Domain unfolding plays a role in superfibronectin formation

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Aggregation

Background:

• Fibronectin (FN) aggregation into superfibronectin (sFN) is crucial for extracellular matrix assembly.
• The precise mechanism of sFN formation mediated by anastellin has remained unclear.

Purpose of the Study:

• To elucidate the molecular mechanism underlying superfibronectin (sFN) formation.
• To identify the binding sites and conformational changes induced by anastellin during FN aggregation.

Main Methods:

• Co-precipitation assays to determine anastellin:FN monomer ratios.
• Protease sensitivity assays (thermolysin digestion) to map binding sites and conformational changes.
• Site-directed mutagenesis (engineered disulfide bond) to assess the role of domain stability.

Main Results:

• Anastellin co-precipitated with FN at an approximate 4:1 ratio (anastellin:FN monomer).
• Anastellin primarily binds to the FN type III domains (III)1-3, inducing a conformational change that exposes a thermolysin-sensitive site.
• Binding of anastellin to (III)11 enhances thermolysin digestion, and stabilizing domain (III)3 inhibits aggregation.

Conclusions:

• A three-step model for sFN formation is proposed: spontaneous FN-III domain unfolding, anastellin binding to unfolded domains, and subsequent aggregation via exposed hydrophobic surfaces and beta-sheet edges.
• Domain stability, particularly of (III)3, is critical for sFN formation.
• The proposed mechanism provides insights into native FN matrix assembly.