Defining the regulatory and inhibitory elements within the prodomain of CsCF-6, a cathepsin F cysteine protease of Clonorchis sinensis

Abstract

CsCF-6 is a member of the multigene family of cathepsin F cysteine proteases of Clonorchis sinensis. Similar to other papain family proteases, CsCF-6 is synthesized as a proenzyme and is converted to the mature form by autocatalytic removal of the prodomain. Here, we analyzed the regulatory and inhibitory elements within the CsCF-6 prodomain to understand the regulatory mechanism of CsCF-6 by its prodomain. The CsCF-6 prodomain played an essential role in the folding of CsCF-6. Particularly, the ERFNAQ motif within the prodomain was essential, and the minimum segment required for this event was the C-terminal part of the prodomain, including Asn58 and downstream residues. The CsCF-6 prodomain effectively inhibited CsCF-6, in which the ERFNAQ motif played a critical role in the inhibition, but the GTFD motif was also required for complete inhibition of CsCF-6. The CsCF-6 prodomain showed broad inhibitory activity against several cysteine proteases. These results suggest that the CsCF-6 prodomain plays bi-functional roles in correct folding and inhibition of its cognate enzyme.