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Abstract
Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.
Highlights
Diphtheria toxin (DT)1 (Mr ϭ 58,342) is secreted by toxigenic strains of Corynebacterium diphtheriae
The crystal structure of DT reveals that DT is composed of three distinct domains, the catalytic domain (C-domain), which is A-fragment itself, the transmembrane domain (T-domain), which is essential for the translocation of the C-domain to the cytoplasm, and the receptor-binding domain (R-domain), which binds to DT receptor
DT receptor (DTR) is identical to the precursor form of heparin-binding EGF-like growth factor [20, 21] that was originally identified as the heparin-binding member of the epidermal growth factor family [22, 23]
Summary
Materials—DT was prepared as described previously [6]. Rabbit antihuman proHB-EGF antibody (H-6) was produced as described previously [21] and affinity-purified using a synthetic peptide corresponding to amino acid residues 54 –73 of human DTR/proHB-EGF conjugated to Sepharose CL-6B (Pharmacia Biotech Inc., Tokyo, Japan). PTMHG-1, pTMHGDS-1, and pTMHGDK-1, which encode mouse proHB-EGF, human/mouse HB-EGF chimera H(106 –136), and human/mouse HB-EGF chimera H(106 –186), respectively [33], were digested with PvuII and BalI, and the PvuII-BalI fragment of human DTR/proHB-EGF cDNA was inserted. This segment encodes the recognition site for anti-human proHB-EGF antibody (H-6) [21], which can be used as an immunological tag to measure the surface expression of proHB-EGF.
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