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

To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔ I 1–5, FNΔ III 1–3, FNΔ III 4–8, and FNΔ III 11–14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA–D was generated by swapping FNIII domains 1–8 in FNΔ III 11–14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA–D. When we tested these mutants in cell culture, FNΔ I 1–5, FNΔ III 1–3, and FNtnA–D were unable to form a matrix. Interestingly, FNΔ III 1–3 and FNtnA–D were capable of co-assembly with full-length FN, while FNΔ I 1–5 was not. This indicates that the segment I 1–5 is crucial for matrix assembly and segment III 1–3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.