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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.

Ventura E, Sassi F, Parodi A, Balza E, Borsi L, Castellani P, Carnemolla B, Zardi L - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Recombinant Therapeutic Proteins, CBA - Advanced Biotechnology Centre, Genoa, Italy.

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.

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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.
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pone-0009145-g001: 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.

Mentions: Fibronectins (FNs) are high-molecular-mass adhesive glycoproteins (for reviews see [1]–[3]) present in the extracellular matrix (ECM) and body fluids, constituted by a dimer composed of two subunits of about 250 kDa linked at the C-termini by two disulfide bonds. Each monomer consists of three types of repeating units: 12 FN repeats of type I (about 40 amino acids each), two type II (about 60 amino acids) and 15–17 type III (about 90 amino acids) (see Fig. 1A). Fibronectin mediates a wide variety of cellular interactions and is involved in a number of processes, such as cell adhesion, the establishment and maintenance of normal cell morphology, cell migration, growth and differentiation. It interacts with several other ECM and cell surface proteins, including collagen, heparin, fibrin, and cell membrane receptors. Finally, FN can be a ligand for numerous integrins, including the classic FN receptor alpha5-beta1 on the RGD sequence of repeat III-10 [4]. Fibronectins are the product of a single gene localized on chromosome 2 [5], but different isoforms arise from the alternative splicing of the pre-mRNA, a process that for some ECM proteins is modulated by cytokines and extracellular/intracellular pH [6], [7] in three sites: the type III connecting sequence (IIICS), a complete type III repeat, extra domain A (ED-A), and a complete type III repeat, extra domain B (ED-B) (Fig. 1A).


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.

Ventura E, Sassi F, Parodi A, Balza E, Borsi L, Castellani P, Carnemolla B, Zardi L - PLoS ONE (2010)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818841&req=5

pone-0009145-g001: 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.
Mentions: Fibronectins (FNs) are high-molecular-mass adhesive glycoproteins (for reviews see [1]–[3]) present in the extracellular matrix (ECM) and body fluids, constituted by a dimer composed of two subunits of about 250 kDa linked at the C-termini by two disulfide bonds. Each monomer consists of three types of repeating units: 12 FN repeats of type I (about 40 amino acids each), two type II (about 60 amino acids) and 15–17 type III (about 90 amino acids) (see Fig. 1A). Fibronectin mediates a wide variety of cellular interactions and is involved in a number of processes, such as cell adhesion, the establishment and maintenance of normal cell morphology, cell migration, growth and differentiation. It interacts with several other ECM and cell surface proteins, including collagen, heparin, fibrin, and cell membrane receptors. Finally, FN can be a ligand for numerous integrins, including the classic FN receptor alpha5-beta1 on the RGD sequence of repeat III-10 [4]. Fibronectins are the product of a single gene localized on chromosome 2 [5], but different isoforms arise from the alternative splicing of the pre-mRNA, a process that for some ECM proteins is modulated by cytokines and extracellular/intracellular pH [6], [7] in three sites: the type III connecting sequence (IIICS), a complete type III repeat, extra domain A (ED-A), and a complete type III repeat, extra domain B (ED-B) (Fig. 1A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Recombinant Therapeutic Proteins, CBA - Advanced Biotechnology Centre, Genoa, Italy.

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.

Show MeSH
Related in: MedlinePlus