Longistatin is an unconventional serine protease and induces protective immunity against tick infestation

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37 °C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC50 of 278.57 μM, 0.35 μM, 41.56 μM and 198.86 μM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn2+) in a concentration-dependent manner with an IC50 value of 275 μM, and the inhibitory effect of Zn2+ was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by ∼34%; thus, the vaccination trial was ∼73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Graphical abstractLongistatin degraded fibrinogen and hydrolyzed serine protease-specific substrates. Longistatin was inhibited by serine protease inhibitors and Zn2+. Longistatin induced protective immunity against tick infestation.Figure optionsDownload full-size imageDownload high-quality image (187 K)Download as PowerPoint slideHighlights► Longistatin was detected in salivary glands and ducts of ticks, and in host tissues. ► Longistatin hydrolyzed several serine protease-specific substrates and degraded fibrinogen. ► Longistatin showed specificity for the amide bond of Arg, indicating trypsin like substrate specificity. ► Longistatin was efficiently inhibited by various serine protease inhibitors and Zn2+ and functions like a potent atypical serine protease. ► Longistatin induces protective immunity against ticks and may be a suitable candidate for an effective anti-tick vaccine.