TOXIN BINDING-SITE OF THE DIPHTHERIA-TOXIN RECEPTOR - LOSS AND GAIN OF DIPHTHERIA-TOXIN BINDING OF MONKEY AND MOUSE HEPARIN-BINDING, EPIDERMAL GROWTH FACTOR-LIKE GROWTH-FACTOR PRECURSORS BY RECIPROCAL SITE-DIRECTED MUTAGENESIS

The transmembrane precursor of the monkey (Mk) heparin-binding, epider mal growth factor-like growth factor (proHB-EGF) functions as a diphth eria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. P reviously, using chimeric Ms/Mk precursors, we have shown that DT resi stance of cells bearing Ms proHB-EGF may be accounted for by several a mino acid substitutions between residues 122 and 148 within the EGF-li ke domain and that Glu-141 is an important amino acid residue for DT b inding. In this study, reciprocal site-directed mutagenesis was perfor med on the major non-conserved residues in the region of 122-148, alon e or in combination, between Mk and Ms precursors to identify more pre cisely which amino acid residues are important for DT binding. Two app roaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB-EGF by substitution(s) with th e corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu-141His (approximately 4000-fold) and the next greatest with Mk/IIe-133Lys (approximately fo urfold). The double mutations Mk/Leu-127Phe/Glu-141His, Mk/Ile-133Lys/ Glu-141His and Mk/His-135Leu/Glu-141His resulted in complete toxin res istance (> 100000-fold). The second approach, both novel and complemen tary, was to gain DT binding and sensitivity of Ms proHB-EGF by substi tution(s) with the corresponding Mk residue(s). Surprisingly, the sing le mutation Ms/His-141Glu resulted in the gain of moderate DT sensitiv ity (> 260-fold). The double mutation Ms/Lys-133Ile/His-141Glu and the triple mutation Ms/Lys-133Ile/Leu-135His/His-141Glu resulted in a pro gressive gain in toxin sensitivity (> 4700-fold and > 16000-fold respe ctively) and affinity. This triple mutant cell line is essentially as sensitive (IC50 = 3.1 ng ml(-1)) as the highly toxin-sensitive monkey Vero cell line (IC50 = 4 ng ml(-1)), indicating that these three Mk re sidues enable the Ms proHB-EGF to act as a fully functional DT recepto r. Taken together, these results indicate that Glu-141 plays the most critical role in DT binding and sensitivity and that two additional am ino acid residues, Ile-133 and His-135, also play significant roles.