CD spectra of natural Bla g 2 (black line) and rBla g 2-N93Q (gray dashed).

<p>CD spectra of natural Bla g 2 (black line) and rBla g 2-N93Q (gray dashed).</p

Overlap of 7C11 mAb and IgE antibody epitopes.

<p>Inhibition of IgE antibody binding to Bla g 2 by mAb 7C11 (0.001–10 µg/ml) and a rabbit anti-Bla g 2 polyclonal antibody.</p

Structural environment of the lysine residues involved in cation-π interactions important for antibody binding.

<p>A) Lysine 132 interacting with the tyrosine 33 of the heavy chain of the mAb 7C11. B) Lysine 251 interacting with the tyrosine 32 of the light chain of the mAb 4C3.</p

Binding of IgE antibodies from cockroach allergic patients to rBla g 2-N93Q and epitope mutants.

<p>A) IgE binding to mAb 7C11 epitope mutants, and B) IgE binding to mAb 4C3 epitope mutants. In A and B scatter plots show median of percentages (± interquartile ranges) of IgE binding versus rBla g 2-N93Q for the sera tested (n = 8–11) Each data point was calculated as average of percentage of IgE binding (± SEM) versus the rBla g 2-N93Q for 1–4 tests per serum depending on serum availability.</p

Epitopes for mAb 7C11 and 4C3 are located on opposite lobes of Bla g 2.

<p>Ribbon diagram of rBla g 2 (gold) in complex with a Fab fragment of the mAb 4C3 (blue) superimposed to the Fab′ of mAb 7C11 in complex with Bla g 2 (allergen not shown). Monoclonal Ab 7C11 binds to the N-terminal lobe of the allergen and mAb 4C3 to the C-terminal lobe. Antibody fragments consist of a heavy and a light chain, in green and light lilac for 7C11, and in marine blue and cyan for 4C3, respectively. The carbohydrates in position N268 are shown in red.</p

Dose-response curves of Bla g 2 and epitope mutants by ELISA.

<p>A) Binding of the anti-Bla g 2 polyclonal antibody to mAb 7C11 epitope mutants. B) Binding of the anti-Bla g 2 polyclonal antibody to mAb 4C3 epitope mutants. C) Binding of mAb 7C11 to mAb 7C11 epitope mutants, D) Binding of mAb 4C3 to mAb 4C3 epitope mutants. Data are mean ± standard deviation of duplicates from one representative experiment out of two performed for each panel. Legends in panels C and D also apply to panels A and B, respectively.</p

RC1339/APRc from <i>Rickettsia conorii</i> Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

<div><p>Members of the species <i>Rickettsia</i> are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many <i>Rickettsia</i> species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 <i>Rickettsia</i> genomes. Using <i>R. conorii</i> gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of <i>rc1339</i> APRc (for <u>A</u>spartic <u>P</u>rotease from <i><u>R</u>ickettsia <u>c</u>onorii</i>) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as <i>Rickettsia</i>, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in <i>R. conorii</i> and <i>R. rickettsii</i> and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the <i>in vitro</i> processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel <i>bona fide</i> member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.</p></div

Inhibition of biotinylated mAb binding to rBla g 2 by allergen mutants using ELISA.

<p>A) Inhibition of binding of biotinylated-mAb 7C11 to rBla g 2-N93Q by mAb 7C11 epitope mutants. B) Inhibition of binding of biotinylated-mAb 4C3 to rBla g 2-N93Q by mAb 4C3 epitope mutants. Data are mean ± standard deviation of duplicates from one representative experiment out of two performed for each panel.</p

Rational design for mutagenesis.

<p>A) Principal residues involved in the the mAb 7C11 epitope on the N-terminal lobe of Bla g 2. The longest consecutive part of the epitope (shown in yellow) interacts with all three CDR of the light chain (surface in green) and has 9 residues (starting at 60) from which two were mutated to alanine (K65 and D68a). K65, R83 and K132 form cation-π interactions with tyrosines Y53, Y92 and Y33 from the mAb 7C11, respectively. The heavy chain of the antibody is shown in lilac. B) Main residues involved in the mAb 4C3 epitope on the C-terminal lobe of Bla g 2. The surface of the allergen, with the main residues involved in antibody binding is shown in gold. The third loop of the heavy chain of the antibody (in blue) includes: a) R103 attracted to the negatively charged E233, and b) W105 that docks into a hydrophobic pocked formed by A234, I199 and P256 in Bla g 2. Residues K251 and Y32 involved in a cation-π interaction are also shown.</p

Close-up of the interactions of EcTI and trypsin.

<p>(a) Steroview of the region of interactions, with residues belonging to trypsin shown as cyan sticks and the disulfide bond colored yellow. The residues of EcTI are shown as green sticks and the gray spheres represent water molecules. Hydrogen bonds are indicated by red dashed lines. (b) Interface of the EcTI-trypsin complex, with EcTI shown as a green ribbon with selected side chains in stick representation, whereas the surface of trypsin is colored according to charge (blue positive, red negative, gray uncharged).</p