Structure and function study of peptides derived from E. Coli alpha hemolysin for the construction of an immunotoxin

GUZMÁN F; CANÉ L; HERLAX S; MATÉ S

Quilmes

Simposio; II Simposio de Jóvenes Biofísicos; 2017

Sociedad Argentina de Biofísica

STRUCTURE AND FUNCTION STUDY OF PEPTIDES DERIVED FROM E. Coli ALPHA HEMOLYSIN FOR THE CONSTRUCTION OF AN IMMUNOTOXINAbstract: Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the family of 'Repeat in toxins'(RTX). On the basis of experimental data and structural predictions, four peptides derived from HlyA were synthesized: PEP 1: transmembrane domain described as hemolytically active; PEP 2: also a transmembrane domain which sequence corresponds to a cholesterol binding domain (CARC); PEP3: similar to PEP2 but with residue Y347 substituted by A and PEP4: similar to PEP2 but with a CRAC sequence (?cholesterol recognition/interaction amino acid consensus domain?). The aims of this work were to study the participation of CRAC and CARC in the stabilization 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. Peptides were synthesized by the solid phase peptide synthesis method (Fmoc strategy), purified by HPLC (C-18 column), the molecular mass was determined by mass spectrometry and peptide structure by circular dichroism. The hemolytic activity of peptides was measured using human erythrocytes, and cytotoxicity using the MTT reduction method on the breast cancer cell line MCF-7. Results describe PEP2 as hemolytic and cytotoxic, which is promising and encourage us to use it in the design of immunotoxins. PEP3 was found not to be cytotoxic suggesting residue Y347 is fundamental for the interaction of HlyA with lipidic membranes. PEP4 was found not to be hemolytic nor cytotoxic, which implicates the CRAC sequence added was unfavorable for peptide activity.In conclusion, we synthesized, and study the structure and activity, of four peptides derived from HlyA of E. coli. Also, we found a peptide that it is both cytotoxic and hemolytically active, which leads our group to the design and construction of an immunotoxin comprising PEP2 and an antibody that specially recognizes tumoral cells.