Ixochymostatin, a trypsin inhibitor-like (TIL) protein from Ixodes scapularis, inhibits chymase and impairs vascular permeability

Ticks, as pool feeders, obtain a blood meal by lacerating small blood vessels and ingesting the blood that flows to the feeding site, which triggers various host-derived responses. However, ticks face the challenge of wound healing, a process involving hemostasis, inflammation, cell proliferation and migration, and remodeling, hindering blood acquisition. To overcome these obstacles, tick salivary glands produce a diverse array of bioactive molecules. Here, we characterize ixochymostatin, an Ixodes scapularis protein belonging to the trypsin inhibitor-like (TIL) family. It is expressed in multiple developmental stages and in tick salivary glands and acts as a slow and tight-binding inhibitor of chymase, cathepsin G, and chymotrypsin. Predictions for the tertiary structure complex between ixochymostatin and chymase suggest a direct interaction between the inhibitors reactive site loop and protease active sites. In vitro, ixochymostatin protects the endothelial cell barrier against chymase degrading action, decreasing cell permeability. In vivo, it reduces vascular permeability induced by chymase and compound 48/80, a mast cell degranulator agonist, in a mouse model. Additionally, ixochymostatin inhibits the chymase-dependent generation of vasoconstrictor peptides. Antibodies against ixochymostatin neutralize its inhibitory properties, with epitope mapping identifying potential neutralization regions. Ixochymostatin emerges as a novel tick protein modulating host responses against tick feeding, facilitating blood acquisition. HighlightsO_LIIxochymostatin is a newly described trypsin inhibitor-like (TIL) protein from Ixodes scapularis. C_LIO_LIIxochymostatin inhibits chymase and reduces vascular permeability. C_LIO_LIIxochymostatin inhibits chymase-dependent generation of vasoconstrictive peptides. C_LIO_LIAntibodies generated against ixochymostatin neutralize its inhibitory functions. C_LIO_LIPotential epitopes responsible for anti-ixochymostatin neutralization were mapped. C_LI