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Glutamic acid 141 of the diphtheria toxin receptor (HB-EGF precursor) is critical for toxin binding and toxin
1Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas 75235-9048, USA.
Abstract:
The transmembrane precursor of the monkey heparin-binding EGF-like growth factor also functions as a diphtheria toxin receptor. The mouse precursor does not bind the toxin. Previously, the most important region for binding the toxin in the monkey precursor was narrowed down to residues 122-148 through the expression of chimeric mouse/monkey precursors and subsequent toxin-sensitivity assays. To define further the toxin binding domain of the monkey precursor, distinct monkey/mouse chimeric precursors were expressed and assayed. The region between monkey residues 136-148 was found to be absolutely necessary for the retention of toxin sensitivity. Within this region, the monkey and mouse precursors differ in only two residues (residues 141 and 147). A toxin-insensitive monkey/mouse chimera that contained monkey residues 1-136 was converted to a toxin-sensitive chimera by the mutation of a single residue (His141 to Glu141). Expression of a mutant monkey precursor in which a single monkey residue (Glu141) was converted to the mouse residue (His141) yielded a cell line that was approximately 100-fold less sensitive to the toxin and the mutant precursor bound the toxin approximately 12-fold less tightly than the wild-type monkey precursor. Taken together, these results indicate that Glu 141 plays a critical role in toxin binding and toxin sensitivity.
Insights
The monkey heparin-binding EGF-like growth factor precursor binds diphtheria toxin, unlike the mouse version. A specific residue, Glu 141, is critical for this toxin binding and sensitivity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The transmembrane precursor of monkey heparin-binding EGF-like growth factor (HB-EGF) acts as a diphtheria toxin receptor.
- Mouse HB-EGF precursor lacks this toxin-binding capability.
- Previous studies localized the toxin-binding region to residues 122-148 in the monkey precursor.
Purpose of the Study:
- To further delineate the specific amino acid residues responsible for diphtheria toxin binding and sensitivity in the monkey HB-EGF precursor.
- To investigate the role of individual residues within the 136-148 region in mediating toxin interaction.
Main Methods:
- Construction and expression of distinct monkey/mouse chimeric HB-EGF precursors.
- Toxin-sensitivity assays to evaluate the functional consequence of chimeric constructs.
- Site-directed mutagenesis to alter specific amino acid residues (e.g., His141 to Glu141) and assess their impact on toxin binding and sensitivity.
Main Results:
- The region between monkey residues 136-148 was identified as essential for diphtheria toxin sensitivity.
- A single amino acid substitution from His141 to Glu141 in a chimera restored toxin sensitivity.
- Mutating Glu141 to His141 in the monkey precursor significantly reduced both toxin sensitivity (approx. 100-fold) and toxin binding affinity (approx. 12-fold).
Conclusions:
- Glutamate at position 141 (Glu141) in the monkey HB-EGF precursor is a critical determinant for diphtheria toxin binding.
- This specific residue plays a pivotal role in mediating cellular sensitivity to diphtheria toxin.