Activity-Modulating Monoclonal Antibodies to the Human Serine Protease HtrA3 Provide Novel Insights into Regulating HtrA Proteolytic Activities

<div><p>Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential <i>in vivo</i> utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders.</p></div

Schematic representation of HtrA3 domain organization, mAb epitope locations and confirmation of mAbs specificity.

<p>(A) The domain structure of HtrA1, HtrA3, HtrA4 and HtrA2. (B) The domain structure of HtrA3-L and HtrA3-S. The solid bars above or below the protein domains denote the locations of epitope residues of each mAb identified by the linear peptide library mapping assay. “X” indicates a peptide deemed likely to be a false positive. SP, signal peptide; IGFB, IGF-binding domain; Kazal, Kazal-type S protease inhibitor domain; trypsin, trypsin-like serine protease domain; PDZ, PDZ domain; TM, transmembrane; TP, transient peptide. (C) An equal amount (50 ng) of recombinant human HtrA proteins HtrA1, HtrA2, HtrA3 (HtrA3-L-S305A) and HtrA4 were separated on reducing 12% SDS-PAGE gels and analyzed by Western blot with HtrA3 mAbs (3E6, 6G6, 2C4, 10H10 and 9C9), and an HtrA4-specific antibody.</p

Enhancement of trophoblast migration by mAb 10H10.

<p>(A) Representative cell index curves of HTR8 cells for migration in the absence or presence of 10H10 or control mAb (5 µg/ml) measured with xCELLigence system. (B) Exogenous addition of mAb 10H10 (5 µg/ml) significantly increased HTR8 cell migration at 12–32 hours, compared to untreated controls. Data are mean ± SEM from 3 independent experiments, *, P&lt;0.05.</p

The trimeric HtrA3 homology model and location of the epitopes for mAbs 10H10, 2C4 and 3E6.

<p>(A) Cartoon representation of the trimeric HtrA3 (HtrA3-L) homology model, with each of the three HtrA3 monomers colored differently (grey, light green and light pink). Individual domains within each monomer are labeled: I-K; N-terminal combined IGFB-Kazal domains; Prot; central protease domain, and PDZ; the PDZ domain. Red asterix indicates the location of the catalytic site in each monomer. Epitope for the inhibitory mAb 10H10, residues 278–292 in the protease domain, is colored dark blue and corresponds to the putative HtrA3 sensor loop L3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108235#pone.0108235-Truebestein1" target="_blank">[28]</a>. Epitopes for mAbs 2C4 (residues 230–239) in each protease domain and 9C9 (residues 403–417) located in the PDZ domain are colored yellow and red, respectively. View directly above the protease catalytic sites. (B) Close up view of the 10H10 inhibitory epitope (dark blue) and modeled protease catalytic triad (orange sticks) for one HtrA3 monomer (grey cartoon). Catalytic triad residues His-191, Asp-227 and Ser-305 are displayed as orange sticks; Glu-280, Arg-282 and Asp-288 of the putative L3 sensor loop are displayed as dark blue sticks. Salt bridge/hydrogen bond interactions between Glu-280 and Arg-282 are shown as dashed black lines. The adjacent HtrA3 monomer is shown as a light pink cartoon. (C) Same view as in panel (A), protein is depicted as a molecular surface. (D) View of the non-catalytic face of HtrA3, i.e. a rotation of +180^o about the Y-axis from the view shown in panels (A) and (C). (E) Side view of the grey monomer [−90^o rotation about the X-axis, followed by a +120^o rotation about the Y-axis from the view shown in panel (A)]. One letter amino acid codes have been used for labels.</p

Effects of HtrA3 mAbs on substrate cleavage and dose-dependent modulation of HtrA3 activity by mAbs.

<p>(A) Representative real-time progressive curves of substrate cleavage by recombinant wild type human HtrA3 (HtrA3-L) in the presence of 20 µg/ml individual HtrA3 mAb (6G6, 9C9, 10H10, 3E6 and 2C4) or control mAb. (B) Inhibition of HtrA3 activity by mAb 10H10. (C) Enhancement of HtrA3 activity by mAb 6G6. (D) Inhibition of HtrA3 activity by mAb 10H10 subsequent to 6G6 stimulation. The data are expressed as changes in the rate of substrate cleavage relative to the control (B &amp; C: control  =  HtrA3 with IgG control mAb, D: control  =  HtrA3 with mAb 6G6 at 20 µg/ml). Data are mean ± SEM from 3 independent experiments, *, P&lt;0.05, **, P&lt;0.01, ***, P&lt;0.001.</p

Location of the epitopes for mAbs 9C9 and 6G6.

<p>(A) Same view as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108235#pone-0108235-g006" target="_blank">Figure 6A &amp; C</a>. The epitope regions for mAb 9C9 are shown: residues 283–297 (colored brown), residues 224–227 (from the 223–242 peptide, colored dark green) and residues 320–322 (from the 308–322 peptide, colored magenta). (B) A +180^o rotation about the Y-axis from view shown in panel (A), showing the non-catalytic face of HtrA3. Residues 228–242 are colored dark green and 308–319 are colored magenta. (C) The epitope regions for mAb 6G6 are shown: residues 73–78 (from the 73–92 peptide, colored dark grey), residues 288–302 (colored blue), residues 322–327 (from the 313–327 peptide, colored purple), and residues 398–412 (colored orange). (D) A +180^o rotation about the Y-axis from view shown in panel (C), showing the non-catalytic face of HtrA3. Residues 79–92 are colored dark grey and 313–321 are colored purple. The 6G6 epitope residues 133–147 (colored white) are now visible on the protease domain surface. (E) Close up view of panel (D) showing the 6G6 epitope regions on the non-catalytic face of HtrA3. Distances between some of the epitope regions are shown in Å. The arrow indicates the direction the PDZ domain would need to move to place the 6G6 epitope residues 398–412 (colored orange) in closer to the residues 313–321 (colored purple), 133–147 (colored white) and 79–92 (colored dark grey). In panels (A) and (C), the location of the catalytic site in each monomer is indicated by a yellow asterix.</p

Enhancement of trophoblast invasion by mAb 10H10.

<p>(A) Representative cell index curves of HTR8 cells for invasion in the absence or presence of 10H10 or control mAb (5 µg/ml) measured with xCELLigence system. (B) Exogenous addition of mAb 10H10 (5 µg/ml) significantly increased HTR8 cell invasion at 12–56 hours, compared to untreated controls. Data are mean ± SEM from 3 independent experiments, *, P&lt;0.05.</p

Selected bond lengths (Å) of coelenterazine obtained from the MD simulation and from the QM/MM studies of the deprotonated form.

<p>Selected bond lengths (Å) of coelenterazine obtained from the MD simulation and from the QM/MM studies of the deprotonated form.</p

QM/MM simulations provide insight into the mechanism of bioluminescence triggering in ctenophore photoproteins

<div><p>Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs <i>via</i> the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C₃ position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.</p></div

Induced fit docking study on substrate binding mode.

<p>A) Cutaway view of the berovin cavity showing coelenterazine occupied in. B) Close-up of the amino acid residues around the 2- hydroperoxy group of coelenterazine.</p