A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

The mechanism of fibronectin (FN) assembly and the selfassociation 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 Fibronectin (FN)2 is a secreted protein expressed in the liver and circulated in blood as a soluble dimer (1). In addition to this plasma FN, some cell types, such as fibroblasts and endothelial cells, secrete FN and assemble it into extracellular matrix fibrils. FN is a modular protein containing 12 FN type I (FNI) domains, two FN type II domains, and 15-17 FN type III (FNIII) domains (2, 3). Two cysteines at the C terminus asymmetrically form interchain disulfide bonds (4, 5). Dimeric FN molecules interact with each other on the cell surface to form matrix fibrils. This process requires cell surface integrins (6 -9). Live cell imaging showed that integrin translocation by the actin cytoskeleton is crucial for the early stages of FN matrix assembly (10, 11). More recently, it has been reported that cell-to-cell adhesion via cadherin controls tissue tension and affects FN matrix formation during embryogenesis (12).The FN matrix had long been thought to be formed by disulfide-bonded FN multimers because the protein migrated at the top of an SDS gel under nonreducing conditions (13-16). However, studies by Chen and Mosher (17) A recent NMR study showed a unique structure for the domain pair III 1-2 (32). It had been recognized from sequence analysis that there is an 18-amino acid linker between these domains. The NMR structure showed that the A strand of III 2 was disordered, giving a total length of ϳ35 amino acids for the linker, and further indicated that III 1 and III 2 formed a closed compact structure, with a potential salt bridge between Lys-669 in III 1 and Asp-767 in III 2. These two amino acids were mutated to alanine, giving the mutant designated KADA. The native III 1-2 was found not to bind I 1-5, but the KADA mutation was reported to dramatically enhance binding of I 1-5, as measured by surface plasmon resonance.

Section: Discussionsupporting

confidence: 92%

Section: Fibronectin (Fn)mentioning

confidence: 87%

See 1 more Smart Citation

Fibronectin Aggregation and Assembly

Ohashi

Erickson

2011

“…One possible explanation is that domain III-1 is opened in fibrils, but the cysteine becomes buried in an FN-FN bond. Previous work has implicated III-1 in FN fibril assembly (27)(28)(29). Conversely, domains III-6 and III-12 showed no labeling in solution but showed significant labeling in stretched fibrils, suggesting that these domains are unfolded specifically in matrix fibrils.…”

Section: Table 2 Relative Rates Of Fn-iii Domain Openingmentioning

confidence: 84%

Probing the Folded State of Fibronectin Type III Domains in Stretched Fibrils by Measuring Buried Cysteine Accessibility

Lemmon

Ohashi

Erickson

2011

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils. Fibronectin (FN)2 is a dimer of a 250-kDa protein that forms insoluble fibrils that are a critical component of the extracellular matrix. It is made up of a series of three domain types connected end-to-end (see Fig. 1A) (1). The N terminus of FN consists of nine FN-I and two FN-II domains, each of which contains an internal disulfide bond. The C terminus of FN consists of three FN-I domains and contains intermolecular disulfides that link the two FN monomers to form the dimer. The central portion of the FN molecule is made up of 15 tandem FN-III domains; these domains each consist of seven ␤-strands folded into a ␤-sandwich structure and contain no internal disulfides (see Fig. 1B). FN in solution adopts a compact conformation in which the second to fourth FN-III domains (III-2-4) of one subunit interact with the 12 th to 14 th FN-III domains (III-12-14) of the other, causing the FN dimer to fold onto itself (see Fig. 1A) (2).FN interacts with both cells and other extracellular matrix proteins. The N-terminal domains contain binding sites for the extracellular matrix proteins collagen and fibrin, whereas the ninth and tenth FN-III domains bind to integrins at the cell surface (3-6). Upon attachment to cells, FN is stretched and assembled into fibrils by cell contraction: matrix assembly is inhibited if cell contractility is inhibited (7) or if tension within the matrix is released (8). Although the exact mechanism of FN fibril formation is still unknown, studies have shown that FN fibrils are stretched to up to 4 times their resting length by cells and are highly elastic structures (9 -12). This elasticity requires cell contraction; disruption of cytoskeletal contraction relaxes stretched fibrils (10).Two different mechanisms have been proposed to explain the elasticity of FN matrix fibrils (see Fig. 1A) (13). One theory contends that the elasticity is a function of FN-III domains unfolding under tension (see Fig. 1A, mechanism 2) (14, 15). These domains contain no internal disulfides and can be mechanically unfolded by atomic force microscopy (16, 17). It is unclear how relevant these atomic force microscopy pulling force...

“…This suggests either that the critical event for LTBP1 assembly is the formation of a fibrillar fibronectin network and/or that multiple interacting sites on the fibronectin molecule are required for LTBP1 incorporation and that the individual fragments tested were lacking one or more of these sites. Future studies will therefore require the use of fibronectin constructs that contain all the necessary domains for assembly into fibrils but contain deletions in other internal domains (36). The ability of the 70-kDa fibronectin fragment to inhibit incorporation of LTBP1 in primary osteoblasts favors the hypothesis that fibronectin fibril assembly is the critical event, since this fragment works by blocking fibronectin selfinteracting domains but does not inhibit binding of fibronectin to cell surface integrins.…”

Section: Discussionmentioning

confidence: 99%

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Dallas

Sivakumar

Jones

et al. 2005

278

233

Latent transforming growth factor-␤-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF␤ in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-␤ (TGF␤) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGF␤ into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates ␣5␤1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGF␤ via LTBP1 interactions.