Dimeric Fibronectin Research Articles

Isolation and characterization of cDNA clones for human liver fibronectin

Cellular and plasma fibronectin dimers are constituted by similar but not identical polypeptides. Their differences are the consequence of internal primary sequence variability due to alternative splicing in at least 2 regions (ED and IIICS) of the pre mRNAs [1–8]. A detailed analysis of human liver fibronectin mRNA in these regions was carried out by isolating cDNA clones and determining their nucleotide sequence. A novel type of IIICS segment (coding for 64 amino acids) was present in the two cDNA clones studied and, as expected from previous Sl mapping studies [6], the ED segment was absent in both.

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190–235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 m m dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 m KSCN gradient. Residual dimeric species (10–20%) could be separated from monomers in high yield by gelsieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 m KSCN. Most new SH groups (74–81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatinbinding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Role of fibronectin in human monocyte and macrophage bactericidal activity.

Fibronectin is a high-molecular-weight glycoprotein found as a soluble dimer in plasma and as an insoluble multimer in tissues. It has been proposed that plasma fibronectin facilitates phagocytic removal of lysed cells and damaged tissues. Fibronectin binds avidly to several species of gram-positive bacteria and enhances staphylococcal and streptococcal attachment to cultured cells. Determination of whether fibronectin will enhance the bactericidal activity of monocytes and macrophages has not been reported. The bactericidal activity of freshly isolated monocytes, cultured monocytes, or lymphokine-activated macrophages was tested in the presence of either dimeric or multimeric fibronectin. Freshly isolated monocytes and lymphokine-activated macrophages killed Staphylococcus aureus effectively in the absence of fibronectin or whole serum. In contrast, monocytes cultured for 7 to 10 days had diminished staphylocidal capacity. When the monocytes were cultured with either dimeric or multimeric fibronectin, however, bactericidal capacity was maintained. Thus, although fibronectin did not enhance the bactericidal activity of mononuclear phagocytes, both multimeric and dimeric fibronectin were effective at maintaining the bactericidal capacity.

Effect of fibronectin fragments on macrophage phagocytosis of gelatinized particles.

Rat plasma fibronectin enhances the binding and ingestion of gelatin-coated, formalin-fixed, or tanned sheep erythrocytes by elicited rat peritoneal macrophages. Fibronectin binding to the gelatinized erythrocytes is required for this enhancement, because macrophages preferentially recognize the surface bound molecule. This enhancement of particle uptake by fibronectin required the presence of a renewable, trypsin-sensitive component(s) on the macrophage surface (fibronectin receptor). When subjected to plasminolysis for 3 hr, fibronectin was degraded into gelatin-binding fragments (170 to 210 kd) and a 25-kd nongelatin binding fragment. The 170 to 210 kd gelatin binding fragments retained uptake-enhancing activity but were less active on a weight and molar basis than intact, dimeric fibronectin. The nongelatin binding 25 kd fragment alone did not enhance uptake. These results indicate that the sites for interaction with both the gelatinized erythrocyte surface and macrophages are retained on 170 to 210 kd fragments. However, the fibronectin dimeric structure is required for maximal expression of opsonic activity.

Studies on fibronectin in the skin. VII. Production in cell cultures from normal human skin

In the present study, cell cultures of fibroblasts from normal skin have been investigated regarding the production of fibronectin. The development of multimeric insoluble fibronectin is demonstrated as small dots at the cell surface, developing into a branched meshwork of fibrous structures in parallel arrays. Soluble dimeric fibronectin is also found in the culture medium.

Separation and analysis of the major forms of plasma fibronectin

Human plasma fibronectin exists in circulation in multiple molecular forms that are distinguishable by SDS-polyacrylamide gel electrophoresis (zone I, approx. 450 kDa dimers; zone II, 190–235 kDa; Zone III, 146–475 kDa). (Chen, A.B., Amrani, D.L. and Mosesson, M.W. (1977) Biochim. Biophys. Acta 493, 310–322). We report here on investigations of plasma fibronectin that had been prufieid from the ‘heparin-precipitable fraction’ of plasma by DEAE-cellulose chromatography using buffers containing a chaotropic salt (KSCN). Zone I fibronectin and zone II fibronectin were subsequently separated by Sepharose CL-6B chromatography in the presence of 0.3 M KSCN. Electrophoresis of reduced zone I fibronectin dimers showed the presence of three types of subunits (i.e., 220 kDa, 215 kDa, 207 kDa), evidently all having the same NH 2-terminal sequence. Subunits of this size were also found in reduced zone II fibronectin, as well as another polypeptide of 190 kDa, the latter amounting to under 5% of the total. Unreduced zone I fibronectin was resolved by gel electrophoresis into a doublet. The upper component amounted to approx. 90% of the total and was comprised of 22 kDa and / or 215 kDa subunits; the lower component contained 207 kDa plus a 220 kDa or 215 kDa subunit. Scanning transmission electron microscopy indicated that under physiologic conditions zone II fibronectin molecules, like those in zone I, exist as pleiomorphic, loosely folded structures (approx. 16 × 8–12 nm) that are somewhat smaller than dimeric zone I molecules (approx. 24 × 16 nm). Circular dichroic spectral analyses suggest that both types have similarly folded local domains. Affinity chromatography experiments revealed a relative decrease in the binding of one II fibronectin to gelatin but no difference from zone I fibronectin with respect to heparin or fibrin binding.

Specificity of fibronectin--fibrin cross-linking.

