NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis

Background: The process of angiogenesis (i.e. the formation of new blood ve ssels from pre-existing ones) is fundamental to physiological processes suc h as reproduction, development and repair, as well;Is to pathological condi tions such as tumor progression, rheumathoid arthritis and ocular disorders . The oncofoetal ED-B domain, a specific marker of angiogenesis, consists o f 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. Results: The NMR structure of the ED-B domain is reported and reveals impor tant differences from other FN type III domains. A comparison of the ED-B d omain with the crystal structure of a four-domain FN fragment shows the nov el features of ED-B to be located in loop regions that are buried at interd omain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potenti al specific recognition site. Epitope mapping with 82 decapeptides that spa n the ED-B sequence revealed that three ED-B-specific monoclonal antibodies , which selectively target newly forming blood vessels in tumor-bearing mic e, bind to adjacent regions on the ED-B surface. Conclusions: The NMR structure enables the identification of a large surfac e area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED -B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the form ation of new blood vessels.