Functional study of CRAC and CARC peptides derived from E.coli α-hemolysin

MATÉ S; GUZMÁN F; CANÉ L; HERLAX V

Congreso; Reunion Anual de la sociedad Argentina de biofisica; 2018

Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the family of 'Repeat in toxins'(RTX). The CRAC domain refers to the Cholesterol Recognition/interaction Aminoacid Consensus sequence. The CARC domain is similar to the CRAC sequence, but exhibits the opposite orientation along the polypeptide chain.The aims of this work were to study the participation of CRAC and CARC in thestabilization of HlyA monomers in membranes by their interaction with cholesterol,to evaluate the role of Y347 in the interaction with membrane, and finally to find a cytotoxic peptide for the construction of an immunotoxin.On the basis of experimental data and structural predictions, six peptides derived from HlyA were synthesized: PEP 1: transmembrane domain described ashemolytically active; PEP 2: also a transmembrane domain which sequencecorresponds to a cholesterol binding domain (CARC); PEP3: similar to PEP2 butwith residue Y347 substituted by A; PEP4: similar to PEP2 but with a CRACsequence; PEP5 and PEP6 correspond to CARC sequences located near theacylation sites.Peptides were synthesized by the solid phase peptide synthesis method (Fmocstrategy), purified by HPLC (C-18 column), the molecular mass was determined bymass spectrometry and peptide structure by circular dichroism. The hemolyticactivity of peptides was measured using human erythrocytes and inhibition ofhemolytic activity assays were performed pre-incubating erythrocytes withpeptides and then adding them to wild type toxin.Results describe PEP2 as hemolytic, which is promising and encourage us to use it in the design of immunotoxins. PEP3 was found not to be hemolytic suggestingresidue Y347 is fundamental for the interaction of HlyA with lipidic membranes.PEP4 was found not to be hemolytic, which implicates the CRAC sequence addedwas unfavorable for peptide activity. PEP 6 competes with HlyA for binding sites in erythrocytes.