Alternative splicing of the angiogenesis associated extra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8

Abstract

Background: Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities.

Methodology: We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6.

Conclusion: We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.

Figure 1. Reactivity of the mAb C6 with different FN fragments.

A. Model of the domain structure of human FN subunit; the three different types of repeats and the specificity of the mAb C6 for the type III fibronectin repeat 8 are shown; B. Comparison of the amino acid sequence of human (h8) and mouse (m8) FNIII 8 domains; the differences in mouse FNIII 8 compared to human FNIII 8 are written in white letters on a black background. We generated four chimerical mutants of the human recombinant fragment FNIII B-8 (mut-1, mut-2, mut-3 and mut-4), substituting various amino acids of the human FNIII 8 with those of the mouse FNIII 8 sequence. The mouse residues introduced in the human sequence are in white letters on a black background. The loop B-C is enclosed in a box. The reactivity of each recombinant fragment with the mAb C6 is shown on the right. C. Reactivity in ELISA of various concentrations of the mAb C6 with human and chimeric FNIII B-8 recombinant fragments: C6 showed reactivity with human FNIII B-8, mut-1 and mut-3, but not with mut-2 and mut-4.

Figure 2. Westernblot of FN fragments using mAb C6 and structural comparison of interfaces FNIII 7/FNIII 8 and ED-B/FNIII 8.

A. Coomassie blue staining of the human FN wild-type and mutated recombinant fragments FNIII 7-B, FNIII 7-B-8-9, FNIII 7-8-9, FNIII 7-8, mut-2, mut-1, FNIII B-8, mut-3 and mut-4 (left panel); reactivity in immunoblotting of the mAb C6 with the generated FN recombinant fragments: C6 reacted with FNIII 7-B-8-9, FNIII B-8, mut-1 and mut-3, but was negative with FNIII 7-B, FNIII 7-8-9, FNIII 7-8, mut-2 and mut-4 (right panel). B. Structural comparison of the interface between the ED-B/FNIII 8 and between the FNIII 7/FNIII 8 domains by superimposition of structures of the ED-B with FNIII 7. The FNIII 7 and ED-B loops A-B and the FNIII 8 loop B-C are indicated (adapted with permission from Fattorusso, R., et al., license number 2247580530397, dated Aug 14, 2009).