Domain unfolding plays a role in superfibronectin formation

J Biol Chem. 2005 Nov 25;280(47):39143-51. doi: 10.1074/jbc.M509082200. Epub 2005 Sep 29.

Abstract

Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of approximately 4:1, anastellin:FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Binding Sites
  • Cattle
  • Fibronectins / biosynthesis*
  • Fibronectins / chemistry*
  • Fibronectins / genetics
  • In Vitro Techniques
  • Kinetics
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Multiprotein Complexes
  • Mutagenesis, Site-Directed
  • Peptide Fragments / biosynthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Transfection

Substances

  • Bacterial Proteins
  • Fibronectins
  • Luminescent Proteins
  • Multiprotein Complexes
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • anastellin
  • yellow fluorescent protein, Bacteria