Interactions of fibrillin-1 with heparin/heparan sulfate, implications formicrofibrillar assembly

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibri ls. Here we identify, characterize, and localize heparin/heparan sulfate-bi nding sites in fibrillin-1 and report on the role of such glycosaminoglycan s in the assembly of fibrillin-1. By using different binding assays, we loc alize two calcium-independent heparin-binding sites to the N-terminal (Arg( 45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Th r(1486)) region of fibrillin-1. Heparin binding to these sites can be inhib ited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demons trating that the heparin binding regions represent binding domains for hepa ran sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture me dium. Inhibition of the attachment of glycosaminoglyeans to core proteins o f proteoglycans by beta -D-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to protcoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.