The fungal‐resistance factors BmSPI38 and BmSPI39 predominantly exist as tetramers, not monomers, in Bombyx mori

Abstract

Previous studies have indicated that trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, BmSPI38 and BmSPI39, suppress conidial germination and integument penetration of entomopathogenic fungi by inhibiting their cuticle-degrading proteases and might functions as fungal-resistance factors in the silkworm. To date, the physiological forms and functional significance of multimerization of BmSPI38 and BmSPI39 remain unknown. In this study, we investigated the physiological forms of BmSPI38 and BmSPI39 in Bombyx mori silkworms using multiple complementary methods, including activity staining, reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, western blotting and immunofluorescence. We found that recombinant BmSPI38 and BmSPI39 tend to form homologous multimers, and their dimers, trimers and tetramers possessed intense inhibitory activity against subtilisin A from Bacillus licheniformis. In contrast, their monomers showed no detectable inhibitory activity. Both BmSPI38 and BmSPI39 also exist mainly as stable tetramers in silkworm tissues, and they also predominantly function as a tetramer in these tissues. This study is the first to demonstrate this preferred quaternary form of a TIL-type protease inhibitor and will likely help to elucidate the mechanisms of BmSPI38 and BmSPI39 in the innate immune response of the silkworm.