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

作者：Ohashi Tomoo^*; Lemmon Christopher A; Erickson Harold P^*

来源：Biochemistry, 2017, 56(34): 4584-4591.

DOI：10.1021/acs.biochem.7b00589

To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FN Delta(I)1-5, FN Delta(III)1-3, FN Delta(III)4-8, and FN Delta(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 FM Delta(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, FN Delta(I)1-5, FN Delta(III)1-3, and FNtnA D were unable to form a matrix. Interestingly, FN Delta(III)1-3 and FNtnA D were capable of co-assembly with full-length FN, while FN Delta(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.

• 出版日期2017-8-29