OC 29.5 - Interaction of fibrin with its novel receptor N-cadherin: identification of amino acid residues critical for this interaction and clarification of its functional role

Background: Our recent study identified N-cadherin as a novel endothelial cell receptor for fibrin and localized the complementary binding sites within the fibrin βN-domains and the 3rd and 5th extracellular (EC3 and EC5) domains of N-cadherin (Yakovlev et al., Biochemistry, 2024, 63:202-211). We have also hypothesized that the (Asp/Glu)-X-(Asp/Glu) motif, which contains two closely spaced negatively charged residues and is present in both fibrin-binding EC3 and EC5 domains of N-cadherin, may be involved in this interaction.

Aims: The major aims of the present study were to test this hypothesis and to clarify the functional role of the fibrin-(N-cadherin) interaction.

Methods: Site-directed mutagenesis, fluorescence spectroscopy, ELISA, SPR.

Results: To test our hypothesis, we replaced Asp/Glu residues in the (Asp/Glu)-X-(Asp/Glu) motif with Ala in the recombinant N-cad(3-5) fragment, which contains both fibrin-binding EC3 and EC5 domains, using site-directed mutagenesis. The mutant fragment, N-cad(3-5)-mut, was expressed in E. coli, purified, and refolded. Fluorescence spectroscopy confirmed that this mutant fragment, as well as wild-type N-cad(3-5), were folded into a compact structure and have a similar conformation. In contrast, ELISA experiments revealed that while wild-type N-cad(3-5) exhibited significant binding to the recombinant fibrin βN-domains, as expected, the N-cad(3-5)-mut fragment exhibited practically no binding. This finding was confirmed by SPR. These results clearly indicate that the (Asp/Glu)-X-(Asp/Glu) motif of N-cadherin plays a critical role in fibrin binding, confirming our hypothesis. Our preliminary experiments with human macrovascular endothelial cells devoid of N-cadherin revealed that fibrin-induced inflammation and angiogenesis are affected by the loss of N-cadherin.

Conclusion(s): We have identified amino acid residues in the EC3 and EC5 domains of N-cadherin critical for the interaction of fibrin with N-cadherin. Our experiments also suggest the involvement of this interaction in fibrin-dependent inflammation and angiogenesis. Further experiments are required to confirm this suggestion.