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Hamster diphtheria toxin receptor: a naturally occurring chimera of monkey and mouse HB-EGF precursors
J H Cha1, J S Brooke, L Eidels
1Department of Microbiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas, 75235-9048, USA.
Abstract:
The sensitivity of mammalian cell lines to diphtheria toxin (DT) varies between species. Monkey (Mk) Vero cells are highly sensitive to DT, whereas rat and mouse (Ms) cells are resistant; hamster (Hm) cells display moderate DT sensitivity. The precursor of the Mk heparin-binding epidermal growth factor-like growth factor (proHB-EGF) functions as a DT receptor but the Ms proHB-EGF does not. In this study we have cloned, expressed, and characterized the Hm proHB-EGF/DT receptor. The expression of Hm proHB-EGF confers moderate DT sensitivity to normally DT-resistant mouse cells. The amino acid sequence of Hm preproHB-EGF shows that, overall, it more closely resembles the Ms preproHB-EGF sequence, except in the DT-binding region where it more closely resembles the Mk sequence. In the DT-binding region the Hm proHB-EGF sequence differs from the Mk proHB-EGF in only four amino acid residues (124, 126, 133, and 147); one of these residues, Ile133 in Mk proHB-EGF, has been previously reported to be important for DT binding and sensitivity. Analysis of Mk proHB-EGF mutants with residues substituted for Ile133 suggests that Asn133 in Hm proHB-EGF may be responsible for the moderate DT sensitivity of Hm proHB-EGF-expressing cells.
Insights
Mammalian cell sensitivity to diphtheria toxin (DT) varies by species. Hamster proHB-EGF confers moderate DT sensitivity to resistant mouse cells, revealing key differences in the DT-binding region.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- Mammalian cell lines exhibit differential sensitivity to diphtheria toxin (DT).
- Monkey (Mk) Vero cells are highly sensitive, rat and mouse (Ms) cells are resistant, and hamster (Hm) cells show moderate sensitivity.
- The Mk heparin-binding epidermal growth factor-like growth factor (proHB-EGF) acts as the DT receptor, while Ms proHB-EGF does not.
Purpose of the Study:
- To clone, express, and characterize the hamster (Hm) proHB-EGF, the DT receptor in hamster cells.
- To investigate the role of Hm proHB-EGF in conferring DT sensitivity to resistant cells.
- To elucidate the molecular basis for differential DT sensitivity across species.
Main Methods:
- Cloning and expression of hamster proHB-EGF.
- Characterization of Hm proHB-EGF function as a DT receptor.
- Amino acid sequence analysis of proHB-EGF from different species, focusing on the DT-binding region.
- Site-directed mutagenesis of Mk proHB-EGF to analyze specific residue contributions.
Main Results:
- Expression of Hm proHB-EGF rendered normally DT-resistant mouse cells moderately sensitive to DT.
- Sequence analysis revealed Hm proHB-EGF resembles Ms proHB-EGF overall but shares similarities with Mk proHB-EGF in the DT-binding region.
- Four amino acid differences were identified in the DT-binding region between Hm and Mk proHB-EGF.
- A specific residue, Ile133 in Mk proHB-EGF, known to be crucial for DT binding, was found to be Asn133 in Hm proHB-EGF.
Conclusions:
- Hamster proHB-EGF confers moderate DT sensitivity to mouse cells, indicating its role as a functional DT receptor.
- The differential DT sensitivity is linked to specific amino acid residues within the DT-binding region of proHB-EGF.
- Asn133 in Hm proHB-EGF is likely responsible for the observed moderate DT sensitivity in hamster cells.