Domain Unfolding Plays a Role in Superfibronectin Formation

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 ϳ4:1, 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 ␤-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. Fibronectin (FN)2 is an extracellular matrix protein and is also present in a soluble form in blood and tissue fluid (1). Soluble, dimeric FN molecules assemble into an insoluble supramolecular structure known as the FN matrix, which appears during embryonic development, wound healing, and also in cell culture (1). The biological importance of FN during development and wound healing has been demonstrated by generating conventional and conditional FN knock-out mice and alternative splicing domain (EDA) knock-out and knock-in mice (2-4).FN matrix fibrils may be one of the least understood macromolecular protein assemblies. Whereas a large number of protein structures can be self-assembled from purified subunits (e.g. microtubules, actin filaments, collagen fibrils, and viruses capsids), FN fibrils have only been produced by living cells in culture. The cells in tissue culture can assemble FN matrix fibrils either from FN that they synthesize or from soluble FN added exogenously. Fibril assembly takes place on the cell surface and requires integrins (5-9).A fundamental deficit is our lack of knowledge of the structure of FN fibrils. FN molecules must be attached to each other to form the fibrils, but we do not know even the sites of contact between molecules nor the types of bonds that hold them together. Several attempts at in vitro assembly of FN have resulted in aggregates that may be related to FN matrix fibrils. For instance, aggregates could be formed when FN was partially denatured in guanidine HCl and incubated over time (10, 11). However, these aggregates seemed to be mediated by disulfide bonding of two f...

Fibronectin Aggregation and Assembly

“…Anastellin binds FN to form a large aggregate, called superFN, which has FN matrix-like fibrillar structures when observed by light microscopy. Our previous studies indicated that the opening or unfolding of FNIII domains is important for anastellin binding and aggregation (24,37). We also found that the N-terminal FN fragment, I 1-9, which prevented FN matrix formation in cell culture, did not inhibit superFN aggregation.…”

“…III [7][8][9][10] and the variable domain seem to play roles as integrin binding sites. III 4 -5, III 12-14, and I 10 -12 have contradictory reports (24,26,30), so they may only be required for particular cell types or under some conditions. On the other hand, many studies consistently show that I 1-9 is crucial for FN matrix assembly.…”

“…For the past three decades, FN matrix assembly has been studied with FN fragments, antibodies, and deletion mutants. These studies indicate that several domains are crucial for FN matrix formation: I 1-9 (19 -24), III 1 and/or III 2 (20,25,26), , 23,24), III 12-14 (28), the variable domain (8), I 10 -12 (29), and the interchain disulfide bonds at the C terminus involved in dimerization (20). III [7][8][9][10] and the variable domain seem to play roles as integrin binding sites.…”

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

“…We suggest that these domains are actually folded, as indicated by tryptophan fluorescence, but are transiently unfolding and refolding. We previously suggested that domains III-1, III-2, III-3, and III-11 transiently open and bind anastellin, a fragment of domain III-1, when forming the in vitro induced FN matrix known as superfibronectin (18,19). We suggest that the FN-III domains (n ϭ 3).…”

“…In cell-assembled fibrils, the pulling speed is nearly zero, so it is difficult to extrapolate the cell-applied force that would be needed to unfold these domains. In addition to mechanical unfolding of FN-III domains, transient opening of certain FN-III domains has been demonstrated in the absence of applied force during the assembly of in vitro assembled super-fibronectin (18,19).The second theory to explain fibril elasticity contends that FN molecules are in a compact conformation in relaxed fibrils and are stretched to the extended conformation in elongated fibrils (see Fig. 1A, mechanism 1) (20).…”