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

Abstract 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 inhibitor’s 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. Highlights Ixochymostatin is a newly described trypsin inhibitor-like (TIL) protein from Ixodes scapularis . Ixochymostatin inhibits chymase and reduces vascular permeability. Ixochymostatin inhibits chymase-dependent generation of vasoconstrictive peptides. Antibodies generated against ixochymostatin neutralize its inhibitory functions. Potential epitopes responsible for anti-ixochymostatin neutralization were mapped.