Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

5-POS Board 5 GLYCOPHORIN IS NOT A RECEPTOR FOR HLYA IN HUMAN ERYTHROCYTES Lucia Cané 1 ; Sandrine Genetet 2 ; Isabelle Moure-Chanteloup 2 ; Mariano Ostuni 2 ; Pablo Schwarzbaum 3 ; Vanesa Herlax 1 ; 1 Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Facultad de Ciencias Médicas. Universidad Nacional de La Plata., La Plata, Argentina 2 Inserm, INTS, Unité Biologie Intégrée du Globule Rouge., Université Paris Diderot., Paris, France 3 Instituto de Química y Fisico-Química Biológicas (IQUIFIB) "Prof. Alejandro C. Paladini"., Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Capital Federal, Argentina Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. HlyA acts on a variety of cell types from several species, including human red blood cells (RBCs); and also binds to and disrupts protein-free liposomes. Studies that have explored the binding of HlyA to membranes and the characterization of a putative toxin-specific receptor have produced contradictory results. Regarding RBCs, glycophorins were characterized as putative receptors in horse RBCs, though other studies indicated that the binding of HlyA to rabbit RBCs occurred in a nonsaturable manner and that the toxin did not interact with a specific protein receptor. On the other hand, we have previously demonstrated that HlyA induces a diminish in human RBCs deformability, and the release of lytic and nonlytic ATP, effects that have been associated to the interaction of ligands with glycophorin.In this context, the aim of our study was to characterize if the interaction of HlyA with human glycophorins mediate the hemolytic and sublytic effects of the toxin. For this purpose we measure the hemolytic activity of HlyA on RBCs pretreated with different antibodies (iH4 (nanoantibody against Y 52 PPE 55 ), anti- GPA/GPB, anti-GPA) and on GPA/GPB null RBCs. For our surprise, the hemolytic activity of the toxin was similar to control RBCs in all the conditions tested, indicating that glycophorin is not necessary for HlyA lytic activity. Moreover, we performed FACS assays using antibodies anti-GPA (R18) and anti-band3 complexed with glycophorin (Wr), on treated and untreated RBCs. Results showed a slight increment in fluorescence intensity when toxin interacts with RBCs, indicating conformational changes of both GPA and Band 3 when the toxin interact with RBCs. In conclusion, results indicate that HlyA-GPA or GPB interaction is not necessary for HlyA lytic activity.

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