Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II, a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while imp...

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Bibliographic Details
Published in:	Biochemistry and biophysics reports 2021-07, Vol.26, p.101008-101008, Article 101008
Main Authors:	Rojas, Laritza, Cabrera-Muñoz, Aymara, Gil Pradas, Dayrom, González, Jessica B., Alonso-del-Rivero, Maday, González-González, Yamile
Format:	Article
Language:	eng
Subjects:	Elastase Inhibitor Kazal-family Protease Site-directed mutagenesis Subtilisin Trypsin
Online Access:	Get full text
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Summary:	CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine. •The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition.•Besides P1 site, other residues contribute to CmPI-II:SUBTA/elastases interaction.•R12 change at P1 site on CmPI-II improves the selectivity of the inhibitor.•CmPI-II scaffold is a source of selective inhibitors with biomedical applications.
ISSN:	2405-5808 2405-5808