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

dc.contributor.author Ohashi, Tomoo
dc.contributor.author Erickson, Harold P
dc.date.accessioned 2018-04-01T15:12:10Z
dc.date.available 2018-04-01T15:12:10Z
dc.date.issued 2011-11
dc.identifier.issn 0021-9258
dc.identifier.issn 1083-351X
dc.identifier.uri https://hdl.handle.net/10161/16466
dc.description.abstract 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.
dc.language eng
dc.publisher Elsevier BV
dc.relation.ispartof The Journal of biological chemistry
dc.relation.isversionof 10.1074/jbc.m111.262337
dc.subject Humans
dc.subject Disulfides
dc.subject Fibronectins
dc.subject Gelatin
dc.subject Protein Structure, Tertiary
dc.subject Protein Folding
dc.subject Mutation
dc.subject Protein Stability
dc.subject HEK293 Cells
dc.title Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.
dc.type Journal article
duke.contributor.id Ohashi, Tomoo|0103382
duke.contributor.id Erickson, Harold P|0114747
dc.date.updated 2018-04-01T15:12:09Z
pubs.issue 45
pubs.organisational-group School of Medicine
pubs.organisational-group Duke
pubs.organisational-group Duke Cancer Institute
pubs.organisational-group Institutes and Centers
pubs.organisational-group Biochemistry
pubs.organisational-group Basic Science Departments
pubs.organisational-group Cell Biology
pubs.publication-status Published
pubs.volume 286


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