Fibrillin-1 Interactions with Heparin: IMPLICATIONS FOR MICROFIBRIL AND ELASTIC FIBER ASSEMBLY

Stuart A Cain,Clair Baldock,John Gallagher,Amanda Morgan,Daniel V Bax,Anthony S Weiss,C Adrian Shuttleworth,Cay M Kielty

doi:10.1074/jbc.m501390200

Abstract

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.

Highlights

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans
We have investigated the nature of the specific fibrillin-1 interactions with heparin in order to determine how these glycosaminoglycans influence microfibril and elastic fiber assembly, and to obtain insights into the molecular basis of physiological heparan sulfate interactions with fibrillin-1
We have shown that there are four distinct high affinity heparin-binding sites on fibrillin-1, and we have localized three of these sites to the N terminus, the “neonatal Marfan” region, and to a site downstream from the Arg-Gly-Asp (RGD) cell attachment motif

Summary

Introduction

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly. Heparin and heparan sulfate interacted in a calcium-independent manner to a large fibrillin-1 N-terminal fragment (Arg45– Thr450) and to the C-terminal half of fibrillin-1 (Asp1528– Arg2731), and in a calcium-dependent manner to a large central (Asp1028–Thr1486) fragment of fibrillin-1 (9) These heparan sulfate-binding sites had a selective specificity for sulfated L-iduronate-rich heparan sulfate. Heparan sulfate chains are synthesized by enzymes that initially polymerize a chain of repeating GlcA and GlcNAc units, which are variously modified by N- and O-sulfation and uronate epimerization (17, 18) These modifications occur in clusters, creating sulfation domains (Sdomains) that form the principal recognition site for heparan sulfate-binding proteins (17, 18). Interactions between proteins and S-domains can be dependent on a critical sequence of sulfate-modified main; cbEGF, calcium-binding EGF; HS, heparan sulfate; PF, protein fragment; BSA, bovine serum albumin; RU, response units; MES, 4-morpholineethanesulfonic acid; dp, degree of polymerization

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