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Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.

Publication ,  Journal Article
Ohashi, T; Erickson, HP
Published in: J Biol Chem
November 11, 2011

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.

Duke Scholars

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

November 11, 2011

Volume

286

Issue

45

Start / End Page

39188 / 39199

Location

United States

Related Subject Headings

  • Protein Structure, Tertiary
  • Protein Stability
  • Protein Folding
  • Mutation
  • Humans
  • HEK293 Cells
  • Gelatin
  • Fibronectins
  • Disulfides
  • Biochemistry & Molecular Biology
 

Citation

APA
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Ohashi, T., & Erickson, H. P. (2011). Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain. J Biol Chem, 286(45), 39188–39199. https://doi.org/10.1074/jbc.M111.262337
Ohashi, Tomoo, and Harold P. Erickson. “Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.J Biol Chem 286, no. 45 (November 11, 2011): 39188–99. https://doi.org/10.1074/jbc.M111.262337.
Ohashi T, Erickson HP. Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain. J Biol Chem. 2011 Nov 11;286(45):39188–99.
Ohashi, Tomoo, and Harold P. Erickson. “Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.J Biol Chem, vol. 286, no. 45, Nov. 2011, pp. 39188–99. Pubmed, doi:10.1074/jbc.M111.262337.
Ohashi T, Erickson HP. Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain. J Biol Chem. 2011 Nov 11;286(45):39188–39199.

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

November 11, 2011

Volume

286

Issue

45

Start / End Page

39188 / 39199

Location

United States

Related Subject Headings

  • Protein Structure, Tertiary
  • Protein Stability
  • Protein Folding
  • Mutation
  • Humans
  • HEK293 Cells
  • Gelatin
  • Fibronectins
  • Disulfides
  • Biochemistry & Molecular Biology