Krynina O. Biological role of heparin-binding site in the structure of heparin-binding EGF-like growth factor.

The purpose of this thesis was to investigate the role of the heparin-binding domain in the structure of human HB-EGF and its importance for the biological activity of growth factor, namely the binding of diphtheria toxin and the formation of sHB-EGF complexes with EGFR and their subsequent internalization. Using full-length and truncated (with deletion of the heparin-binding domain) forms of recombinant sHB-EGF, it has been shown later internalization of sHB-EGF, compared to the truncated form. Western blot analysis of A431 cell lysates stimulated with full-length or truncated sHB‑EGF showed that the lack of interaction of sHB-EGF with HSPGs causes earlier phosphorylation of EGFR, but it is less potent and prolonged. To investigate the role of the heparin-binding domain in the reception of diphtheria toxin (DT), we created genetic constructs encoding fluorescently labeled proHB-EGF and wild-type sHB-EGF and with two types of mutations: amino acid substitutions of positively charged amino acids to alanine and the whole domain deletion. sHB-EGF various forms binding of derivative DT - subunit B (SbB) - was assessed by enzyme-linked immunosorbent assay. It was found that sHB‑EGF with deletion of the heparin-binding site had a higher Kd value compared to the normal or mutated form of growth factor. Using MDA-MB-231 cells and the CRISPR/Cas9 system subline with the lack of HB-EGF expression was created. Genetic constructs encoding proHB-EGF with and without mutations were used for MDA-MB-231HB-EGF(-) cell transfection. Intracellular transport of a recombinant DT derivative mediated by proHB-EGF with a mutated heparin-binding domain was demonstrated for the first time. It was also found that the association of proHB-EGF with HSPGs on the cell surface reduced the stability of the complex with the DT derivative, probably due to conformational rearrangements within the growth factor molecule itself. Within this dissertation, HB-EGF-neutralizing polyclonal antibodies were also obtained and their antiproliferative effect on A431 cells was tested.