Fibronectin Aggregation and Assembly

Abstract

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.

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants

“…We also found previously that introducing a disulfide bond in III 2, which stabilized folding, significantly reduced the level of the FN matrix. 16 The binding of the N-terminal region, I 1–5, to III 2 also agrees with the N-terminal 30–40 nm overlap between FN molecules in matrix fibrils observed by super-resolution microscopy. 33 …”

“…We previously reported that the folding and stability of FNIII domains were crucial for anastellin-induced FN aggregation. 15,16 We suggest that the GFND mutations destabilize the FNIII domains and these unstable FNIII domains may slowly form nonfibrillar aggregates in the kidney. In the case of the Y973C mutant, it is also possible that unusual disulfide bond formation, even in the case of a heterozygous mutation, enhances nonfibrillar aggregation.…”

“…16 This vector has an engineered signal sequence that gives an additional three amino acids (EGS) at the N terminus of the secreted protein. To generate the various deletion mutants, shortened FN fragments were amplified by polymerase chain reaction and cloned back into the FN-YPet/pHLSec2 vector: FNΔ I 1–5 (deletion; Q 32 AQQ⋯KCER 272 ), FNΔ III 1–3 (deletion; V 614 FIT⋯TTGT 901 ), FNΔ III 4–8 (deletion; P 902 RSD⋯RQKT 1447 ), FNΔ III 11–14 (deletion; P 1636 SQM⋯RKKT 2082 ), and FNmono (deletion; C 2458 PIE⋯DSRE 2477 ).…”

“…21 These constructs were expressed in a transient mammalian cell expression system as reported previously. 16,21,22 Briefly, purified constructs were transfected into HEK293 cells with PEI (polyethylenimine-25 kDa branched, Aldrich). The conditioned medium was collected after transfection for 6–7 days.…”

“…We have previously proposed that FN aggregation and assembly require the unfolding of FNIII domains. 15,16 In this model, high local FN concentrations on the cell surface could enhance the probability of interactions following the spontaneous unfolding and/or opening of FNIII domains.…”