Biochemical analysis of the interaction of fibronectin with IgG and localization of the respective binding sites

Rostagno, Agueda; Williams, Michael J.; Frangione, Blas; Gold, Leslie I.

doi:10.1016/0161-5890(95)00161-1

Cited by 12 publications

(18 citation statements)

References 48 publications

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“…The estimated K d values for fibrin binding that we have obtained for the N-and C-terminal sites by ELISA contrast with the relative affinities suggested by their binding characteristics to fibrin-Sepharose. Similar discordant results were reported previously for the interaction of Fn and its proteolytic fragments with both fibrin and IgG linked to affinity matrices compared with ELISAs [23,42]. Whereas affinity matrices have proven useful for the isolation and characterization of sites on proteins that interact with specific ligands [1][2][3][15][16][17][18][19][20][21][22][23][24]35,[42][43][44], these matrices may be limited in their usefulness for predicting relative binding affinities\characteristics.…”

Section: Discussionsupporting

confidence: 91%

“…Similar discordant results were reported previously for the interaction of Fn and its proteolytic fragments with both fibrin and IgG linked to affinity matrices compared with ELISAs [23,42]. Whereas affinity matrices have proven useful for the isolation and characterization of sites on proteins that interact with specific ligands [1][2][3][15][16][17][18][19][20][21][22][23][24]35,[42][43][44], these matrices may be limited in their usefulness for predicting relative binding affinities\characteristics. Although the results obtained by affinity chromatography appeared not to vary among individual experiments, the high concentration of protein and random orientation of the protein chemically coupled to the matrix may contribute to the observed artifactual binding.…”

Section: Discussionsupporting

confidence: 91%

“…The 14.4 kDa FBP (100 µg in FBB) was radiolabelled with 2.0 mCi of "#&I-Na in the presence of Iodobeads (Pierce Chemical Co., Rockford, IL, U.S.A.) as described previously [42] (specific activity obtained, 5.0 µCi\µg; 90% trichloroacetic acid precipitable counts).…”

Section: Solid-phase Fibrin-binding Assaysmentioning

confidence: 99%

“…Proteolytic digestion of Fn generates fragments that retain biological activity and can subsequently be isolated by specific ligand affinity chromatography [1][2][3][15][16][17][18][19][20][21][22][23][24]35,37,[42][43][44]. Fn was digested with subtilisin, subjected to gelatin-affinity chromatography to remove fragments containing the gelatin-binding domain, and the unbound effluent fraction from gelatin-Sepharose [43] was incubated with fibrin-Sepharose at 4 mC [23].…”

Section: Isolation Of a Fibrin-binding Fragment From The C-terminus Omentioning

confidence: 99%

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Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin

Rostagno

Schwarzbauer

Gold

1999

Biochemical Journal

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Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1–COOH), expressed in COS-1 cells, and 10F1–12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 91%

Section: Solid-phase Fibrin-binding Assaysmentioning

confidence: 99%

Section: Isolation Of a Fibrin-binding Fragment From The C-terminus Omentioning

confidence: 99%

See 2 more Smart Citations

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin

Rostagno

Schwarzbauer

Gold

1999

Biochemical Journal

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show abstract

“…The VN/FN-containing IC found in our study and the cryoglobulin IC have in common that they contain both immunoglobulins as well as FN. In the opposite of FN, which IgG-binding properties and presence in IC are well-known [24,25], to our knowledge VN has not been described yet to be a constitutive part of cryoglobulins or of IC in general.…”

Section: Discussionmentioning

confidence: 97%

Vitronectin- and Fibronectin-containing Immune Complexes in Primary Systemic Vasculitis

Maehnss

Kobarg

Schmitt

et al. 2002

Journal of Autoimmunity

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Interactions Outside the Antigen-Combining Site

Nezlin

1998

The Immunoglobulins

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Biochemical analysis of the interaction of fibronectin with IgG and localization of the respective binding sites

Cited by 12 publications

References 48 publications

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin

Vitronectin- and Fibronectin-containing Immune Complexes in Primary Systemic Vasculitis

Interactions Outside the Antigen-Combining Site

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