Our experiments indicate that (1) non-covalent binding of fibronectin to fibrin is mediated by sites in a 27 kd NH2-terminal region and a 31 kd COOH-terminal region of fibronectin; (2) the 31 kd region is probably present in both chains of the fibronectin dimer; and (3) covalent (factor XIIIa-mediated) cross-linking of fibronectin and fibrin is between a glutaminyl residue in the 27 kd region of fibronectin and a lysyl residue in the COOH-terminal two-thirds of the fibrin alpha chain.

The effect of the dimeric and multimeric forms of fibronectin on the adhesion and growth of primary glomerular cells

Primary glomerular cells placed in a chemically defined medium containing Waymouth's medium MB 752/1 supplemented with insulin, transferrin, fibroblast growth factor, non-essential amino acids, sodium pyruvate, and antibiotics showed rapid outgrowth of cells which morphologically resembled well differentiated visceral epithelial cells followed by outgrowth of poorly differentiated cells; morphologic evidence suggests these latter cells are precursor cells of the epithelial cell lineage. Whereas the well differentiated glomerular epithelial cells were never observed to divide by sequential phase microscopic observations, a chemically defined medium was developed for optimal growth of the poorly differentiated cell type. This serum-free medium contained Waymouth's medium MB 752/1 supplemented with insulin, transferrin, selenium, and fibronectin (plus non-essential amino acids, sodium pyruvate, and antibiotics). Using this chemically defined medium, we have compared the effects of dimeric and multimeric fibronectin (high molecular weight disulfide-bonded fibronectin produced by incubation of dimeric fibronectin with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11) on the adhesion and growth of the poorly differentiated primary glomerular cell type. Dimeric fibronectin (FN) was twice as effective as multimeric FN in promoting glomerular cell adhesion, although both forms of FN promoted cell adhesion better than an uncoated substratum. In contrast, cell growth studies demonstrated that multimeric FN was a more potent growth stimulant than dimeric FN. The differential effects of dimeric and multimeric forms of FN in vitro suggests that these molecules may have different functions in vivo.

In vitro formation of disulfide-bonded fibronectin multimers.

Fibronectin purified from a plasma protein side fraction in the absence of denaturant contained 1.5 to 1.9 cryptic free sulfhydryl groups per 200- to 250-kDa subunit. Exposure of sulfhydryl groups in physiologic salt solutions required at least 1 M guanidine, and 3 M guanidine was required for optimal exposure. The sulfhydryl groups were not exposed by collagen, a fibronectin-binding collagen fragment, fibrinogen, heparin, hyaluronic acid, calcium ion, EDTA, deoxycholate, or methylamine. One- and two-dimensional gel electrophoresis indicated that a molecule of 40-60 kDa was disulfide-bonded to a minor portion of the fibronectin in whole human plasma and in preparations of purified fibronectin. In addition, traces of disulfide-bonded multimers were present in preparations of purified fibronectin. The proportion of fibronectin in disulfide-bonded multimers increased in guanidine-containing solutions. Compared to dimeric fibronectin, these multimers had limited solubility in physiologic buffers, could be readily cross-linked by Factor XIIIa, and exhibited altered tryptic susceptibility. In free sulfhydryl groups were blocked by prior alkylation with N-ethylmaleimide or iodoacetamide, fibronectin did not form disulfide-bonded multimers in guanidine-containing solutions. The patterns of altered tryptic susceptibility and cyanide cleavage suggested that multimer formation is mediated by both sulfhydryls of fibronectin. The transition from dimeric to multimeric fibronectin can serve as a model for the formation of disulfide-bonded fibronectin multimers in the extracellular matrix.

Transformation-enhancing activity of gelatin-binding fragments of fibronectin.

Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments.

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.

Polypeptides of human plasma fibronectin are similar but not identical

Human plasma fibronectin migrated in electrophoresis after reduction of the disulfide bonds in SDS-polyacrylamide gels as two closely spaced polypeptide bands. These polypeptides, the A chain ( M r 220 000) and the B chain (215 000), were isolated from slices of slab gels. The two isolated chains were immunologically indistinguishable when tested by double immunodiffusion against antiserum to plasma fibronectin. Identical peptides were obtained from both chains after Staphylococcus aureus proteinase digestion or after cyanogen bromide cleavage, respectively. Kinetic analysis of plasmin digestion of isolated dimeric fibronectin, however, suggested that the A chain was cleaved more rapidly than the B chain and that the primary plasmin cleavage products of fibronectin, the 200 000 and 190 000 polypeptides, were derived from the A and B chain, respectively. The basis for the difference between the A and B chains of human plasma fibronectin identified here, is, at present, unknown. Proteolytic or some other posttranslational processing of a common fibronectin polypeptide seems unlikely since also the newly synthesized fibronectin, isolated from human fibroblast cultures pulse-labeled for 5 min, appeared as two closely spaced polypeptide bands in SDS-gel electrophoresis.

Biosynthesis and processing of fibronectin in NIL.8 hamster cells.

Fibronectin is synthesized as a monomeric polypeptide chain. As early as it can be detected inside the cell, it carries carbohydrate side chains. These chains are sensitive to endoglycosidase H, suggesting that they are asparagine linked and high mannose form. The monomeric chains quickly dimerize while still inside the cell. Newly synthesized fibronectin appears as a dimer, both at the cell surface and secreted into the culture medium, about 30 min after commencement of labeling. This exported fibronectin has endoglycosidase H-resistant carbohydrate side chains, indicating processing from the high mannose form to a complex form. Exit of fibronectin to the outside of the cell follows quickly on carbohydrate processing; no large pool of endoglycosidase H-resistant fibronectin exists inside the cell. The dimeric fibronectin at the cell surface is initially deoxycholate soluble but slowly becomes deoxycholate-insoluble and also slowly forms high molecular weight aggregates which require reduction of disulfide bonds for their dissociation.