Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro

Abstract

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

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

“…Structural analysis of Iripin-5 shows the typical serpin fold in the relaxed state that was observed in other known crystal structures of I. ricinus serpins (IRS-2 and Iripin-3; Chmelař et al, 2017;Chlastá ková et al, 2021). The relaxed cleaved state of Iripin-5 was caused by the presence of contaminating proteases, probably during protein storage, and this cleavage has been observed previously (Ková řová et al, 2010).…”

“…It has been shown that the salivary serpin Iris modulates host innate and acquired immunity (Leboulle et al, 2002). Likewise, IRS-2 and Iripin-3 modulated adaptive immune responses (Chmelar et al, 2011;Chlastá ková et al, 2021). Moreover, crystal structures were determined for the last two, which are the only two tick serpins with resolved 3D structures to date.…”

“…Iris, with Met340 at the P1 site, is an inhibitor of leukocyte elastase and elastaselike serine proteases (Prevot et al, 2007), although its inhibition is managed by several exosites in -helices A and D (Prevot et al, 2009). However, IRS-2 has Tyr341 at its P1 site, which signifies the inhibition of chymotrypsin-like proteases (Chmelař et al, 2017), and Iripin-3 has Arg342 at the P1 site, indicating its trypsin-like protease inhibition (Chlastá ková et al, 2021). Nevertheless, diverse RCL residues can represent potential cleavage sites, but only a few residues (16-17 residues from the C-terminal -sheet) manage to successfully inhibit the target protease (Gettins, 2002).…”

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

“…Iripin-8 also inhibited TT in a dose-dependent manner and blocked fibrin clot formation completely at concentrations of 800 nM and higher (Figure 3B). The other serpins presented for comparison in Figure 3C did not have any effect on blood coagulation except the inhibition of PT by Iripin-3, which we published elsewhere [32].…”

“…However, according to numerous transcriptomic studies, the total number of tick serpins is significantly higher [13,[24][25][26][27]. In I. ricinus, at least 36 serpins have been identified based on transcriptomic data, but only 3 of them have been characterized at the biochemical, immunomodulatory, anticoagulatory, or antitick vaccine levels [13,[28][29][30][31][32].…”