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 (*)
- Yuji Shishido(1),
- Kailash D. Sharma(1),
- Shigeki Higashiyama(2),
- Michael Klagsbrun(3) and
- Eisuke Mekada(1)(§)
- From the (1)Institute of Life Science, Kurume University, Kurume, Fukuoka, 830, Japan
- (2)Department of Biochemistry, Osaka University Medical School, Suita, Osaka, 565, Japan
- (3)Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115
- §To whom correspondence should be addressed: Division of Cell Biology, Institute of Life Science, Kurume University, Kurume, Fukuoka 830, Japan; Tel.: 81-942-376317; Fax: 81-942-313320.
Abstract
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 
I-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.
Footnotes
-
↵(*) This work was supported by grants from Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- DT
-
diphtheria toxin
- DTR
-
diphtheria toxin receptor
- CHO
-
Chinese hamster ovary
- EGF
-
epidermal growth factor
- HB-EGF
-
heparin-binding EGF-like growth factor
- FGF
-
fibroblast growth factor
- bFGF
-
basic FGF
- HS
-
heparan sulfate
- HSPG
-
heparan sulfate proteoglycan
- GAG
-
glycosaminoglycan
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophroesis
- FITC
-
fluorescein isothiocyanate.
-
- Received July 7, 1995.
- Revision received September 19, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
