A Heme-binding Aspartic Proteinase from the Eggs of the Hard TickBoophilus microplus

Abstract

An aspartic proteinase that binds heme with a 1:1 stoichiometry was isolated and cloned from the eggs of the cattle tick Boophilus microplus. This proteinase, herein named THAP (tick heme-binding aspartic proteinase) showed pepstatin-sensitive hydrolytic activity against several peptide and protein substrates. Although hemoglobin was a good substrate for THAP, low proteolytic activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme-toglobin 1:1 ratio. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. The addition of heme did not change THAP activity toward a synthetic peptide or against ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin (VT), the probable physiological substrate of THAP, is a hemeprotein. Hydrolysis of VT by THAP was also inhibited by the addition of heme to the incubation media. Taken together, our results suggest that THAP uses heme bound to VT as a docking site to increase specificity and regulate VT degradation according to heme availability.

New approaches toward anti-Rhipicephalus (Boophilus) microplus tick vaccine

“…Therefore, a critical adaptation is necessary, which may prevent excessive reactive oxygen species production during several physiologic conditions. This finding supports the hypothesis that an egg VT degradation aspartic protease called Tick Heme-binding Aspartic Proteinase -THAP present in eggs has its activity inhibited by heme through a site that is distinct from the catalytic site (SORGINE et al, 2000;POHL et al, 2008). The increase in egg free heme content would generate a feedback inhibition in VT degradation.…”

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixusStahl

“…Newly laid eggs are therefore able to initiate VT degradation. In vitro, measurements of hydrolase activities and yolk degradation using fresh eggs have been investigated by many groups (Nussenzveig et al, 1992;Logullo et al, 1998;Sorgine et al, 2000;Fialho et al, 2002). However, even though freshly laid eggs carry all the yolk mobilization machinery, the massive yolk proteolysis does not occur in vivo immediately after the egg is laid, but starts at a certain time later, during early embryogenesis.…”

Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

“…Briefly, hemoglobin (10 g) was incubated with SmCB1 (0.25 g) in 25 mM sodium acetate, pH 3-6, including 2.5 mM DTT in a total volume of 35 l for 1-4 h at 37°C. Aliquots of the digest were subjected to derivatization with fluorescamine to quantify the newly formed N-terminal ends (49). The fluorescence signal was measured using an Infinite M200 microplate reader (Tecan) at 370 nm excitation and 485 nm emission wavelengths.…”