The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.
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 approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 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.
Whitelock
J M
JM
Commonwealth Scientific and Industrial Research Organization, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, Sydney, New South Wales 2114, Australia.
Murdoch
A D
AD
Iozzo
R V
RV
Underwood
P A
PA
D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism*; D014945:Wound Healing; D000099175:Heparanase
D017364:Collagenases; D005341:Fibrinolysin; D016222:Fibroblast Growth Factor 2; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D046911:Macromolecular Substances; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases; D011509:Proteoglycans; D000099175:Heparanase; C071927:perlecan