1. Study on activation mechanism and cleavage sites of recombinant butelase-1 zymogen derived from Clitoria ternatea.
- Author
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Zhao J, Ge G, Huang Y, Hou Y, and Hu SQ
- Subjects
- Amino Acid Sequence, Chromatography, Liquid, Enzyme Precursors genetics, Enzyme Precursors metabolism, Peptides chemistry, Tandem Mass Spectrometry, Clitoria metabolism
- Abstract
Asparagine endopeptidases (AEPs) were synthesized as a zymogen and were known to undergo pH-dependent autoproteolytic activation using their endopeptidase activity. Butelase-1, one of the few AEPs with ligation activity, can also be synthesized as a zymogen and activated at acidic pH in vitro, but the detailed activation process and potential activation sites of its zymogen are not fully understood. In this study, recombinant butelase-1 exhibited high ligation activity and ineffective endopeptidase activity, and its activities were strictly pH-dependent. The endopeptidase activity caused the activation of butelase-1 zymogen at acidic pH, which was autocatalytic, required sequential removal of C- and N-terminal pro-peptides, and was a bimolecular reaction. The pro-peptides were critical to the stability of butelase-1. Once the pro-peptides left the active domain, butelase-1 was quickly inactivated at pH 7.0. Based on the LC-MS/MS sequencing of activation products, Asp319 and Asn322 were identified as potential C-terminal pro-region hydrolysis sites of the butelase-1 zymogen, which was validated by site-directed mutagenesis. Our results provided a reasonable explanation for the self-activation of butelase-1 zymogen in vitro and provided supplementary information for the activation of AEP ligase zymogen., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
- Published
- 2022
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