BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae

BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271 Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (Ki 0.4 and 0.3 nM) and subtilisin A (Ki 1.4 nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (Ki 50 nM) and BmSI-6 (Ki 2.2 nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.