Heparan Sulfate Regulates Fibrillin-1 N- and C-terminal Interactions

Andrew K Baldwin,Yashithra Mahalingam,John R Couchman,C Adrian Shuttleworth,Cay M Kielty,Thomas A Jowitt,Bertrand Raynal,Stuart A Cain

doi:10.1074/jbc.m803373200

Abstract

Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized within the domain encoded by exon 7 after the first hybrid domain. Rodent embryonic fibroblasts adhered to PF1 and deletion fragments, and, when cells were plated on fibrillin-1 or fibronectin Arg-Gly-Asp cell-binding fragments, cells showed heparin-dependent spreading and focal contact formation in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions.

Highlights

It is increasingly clear that fibrillin-1 is a major extracellular heparin/heparan sulfate-binding molecule (6 – 8)
We have identified a novel high affinity C-terminal heparin binding site that competes with tropoelastin
rat embryonic fibroblasts (REFs) [29], wildtype and syndecan-4 null mouse embryonic fibroblasts (MEFs), and CHOK1 and CHO761 cells, all plated on the fibrillin-1 ArgGlyAsp-containing (RGD) fragment PF8 or on fibronectin (FN) or the 110-kDa integrinbinding domain of fibronectin (110FN), were analyzed for focal plaques and actin filaments as reported [9, 29], in the presence or absence of PF1, PF13, or hepII

Summary

EXPERIMENTAL PROCEDURES

Recombinant Fibrillin-1 and MAGP-1—The cloning, expression, and purification of recombinant fibrillin-1 fragments PF1, PF2, PF4, PF8, PF12, and PF13 (Fig. 1), using the mammalian expression vector pCEP-pu/AC7 and 293-EBNA cells, has been described [6, 11, 12]. Fragments—For kinetic binding studies of dp heparin sac- the equilibrium constant, equilibrium response was plotted charides with fibrillin-1 by surface plasmon resonance, a Bia- against concentration, in the same method as described above core biosensor was used (Biacore 3000; GE Healthcare). Any data for all of the protein fragments very well, apart from PF1 nonspecific fibrillin-1 binding was detected by blocking wells and PF4, with low ␹2 values. REFs [29], wildtype and syndecan-4 null MEFs, and CHOK1 and CHO761 cells, all plated on the fibrillin-1 ArgGlyAsp-containing (RGD) fragment PF8 or on fibronectin (FN) or the 110-kDa integrinbinding domain of fibronectin (110FN), were analyzed for focal plaques and actin filaments as reported [9, 29], in the presence or absence of PF1, PF13, or hepII.

RESULTS

KD nM

Measured mass

DISCUSSION

ADDITIONS AND CORRECTIONS