The component of Carica papaya seed toxic to A. aegypti and the identification of tegupain, the enzyme that generates it

As Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. The aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17 μg mL−1 tegument extract and 27 μg mL−1 cotyledon extract caused 100% larval mortality in a bioassay. The mixture was no longer larvicidal after the tegument extract was pre-treated at 100 °C for 10 min. The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km 58.8 μM, Kcat 28020 s−1, Kcat/Km 5 × 108 M−1 s−1), and its activity increased with 2 mM dithiothreitol (DTT), at 37 °C, pH 5.0. The chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (Kiapp 2.43 nM), E64 (3.64 nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds.

► A new cysteine proteinase, named tegupain, was purified from Carica papaya tegument. ► The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km 58.8 μM). ► Tegupain is responsible for the generation of toxic compound present in the cotyledon of Carica papaya against Aedes aegypti.