epidermal-growth-factor and Diphtheria

Topics: alpha-Macroglobulins; Animals; Bacterial Toxins; Cell Membrane; Cells, Cultured; Clathrin; Coated Pits, Cell-Membrane; Diphtheria; Endocytosis; Endosomes; Epidermal Growth Factor; Golgi Apparatus; Lysosomes; Microscopy, Electron; Pseudomonas; Receptors, Cell Surface; Viruses

Although equine diphtheria antitoxin may be an effective therapy for human diphtheria, its use often induces serum sickness. We describe here a strategy for developing an alternative treatment based on the human diphtheria toxin (DT) receptor/heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor. Recombinant mature human HB-EGF acts as a soluble receptor analog, binding radioiodinated DT and preventing its binding to the cellular DT receptor/HB-EGF precursor. However, the possibility existed that radioiodinated DT-HB-EGF complexes associate with cells due to the binding of the heparin-binding domain of recombinant HB-EGF to cell surface heparan sulfate proteoglycans. This possibility was confirmed by performing DT binding studies in the presence of heparin. A recombinant truncated HB-EGF (residues 106 to 149), which lacks most of the heparin-binding domain, showed an essentially heparin-independent binding of radioiodinated DT to cells. Furthermore, it was a more effective inhibitor of DT binding than was recombinant mature HB-EGF. Since mature HB-EGF is a known ligand for the EGF receptor and is thus highly mitogenic (tumorigenic), we then changed amino acid residues in the EGF-like domain of the recombinant truncated HB-EGF and demonstrated that this DT receptor analog (I117A/L148A) displayed a low mitogenic effect. The truncated (I117A/L148A) HB-EGF protein retained high DT binding affinity, as confirmed by using surface plasmon resonance. Our results suggest that the truncated (I117A/L148A) HB-EGF protein could be an effective, safe antidote for human diphtheria.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antidotes; Diphtheria; Diphtheria Toxin; Epidermal Growth Factor; Heparin; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Mice; Mitogens; Molecular Sequence Data; Receptors, Cell Surface

Topics: Animals; Anti-Bacterial Agents; Diphtheria; Epidermal Growth Factor; Escherichia coli; Guinea Pigs; Peptide Fragments; Protein Precursors; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin