Helmut Hörmann

Fibronektin — Mittler zwischen Zellen und Bindegewebe

SummaryFibronectin, previously also termed LETS-protein, is a high-molecular-weight protein (mol. w. ca. 450,000) present in the form of thin fibrils in the pericellular space of fibroblasts and other adherent cells, as well as in distinct areas of the connective tissue. A soluble form, immunologically identical and chemically at least very similar to the cell-attached protein, is found in plasma in a concentration of about 300 µg/ml. It is also denominated cold-insoluble globulin. The protein has affinity both to cell surfaces and to various matrix substances such as fibrin and collagen and, therefore, is capable of mediating cell attachment to these substrates. In addition, it serves as an opsonin for the phagocytosis of gelatin-containing compounds and probably is essential for the removal of soluble fibrin from the circulating blood by the reticulo-endothelial system. Bacterial cell walls are also recognized by fibronectin.A conversion of soluble fibronectin to fibrils is achieved by heparin which also enhances the binding of soluble fibronectin to cells. Heparin or, as suggested, the related heparansulfate present on the surface of various cells, appears to function as a cofactor in the formation of pericellular fibrils. The fibronectin fibrils precipitated with heparin, compared to soluble fibronectin, show a considerably improved affinity to native collagen, especially to type III. Hyaluronic acid has an antagonistic function which, at higher concentrations, prevents the fibronectin fibrils from interacting with collagen and cell surfaces. Masking of fibronectin fibrils was also achieved by sulfated proteoglycans of cartilage.Virus-transformed fibroblasts produce less fibronectin and are less capable of maintaining surface pericellular fibrils. A reasonable explanation is that they have an elevated secretion of hyaluronic acid. The transformed cells attach only weakly to a surface and exhibit a rounded shape in contrast to healthy ones. This phenotype can be corrected to a great extent with fibronectin.It is suggested that fibronectin also influences the formation of connective tissue by accumulating collagen precursors on the surface of fibroblasts and facilitating fibrillogenesis.ZusammenfassungFibronektin, früher auch LETS-Protein genannt, ist ein hochmolekularer Eiweißkörper (Mol-Gew. ca. 450 000), welcher im perizellulären Bereich von Fibroblasten und anderen adhärenten Zellen aber auch sonst im Bindegewebe in Form dünner Fibrillen auftritt. Eine lösliche Form, die mit dem Oberflächenprotein der Zellen immunologisch identisch und chemisch zumindest sehr ähnlich ist, kommt im Blutplasma in einer Konzentration von etwa 300 µg/ml vor. Sie wird auch als Kälte-unlösliches Globulin bezeichnet. Fibronektin besitzt Affinität zu Zelloberflächen und zu verschiedenen Matrixsubstanzen wie Fibrin und Kollagen und vermittelt dadurch das Anhaften von Zellen an diese Substrate. Es wirkt außerdem als Opsonin für die Phagozytose gelatinehaltiger Verbindungen und ist vermutlich wichtig für die Ausschleusung von löslichem Fibrin aus dem Blutkreislauf durch das retikulo-endotheliale System. Auch Bakterienoberflächen werden von Fibronektin erkannt.Eine Umwandlung von löslichem Fibronektin in Fibrillen gelingt mit Heparin, das gleichzeitig die Bindung von löslichem Fibronektin an die Zellen verstärkt. Heparin oder, wie vermutet wird, das an Oberflächen verschiedener Zellen vorhandene Heparansulfat scheinen somit als Cofaktoren für die Ausbildung der perizellulären Fibrillen wesentlich zu sein. Die mit Heparin abgeschiedenen Fibronektinfibrillen weisen auch eine bessere Affinität zu nativem Kollagen, besonders zu Typ III, auf als lösliches Fibronektin. Als Antagonist wirkt Hyaluronsäure, die in höheren Konzentrationen die Wechselwirkung der Fibronektinfibrillen mit Kollagen und Zelloberflächen verhindert. Auch sulfatierte Proteoglykane aus Knorpelgewebe maskieren Fibronektinfibrillen.Virus-transformierte Fibroblasten erzeugen weniger Fibronektin und vermögen perizelluläre Fibrillen nur schlecht festzuhalten. Eine Erklärung dafür könnte die erhöhte Abscheidung von Hyaluronsäure sein. Die transformierten Zellen haften nur schlecht an einer Unterlage und weisen im Gegensatz zu gesunden eine runde Form auf. Dieser Phänotyp kann durch Fibronektin zum größten Teil korrigiert werden.Es wird vermutet, daß Fibronektin auch auf die Bildung von Bindegewebe Einfluß nimmt, indem es kollagene Vorstufen an der Oberfläche von Fibroblasten akkumuliert und dadurch die Fibrillogenese beeinflußt.Fibronektin, fruher auch LETS-Protein genannt, ist ein hochmolekularer Eiweiskorper (Mol-Gew. ca. 450 000), welcher im perizellularen Bereich von Fibroblasten und anderen adharenten Zellen aber auch sonst im Bindegewebe in Form dunner Fibrillen auftritt. Eine losliche Form, die mit dem Oberflachenprotein der Zellen immunologisch identisch und chemisch zumindest sehr ahnlich ist, kommt im Blutplasma in einer Konzentration von etwa 300 µg/ml vor. Sie wird auch als Kalte-unlosliches Globulin bezeichnet. Fibronektin besitzt Affinitat zu Zelloberflachen und zu verschiedenen Matrixsubstanzen wie Fibrin und Kollagen und vermittelt dadurch das Anhaften von Zellen an diese Substrate. Es wirkt auserdem als Opsonin fur die Phagozytose gelatinehaltiger Verbindungen und ist vermutlich wichtig fur die Ausschleusung von loslichem Fibrin aus dem Blutkreislauf durch das retikulo-endotheliale System. Auch Bakterienoberflachen werden von Fibronektin erkannt.

Shape, conformation and stability of fibronectin fragments determined by electron microscopy, circular dichroism and ultracentrifugation

Abstract Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K ‡ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.

Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Cold-insoluble globulin (fibronectin), IV[1-35 affinity to soluble collagen of various types.

Zusammenfassung: Die Affinität zwischen Kälte-unlöslichem Globulin (Fibronektin) und nativem sowie hitzedenaturiertem löslichem Kollagen der Typen I, II und III wurde in folgenden Systemen untersucht: 1. Adsorption von I-markierten löslichen Kollagenderivaten an immobilisiertes Fibronektin. 2. Komplexbildung zwischen I-Kollagen und Fibronektin in Lösung und anschließende Fällung des Komplexes mit Anti-FibronektinAntiserum. 3. Hemmung der Komplexbildung von löslichem denaturiertem I-Kollagen, Typ III, und Fibronektin durch nichtmarkierte lösliche Kollagenderivate. Auch in diesem Falle wurden die gebildeten Komplexe durch Anti-Fibronektin-Antiserum gefallt. In allen drei Systemen zeigte Fibronektin die stärkste Affinität zu denaturiertem löslichem Kollagen, Typ III· Es folgten mit etwa gleicher Wirksamkeit denaturiertes Kollagen, Typ I und Typ II. Von den nativen löslichen Kollagenpräparaten wies nur Typ III in allen drei Tests eine gewisse Affinität auf, die deutlich geringer war als die der denaturierten Präparate. Im Inhibitionstest konnte auch für natives Kollagen, Typ I, eine Affinität nachgewiesen werden, die noch geringer war als die von nativem Kollagen, Typ III.

Electron Microscopic Investigations on Soluble and Insoluble Bovine Fibrin

Summary Clots ofsoluble bovine fibrin were dissolved in various agents and reprecipitated by dialysis against phosphate buffer (pH 6.g). Cross-striated fibrils were obtained when the solvent 1 M KBr at pH 5.3 was used. Fibrils without cross-striation precipitated from 2% acetic acid, and more or less unstructured material resulted from 6 M guanidinium chloride or 2 M KCNS. The optical rotation of these fibrin solutions was established. The agents mentioned, besides 2 M KCNS, had a swelling effect in fibrin which was covalently cross-linked by activated Factor XIII. Electron micrographs of stabilized fibrin treated with 1 M KBr revealed subfibrils exhibiting a width equivalent to that of two fibrin monomers. Accordingly, the formation of covalent cross-links is limited to the subfibrils. A three-dimensional covalent network must be rejected. After the removal of KBr cross-striated fibrin fibrils reformed. When enzymatically cross-linked fibrin was treated with 2% acetic acid, 6 M guanidinium chloride or 2M KCNS, cross-striation could not be regained even after the removal of these solutes. ATP precipitated soluble fibrin in globular form. After the removal of ATP fibrin was able to reconvert to cross-striated fibrils.

The role of fibronectin fragments and cell-attached transamidase on the binding of soluble fibrin to macrophages

Proteolytic fragments from the N-terminus of the fibronectin subunit chains were shown to mediate the binding of 125-I-fibrin to macrophages. With increasing molecular weight of the fragments, binding activity decreased and intact plasma fibronectin was inactive. Fibrin binding to macrophages was a time dependent reaction and proceeded considerably faster than binding of fibrinogen. The binding reaction was inhibited by putrescine suggesting the involvement of a transamidase. Pericellular transamidase was demonstrated on macrophages by incorporation of 14-C-putrescine into fibronectin 30 kD-fragment. Expression of this enzyme appeared to be rate-limiting for the binding reaction which was accelerated after loading the cells with placental transamidase.

Evidence for the presence of a free N-terminal fibronectin 30-kDa domain in human plasma by quantitative determination with an indirect immunosorbent assay☆

The free N-terminal 30-kDa domain of the fibronectin subunit chains had previously been shown to mediate binding of soluble fibrin to phagocytic cells. In order to demonstrate whether the fragment is available in plasma in a suitable concentration, an indirect immunoassay procedure for its quantitative evaluation was developed. The free form of the 30-kDa domain was separated from fibronectin and the bulk of the plasma proteins by two-step affinity chromatography on gelatin- and heparin-Sepharose. In the eluate of the heparin-Sepharose the 30-kDa fragment was determined by its capacity to inhibit the immune reaction between a specific antiserum and the 30-kDa fragment immobilized on microtiter wells. The procedure offered reproductibility comparable with other immunoassays (coefficient of variation 4.0 to 8.0%); the lowest amount of detectable 30-kDa fragment was 0.1 microgram/ml. In human plasma this method detected for the first time ca. 5 micrograms/ml 30-kDa fragment. This concentration is in the range required for binding of fibrin to cells.

Dependence of staphylococcal clumping activity of fibrinogen on conformation

Abstract The ability of fibrinogen to clump staphylococci was retained after a transient treatment of the protein with 8 M urea. Cleavage of disulfide bonds by reduction in 8 M urea diminished the clumping activity in urea free solutions considerably. Separated single chains had no clumping activity at all.

The cold-insoluble fibrinogen fraction of bovine and human plasma☆

Abstract A cold-insoluble fibrinogen fraction was prepared from a 2.1 M glycine precipitate of bovine or human plasma. Because of its unfavorable solubility properties this fraction could be further resolved by gel electrophoresis only in the presence of 6 M urea or by gel filtration at an elevated pH. From these investigations the presence of a considerable amount of aggregated material became apparent. The bulk of the aggregates dissociated in 6 M urea, but a small fraction of oligomers was resistant indicating covalent intermolecular cross-linking. Sulfitolysis of the cold-insoluble fraction as well as of separated aggregates yielded single fibrinogen peptide chains devoid of dimers or polymer chains. Therefore, the intermolecular cross-links of the oligomers appear to be sensitive to SO 3 2− cleavage and may represent disulfide bonds rather than cross-links mediated by the action of Factor XIII. Model experiments showed that, besides dissociable aggregates, urea-resistant oligomers were formed in a mixture of fibrinogen and fibrin monomers. The chromatographic and electrophoretic behavior of the mixture in the presence and absence of 6 M urea was similar to that of the cold-insoluble fibrinogen fraction. In bovine cold-insoluble fibrinogen a small amount of a further protein hardly soluble in fibrinogen-free media of neutral pH was detected. It consisted of only one peptide chain. Its molecular weight was found to be 81 000 ± 4000 by sodium dodecylsulfate electrophoresis. Sedimentation equilibrium showed a monomeric form of the protein at pH 10.

Free N-terminal fibronectin 30-kD-domain mediates binding of soluble fibrin to gelfiltered unstimulated thrombocytes

Gelfiltered unstimulated human platelets neither bound 125-I-fibrinogen nor 125-I-fibrin. Fibrin-binding was, however, stimulated by N-terminal fibronectin 30 kD-and 70 kD-fragments while fibronectin was ineffective. The 30 kD-fragment also stimulated some platelet preparations to bind fibrinogen which, however, was suppressed by minute amounts of the thrombin inhibitor PPACK. PPACK hardly influenced fibrin-binding. Fragment-promoted fibrinogen-binding was also inhibited by a monoclonal antibody recognizing the membrane glycoprotein IIb/IIIa complex known to act as fibrinogen receptor. This antibody failed to influence fragment-stimulated fibrin-binding giving evidence that fibrinogen and fibrin were retained by different receptors. In contrast to 125-I-fibrin its plasmin-derived and 125-I-labelled fragment X was not recognized by the platelets in presence of the fibronectin 30 kD-fragment. Fragment-stimulated binding of 125-I-fibrin showed a lag phase and was completely inhibited by 0.25 mM putrescine as well as by 1 mM EDTA or 0.1 mM N-ethylmaleinimide. Evidently, a cell-attached transamidase was involved in fibrin-binding possibly by forming a ternary complex with fibrin and the fibronectin fragment.