The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 µM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis.

The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.