Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus

Abstract

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5–protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement

“…The anticomplement activity of tick saliva or its protein components has been known for decades and is described in numerous publications [14,15,[55][56][57]. Although the active molecules originate from either unique tick protein families [58][59][60][61][62] or lipocalins [16], anticomplement activity has only recently been reported for a tick salivary serpin [44] as the only tick protease inhibitor with such activity. Since complement products might directly damage the tick hypostome or initiate a stronger immune response [11], we propose that the role of Iripin-8 is to attenuate these mechanisms.…”

“…There was a statistically significant reduction in complement activity against erythrocytes when human plasma was incubated with Iripin-8 at concentrations of 2.5 µM and higher (Figure 3C). We also compared Iripin-8 with other two tick salivary serpins: Iripin-3 [32] showed very weak anticomplement activity and was used as a control; compared with Iripin-5 [44], Iripin-8 had lower activity.…”

Immunomodulatory Proteins in Tick Saliva From a Structural Perspective

“…IRS-2 inhibits cathepsin G and chymase produced by neutrophils and mast cells, respectively and blocks the IL-6/STAT-3 signaling pathway (Chmelar et al, 2011;Paĺenıḱováet al, 2015). The latest addition to serpins from Ixodes ricinus are Iripin-3, -5, and -8 (Chlastaḱováet al, 2021;Kascakova et al, 2021;Kotaĺ et al, 2021) Iripin-3 reduces IL-6 production and inhibits the Th1 immune response (Chlastaḱovaé t al., 2021) and Iripin-5 inhibits macrophages migration and blocks activation of the complement (Kascakova et al, 2021).…”

“…IRS-2 inhibits cathepsin G and chymase produced by neutrophils and mast cells, respectively and blocks the IL-6/STAT-3 signaling pathway ( Chmelar et al., 2011 ; Páleníková et al., 2015 ). The latest addition to serpins from Ixodes ricinus are Iripin-3, -5, and -8 ( Chlastáková et al., 2021 ; Kascakova et al., 2021 ; Kotál et al., 2021 ) Iripin-3 reduces IL-6 production and inhibits the Th1 immune response ( Chlastáková et al., 2021 ) and Iripin-5 inhibits macrophages migration and blocks activation of the complement ( Kascakova et al., 2021 ). Although both serpins contain an Arg residue in the P1 position and show anti-trypsin activity, Iripin-3 blocks kallikrein and matriptase whereas Iripin-5 inhibits human neutrophil elastase and proteinase 3.…”

“…Although both serpins contain an Arg residue in the P1 position and show anti-trypsin activity, Iripin-3 blocks kallikrein and matriptase whereas Iripin-5 inhibits human neutrophil elastase and proteinase 3. The crystal structures of IRS-2, Iripin-5, and Iripin-3 ( Figure 2A ), obtained in the relaxed variant of which the cleaved RCL is inserted into the β-sheet, revealed classical serpin fold with three β-sheets and 10 and 9 α-helices, respectively ( Chmelar et al., 2011 ; Chlastáková et al., 2021 ; Kascakova et al., 2021 ). In contrast the crystals of Iripin-8 is obtained in its native form shedding light on the structure of the RCL region ( Kotál et al., 2021 ).…”