Heparin-like molecules on the cell surface potentiate binding of diphtheria toxin to the diphtheria toxin receptor/membrane-anchored heparin-binding epidermal growth factor-like growth factor.
Diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF), has a high affinity for heparin. We studied the effect of heparin-like molecules on the binding of diphtheria toxin (DT) to DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate (HS) proteoglycans were about 15 times less sensitive to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin was added to the culture medium, DT sensitivity of the mutant cells was fully restored. Studies of binding of 125I-labeled DT to HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA indicated that the increased sensitivity to DT after addition of heparin is due to increased binding of DT to cells. Vero cells display a relatively large amount of heparan sulfate residues compared to CHO-K1 cells or L cells. Enhancement of DT binding by the addition of heparin was also observed with CHO-K1 cells and L cells that had been transfected with human DTR/proHB-EGF cDNA, but the degree of enhancement was less than that observed with the HS-deficient CHO cells. Addition of heparin did not affect DT binding or DT sensitivity of Vero cells. Heparin-dependent binding was observed when intact Vero cells were treated with heparitinase or when the cell membrane was solubilized with a neutral detergent. Scatchard plot analysis for the binding of DT to a recombinant HB-EGF in vitro and to L cells expressing human DTR/proHB-EGF revealed that heparin increases the affinity of DTR/proHB-EGF for DT but does not change the number of binding sites. Although DRAP27/CD9 is known to enhance DT binding to DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9 increase DT binding by independent mechanisms. Thus, heparin-like molecules, probably in the form of heparan sulfate proteoglycan on the cell surface, are a third factor required for maximal DT binding activity of cells.
D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D016466:CHO Cells; D002704:Chlorates / Q000494:pharmacology; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000378:metabolism* / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology*; D066257:Heparin-binding EGF-like Growth Factor; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D060245:Tetraspanin 29; D014709:Vero Cells
D015703:Antigens, CD; D002704:Chlorates; D004167:Diphtheria Toxin; D004815:Epidermal Growth Factor; D006493:Heparin; D066257:Heparin-binding EGF-like Growth Factor; D006497:Heparan Sulfate; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D060245:Tetraspanin 29; C554869:CD9 protein, human; C587883:HBEGF protein, human