Biochemistry
Biochemistry
5 years ago

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

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants
Tomoo Ohashi, Christopher A. Lemmon, Harold P. Erickson
To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔI1–5, FNΔIII1–3, FNΔIII4–8, and FNΔIII11–14. 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 FNΔIII11–14 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, FNΔI1–5, FNΔIII1–3, and FNtnA–D were unable to form a matrix. Interestingly, FNΔIII1–3 and FNtnA–D were capable of co-assembly with full-length FN, while FNΔI1–5 was not. This indicates that the segment I1–5 is crucial for matrix assembly and segment III1–3 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.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00589

DOI: 10.1021/acs.biochem.7b00589

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