Imaging and mapping heparin-binding sites on single fibronectin molecules with atomic force microscopy

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fibronectin is a crucial extracellular matrix protein with multiple functional domains.
• Two primary heparin-binding sites, Hep I and Hep II, have been identified in fibronectin.
• Previous research suggested Hep II is the high-affinity heparin-binding site, primarily using fibronectin fragments.

Purpose of the Study:

• To investigate heparin binding to intact dimeric plasma fibronectin molecules.
• To compare the binding affinities of the Hep I and Hep II sites in intact fibronectin.
• To re-evaluate the role of Hep I and Hep II sites in heparin binding.

Main Methods:

• Utilized tapping mode atomic force microscopy (AFM) to image single fibronectin molecules.
• Visualized heparin-coated gold particles bound to intact fibronectin.
• Performed quantitative analysis of heparin-gold particle binding to specific fibronectin domains.

Main Results:

• Heparin-gold particles were observed to bind preferentially to both Hep I and Hep II sites on intact fibronectin.
• Quantitative analysis indicated nearly twice as many heparin-gold particles bound to the N-terminal Hep I site compared to the Hep II site.
• These findings contrast with previous studies using fibronectin fragments.

Conclusions:

• The Hep I site exhibits a higher or comparable binding affinity to heparin than the Hep II site in intact fibronectin molecules.
• This challenges the long-held view that Hep II is the sole high-affinity heparin-binding site.
• The study highlights the importance of examining heparin binding in the context of intact fibronectin structure.