The Degradation of Human Endothelial Cell-derived Perlecan and Release of Bound Basic Fibroblast Growth Factor by Stromelysin, Collagenase, Plasmin, and Heparanases (*)
- From the (1)Commonwealth Scientific and Industrial Research Organization, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, Sydney, New South Wales 2114, Australia and the
- (2)Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
- ¶ To whom correspondence and reprint requests should be addressed: Commonwealth Scientific and Industrial Research Organization, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, Sydney, NSW 2114, Australia. Tel.: 612 886 4847; Fax: 612 886 4805.
Abstract
Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability
to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing,
and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding
site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was
removed from perlecan by heparitinase treatment, and the 
450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of 
300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed
immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate
chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin,
rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate
specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings
provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that
perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of
proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of
the growth factor.
Footnotes
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↵§ Participants in the Federally funded Co-operative Research Centre (CRC) for Cardiac Technology.
-
↵** Recipient of a Faculty Research Award from the American Cancer Society.
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↵* This work was supported in part by National Institutes of Health Grants RO1 CA-39481 and RO1 CA-47282 (to R. V. I.) and a grant from the Australian government under the Co-operative Research Centre scheme. 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.
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↵1 The abbreviations used are:
- HSPG
-
heparan sulfate proteoglycan
- ECM
-
extracellular matrix
- HS
-
heparan sulfate
- PAGE
-
polyacrylamide gel electrophoresis
- HUAEC
-
human umbilical arterial endothelial cell
- FGF
-
fibroblast growth factor
- bFGF
-
basic FGF
- GlcNSO
-
N-sulfated glucosamine
- 2-OSO
-
2-sulfate
- 6-OSO
-
6-sulfate
- ELISA
-
enzyme-linked immunosorbant assay
- MMP
-
matrix metalloproteinase
- ABTS
-
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid
- mAb
-
monoclonal antibody
- TBS
-
Tris-buffered saline
- RAM
-
rabbit anti-mouse IgG
- TIMP-1
-
tissue inhibitor of metalloproteinases.
-
- Received July 27, 1995.
- Revision received February 7, 1996.
