect of 1 μM BBK32 proteins on CP-mediated hemolysis was assessed using a standard assay of complement hemolytic function (CP50). (B) While BBK32-N exhibited no measurable effect, BBK32-FL and BBK32-C abrogated nearly all hemolytic activity in a dose-dependent manner. (C) In contrast, BBK32 proteins fail to inhibit AP-mediated hemolysis. Measures of statistical significance in (A) and (C) were determined by use of an unpaired <i>t</i> test of each experimental series versus buffer control. ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001; ns, not significant. All experiments were performed between two and four times and errors are reported as the mean ± the standard error of the mean (SEM). Calculated IC₅₀ values for the dose-dependent inhibition of CP hemolysis are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005404#ppat.1005404.t002" target="_blank">Table 2</a>.</p

BBK32 _(21–205) induces a conformational change in plasma Fn.

<p>(a) Purified plasma Fn and Fn aggregates induced by anastellin or BBK32 _(21–205) were digested with thermolysin. The digested products were fractionated by SDS-PAGE and peptides that were only found in BBK32 _(21–205) /Fn (lane 3) or anastellin/Fn (lane 2) digests and not present among the peptides obtained from purified plasma Fn (lane 1) were excised and identified by N-terminal sequencing. The sequences obtained, SSPVVID, AVEENQE, and ITETIP, were located to the end of ¹³F3, middle of ³F3, and the start of ¹F3, respectively for both BBK32/Fn and anastellin/Fn. Pre-stained protein standards with indicated M_w are shown. (b) Schematic of Fn. Stars denote cryptic cleavage sites in Fn after aggregation due to both BBK32 and anastellin.</p

BBK32 inhibits the enzymatic cleavage of C1s proenzyme by C1r enzyme.

<p>(A) The ability of BBK32 to inhibit the <i>in vitro</i> proteolytic cleavage of C1s proenzyme by 50 nM of activated C1r enzyme was assessed by monitoring for the presence of C1s proenzyme (single 86-kDa chain; asterisk) or activated C1s enzyme (58-kDa chain 1 and 28-kDa chain 2; arrows) by SDS-PAGE. Overnight reactions were incubated at 37°C in HBS-Ca²⁺ in the presence of various concentrations of BBK32-FL (gel 1), BBK32-C (gel 2), or BBK32-N (gel 3). In the absence of BBK32, C1r enzyme converts 100% of C1s proenzyme to C1s enzyme under the conditions used (see “No BBK32” lanes on gels 1–3). BBK32-FL and BBK32-C inhibited C1r activity in a dose-dependent manner, whereas, BBK32-N failed to inhibit C1r up to a final concentration of 10 μM. All C1r activity assays were conducted a minimum of three times and representative gel images are shown. (B) Densitometry was performed and the normalized peak intensity of the band corresponding to C1s proenzyme was plotted against the concentration of BBK32 present in each reaction. IC₅₀ values were calculated and are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005404#ppat.1005404.t002" target="_blank">Table 2</a> along with statistics for individual fits. (C) The ability of 1 μM BBK32 proteins to inhibit the cleavage of complement C4 by previously activated and purified C1s enzyme was evaluated <i>in vitro</i> by monitoring the conversion of the C4 α-chain to the C4 α’-chain by SDS-PAGE. C1s enzyme activity could not be detected under the conditions used for any BBK32 proteins, indicating that the BBK32 inhibitory activity is specific for C1r. C1s activity assays were performed in duplicate and a gel image from a representative experiment is shown.</p

BBK32 _(21–205) binds F3 modules.

<p>Binding to (a) ¹F3 (b) ^1–2F3 and (c) ³F3 was assessed using ELISA-type assays. F3 modules were immobilized in microtiter wells followed by blocking with ovalbumin. BBK32 _(21–205) or FnbpA peptide D3 was added to wells in increasing concentrations. Anti-BBK32 antibody followed by HRP conjugated anti-rabbit antibody for BBK32 _(21–205), and anti-FnbpA followed by HRP conjugated anti-rabbit antibody were used to demonstrate binding.</p

BBK32 _(21–205) inhibits the proliferation of HUVECs.

<p>Cells were seeded onto tissue culture plates in full media and allowed to adhere for two hours. After two hours, BBK32, anastellin, and FnbpA peptide D3 were added in increasing concentrations (in triplicate) to the wells. Cells were then incubated for 72 hours at 37°C, 5% CO₂. After 72 hours, cells were washed, trypsinized, and counted (three counts/well, three wells/dose) using a hemacytometer.</p

BBK32 _(21–205) effects on Fn matrix assembled by NHDFs.

<p>Cells were incubated for 48 hours and then incubated with 5 µM BBK32 _(21–205), 5 µM anastellin, or 5 µM FnbpA peptide D3. Treated cells were incubated for 20 hours and probed with Alexa Fluor 488-anti-Fn and monoclonal antibody IST 9. Images were taken using LSM 510 Confocal Microscope, objective 63X/1.4 oil. Bar = 10 µm.</p