Ligation of the Fibrin-binding Domain by β-Strand Addition Is Sufficient for Expansion of Soluble Fibronectin

Lisa M Maurer,Wenjiang Ma,Nathan L Eickstaedt,Ian A Johnson,Bianca R Tomasini-Johansson,Douglas S Annis,Deane F Mosher

doi:10.1074/jbc.m111.294041

Lisa M Maurer, Wenjiang Ma + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111.294041

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Abstract

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by β-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit β-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.

Highlights

Conversion of fibronectin from a compact plasma protein to a fibrillar component of extracellular matrix is not understood
To investigate the possibility that heparin binding causes a coupled conformational change encompassing both 10FNIII and the N-terminal FNI modules, we looked at the effect of heparin on the ability of soluble FN to compete with b-Functional upstream domain” (FUD) or b-high affinity downstream domain” (HADD) for binding to adsorbed FN
To investigate how ligation of the N terminus of FN leads to exposure of 10FNIII for processes including assembly and internalization, we utilized a competitive binding assay to monitor accessibility of the mAbIII-10 epitope in 10FNIII that is cryptic in soluble FN at low ionic strength (6)

Summary

Introduction

Conversion of fibronectin from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. “Functional upstream domain” (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel ␤-strand addition to discontinuous sets of N-terminal FN type I modules, 2–5FNI of the fibrin-binding domain and 8–9FNI of the gelatinbinding domain. Soluble N-terminal constructs truncated after 9FNI or 3FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than 3FNIII limit ␤-strand addition to 1–5FNI within intact soluble FN. Allosteric loss of constraining interactions among 1–5FNI, 10FNIII, and 12–14FNIII likely enables assembly of FN into extracellular fibrils

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