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

Laritza Rojas; Aymara Cabrera-Muñoz; Dayrom Gil Pradas; Jessica B González; Maday Alonso-Del-Rivero; Yamile González-González

doi:10.1016/j.bbrep.2021.101008

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

Biochem Biophys Rep. 2021 May 11:26:101008. doi: 10.1016/j.bbrep.2021.101008. eCollection 2021 Jul.

Authors

Laritza Rojas¹, Aymara Cabrera-Muñoz¹, Dayrom Gil Pradas¹, Jessica B González¹, Maday Alonso-Del-Rivero¹, Yamile González-González¹

Affiliation

¹ Centro de Estudio de Proteínas, Universidad de La Habana, Calle 25 # 455, Plaza de La Revolución, CP 10400, La Habana, Cuba.

Abstract

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.

Keywords: Elastase; Inhibitor; Kazal-family; Protease; Site-directed mutagenesis; Subtilisin; Trypsin.