Molecular model of Lox-FB.

<p>Construction of molecular model based on structure of human factor B (PDB 2ok5.1), using the tool SWISS-Model Workspace available on <a href="http://swissmodel.expasy.org/workspace/" target="_blank">http://swissmodel.expasy.org/workspace/</a>. The further analyses were performed using the software SwissPDBViewer.</p

Multiple alignment of the vWFA and serine protease domains from Lox-FB with sequences of FB/C2 proteins from other organisms.

<p>The alignment was performed using MUSCLE algorithm available in MEGA software. Amino acids that are highlighted in bold indicate identical regions. The amino acids residues that are functionally important at Factor D or C1s cleavage site; the metal ion dependent binding site (MIDAS) and the protease active sites are indicated by dark arrows; the three amino acids residues (T⁴³¹, A⁴³³, S⁶¹⁶) that are important on stabilization of catalytic triad are indicated by sign #; the conserved cysteines residues are indicated by asterisks and the two extra cysteines present in Lox-FB, Hd-FB3 and Rd-FB are highlighted in grey. <i>Loxosceles laeta</i> (Lox-FB), <i>Hasarius adansoni</i> (Hd-FB1; HD-FB2; Hd-FB3), <i>Tachypleus tridentatus</i> (Tt-FB1;Tt-FB-2), <i>Ruditapes decussatus</i> (Rd-FB), <i>Nematostella vectensis</i> (Nv-FB), <i>Branchiostoma belcheri</i> (Bb-FB), <i>Strongylocentrotus purpuratus</i> (Sp-FB), <i>Apostichopus japonicus</i> (Aj-FB), <i>Homo sapiens</i> (Hu-C2;Hu-FB).</p

Alignment of Hu-FB and Lox-FB CCPs.

<p>The five CCPs are aligned to each other with the consensus amino acids shown in bold and at the bottom. Two disulfide bonds sustain the CCP domain and are formed between the first and third cysteine, and the second and the fourth cysteine. The alignment was done with CLUSTAL W using Bioedit v. 7.0.9.0 software.</p

cDNA sequence and deduced amino acid sequence of Lox-FB.

<p>The ORF predicts a protein of 651 amino acids with domains of known C2 and Bf proteins. The signal peptide is in italics and comprised of the initial 25 amino acids. The cysteines and the putative N-glycosylated sites are marked in black and grey, respectively.</p

Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).

<p>Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).</p

Overlap of human factor B (Hu-FB) (pink) and the <i>Loxosceles</i> factor B-like (Lox-FB) (grey).

<p>[A] vWA Domain [B] SP Domain [C] amino acids residues that comprise the catalytic triad of Hu-FB (H, D, S) and Lox-FB (S, N, P). The manipulation of models were performed using the software SwissPDBViewer.</p

Proteolytic action of Ps82 on purified human C components C3, C4 and C5.

<p>Samples of 0.1 μg of Ps82 were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) <b>[A]</b>, human C4 (3 μg) <b>[B]</b> and human C5 (3 μg) <b>[C]</b> at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1^st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2^nd lanes: incubation of purified components with Ps82; 3^rd lanes: incubation of the mixture with PMSF and 4^th lanes: incubation of the mixture with Phenanthroline. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.</p

Action of Ps82 on the complement pathways.

<p>Samples (50 μL) of normal human serum (NHS) were pre-incubated with 0.25, 0.33 or 0.5 μg/mL of Ps82 or with 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract. The mixtures were pre-incubated for 30 min for the alternative pathway <b>[A]</b> or 1 h for the classical pathway <b>[C]</b> at 37°C, and then assayed for the residual complement activity in haemolytic tests. For lectin pathway <b>[B]</b>, samples of 50 μL of NHS were pre-incubated, in the presence or absence of 10 mM 1,10-Phenanthroline, with 0.5 μg/mL of Ps82 or 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract for 30 min at 37°C and then evaluated for the residual complement activity by ELISA. Alternatively, samples of NHS incubated with Ps82 or the <i>Premolis semirufa</i>’s bristles extract were evaluated in conditions for activation of the classical pathway, by the deposition of complement component C4b by ELISA <b>[D]</b>. Data are representative for three separate experiments, performed in duplicate, and the results are expressed as percentage of haemolysis (%) ± SD (for A and C) and percentage of C4b deposition (%) ± SD (for B and D) in relation to the control samples (NHS + buffer) and between the treatments. (*) <i>p</i> < 0.05 (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001. The symbol (#) indicates significant differences between the extract and Ps82.</p

Human serum anaphylatoxins generation induced by Ps82.

<p>Samples of NHS (50 μL) were incubated with Ps82 (0.33 μg/mL) or the <i>Premolis semirufa’s</i> bristles extract (175 μg/mL), in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), for 30 min at 37°C. The generation of the anaphylatoxins (C3a, C4a and C5a) was measured using the kits "Human C3a ELISA Kit, Human C4a ELISA Kit and Human C5a ELISA Kit". Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of each anaphylatoxin per mL of human serum (ng/mL) ± SD. (*) <i>p</i> < 0.05 (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the Buffer and the treatments.</p

Terminal Complement Complex (TCC) generation induced by the <i>Premolis semirufa</i>’<i>s</i> bristles extract.

<p>Samples of NHS (50 μL) were incubated with the bristles extract (175 μg/mL) or PBS, in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), at 37°C for 30 min, and SC5b-9 complex present in human serum samples was measured using the MicroVue SC5b-9 Plus EIA Kit. Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of SC5b-9 complex per mL of human serum (ng/mL) ± SD. (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the buffer and between the treatments.</p