Distinct enzyme activities of serine protease p37k in silkworm midgut and molting fluid

Serine proteases possess various biological functions. The serine protease p37k exhibits gelatinolytic activity in the silkworm midgut and degrades cuticular proteins in the molting fluid. In this study, we analyzed the activities changes of recombinant p37k (re-p37k) and p37k in the midgut and molting fluid of Bombyx mori. Firstly, in vitro-expressed re-p37k was activated when a 22 kDa band was observed by Western blot. Re-p37k exhibits strong gelatinolytic activity, with the highest activity observed at pH 7.0–9.0 and 45 °C. Compared to p37k in the midgut, re-p37k loses thermal stability but can be restored by midgut extract or ions. E64, AEBSF, and an inhibitor cocktail inhibited the hydrolytic activity of re-p37k on epidermal proteins, but did not inhibit the gelatinolytic activity. Subsequently, zymography showed that the positions of gelatinolytic band produced by p37k in the midgut and molting fluid were different, 35 kDa and 40 kDa, respectively. Finally, When heated midgut extract was added to re-p37k or molting fluid, the gelatinolytic band shifted from 40 kDa to 35 kDa, and the proteolytic activity of p37k in the molting fluid was inhibited. Collectively, our results demonstrate that p37k exhibits different activities in various tissues, implying its distinct tissue-specific functions during insect metamorphosis.