A Single Heparin Binding Region within the Fibrinogen-like Domain Is Functional in Chick Tenascin-C

doi:10.1074/jbc.270.7.3378

A Single Heparin Binding Region within the Fibrinogen-like Domain Is Functional in Chick Tenascin-C (*)

From the (1)Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel and the
(2)Department of Biophysical Chemistry, Biocenter of the University, CH-4056 Basel, Switzerland

^§ To whom correspondence should be addressed:
Friedrich Miescher Institut, Postfach 2543, CH-4002 Basel, Switzerland.
Tel.: 41-61-6972494; Fax: 41-61-6973976.

Abstract

Tenascin-C binds to cell surface and matrix proteoglycans and to heparin. Two heparin binding regions have recently been localized per tenascin-C monomer, one in the C-terminal fibrinogen-like domain and the other in fibronectin type III repeats 3-5. Here we show that a single region in each subunit is necessary and sufficient for heparin binding by whole tenascin-C at physiological ionic strength. First, native tenascin-C was bound to heparin-agarose and digested with Pronase. A 29-kDa fragment retained on the heparin column was recognized by a monoclonal antibody against the fibrinogen-like domain. In contrast, small fragments labeled by an antibody against fibronectin type III repeats 2-5 were released. Second, mild tryptic digestion of tenascin-C yielded two related fragments of 180 and 170 kDa. The latter missed part of the fibrinogen domain and had lost affinity for heparin, in contrast to the former. Finally, chick tenascin-C constructs were recombinantly expressed in human cells. Whereas the complete protein and a mutant lacking fibronectin type III repeats 1-5 bound to heparin-agarose, recombinant tenascin-C missing the C-terminal fibrinogen-like globe did not. Thus, whole chick tenascin-C contains one essential heparin binding region per subunit, located in the fibrinogen-like domain within 10 kDa from the C terminus.

Footnotes

↵^¶ Supported by grants from the Swiss National Fund (to M. C.).
↵* 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:

EGF

epidermal growth factor

ELISA

enzyme-linked immunosorbent assay

FB

fibrinogen-like domain

FNIII

fibronectin tye III repeat

mAb

monoclonal antibody

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

TN

tenascin.
- Received October 18, 1994.
- Revision received November 28, 1994.