Insights into the activation mechanism of Bm-CPA: Implications for insect molting regulation.

ABSTRACT

Carboxypeptidase A has been found across various animal species, yet its activation mechanism during the insect molting process remains elusive. Our study specifically delved into the activation mechanism of carboxypeptidase A (Bm-CPA), identified in Bombyx mori's molting fluid during metamorphosis. Initially, western blotting identified two forms of Bm-CPA, 65 kDa and 54 kDa, in the epidermis of silkworms during the molting stage. Expressing the complete Bm-CPA sequence in Pichia pastoris allowed the identification, via mass spectrometry analysis, of a 75-amino-acid propeptide for the initial hydrolysis process. Subsequently, a 35 kDa form of Bm-CPA emerged in the molting fluid, confirmed as the active form through in vitro assays, demonstrating potent carboxypeptidase A activity and faint carboxypeptidase B activity. Four potential activation sites (including Lys158/Arg159 and Arg177/Arg178) were identified through mass spectrometry and amino acid mutation analysis. RNAi of Bm-CPA indicates its critical role in molting. Finally, the carboxypeptidase inhibitor (Bm-CPI) from silkworm molting fluid was expressed to explore its role in regulating Bm-CPA activity, demonstrating a direct interaction with the 35 kDa Bm-CPA. Our research implies Bm-CPA's potential involvement in the silkworm molting process, suggesting diverse regulatory roles. These findings highlight intricate protein regulation patterns during insect metamorphosis and development.