Abstract. Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4°C, DS-PG core protein was uniformly distributed over the cell surface. At 37°C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength.Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides conruining either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.
Fibronectin, 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 micrograms/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 heparan sulfate 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.
Kälte-unlösliches Globulin (Fibronektin), JV. Affinität zu löslichem Kollagen verschiedener TypenZusammenfassung: Die Affinität zwischen Kälte-unlösli-chem Globulin (Fibronektin) und nativem sowie hitzedenaturiertem löslichem Kollagen der Typen I, II und III wurde in folgenden Systemen untersucht: 1. Adsorption von 125 I-markierten löslichen Kollagenderivaten an immobilisiertes Fibronektin. 2. Komplexbildung zwischen 12s I-Kollagen und Fibronektin in Lösung und anschließende Fällung des Komplexes mit Anti-FibronektinAntiserum. 3. Hemmung der Komplexbildung von lösli-chem 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.
The precipitation of plasma fibronectin by heparin in dependence on various parameters was investigated. Rising heparin concentration augmented the precipitates up to a maximum beyond which precipitation decreased. Yields close to 80% were obtained at low temperatures, but some precipitation was observed at 37 °C as well. Insolubilization was considerably dependent on the ionic strength, indicating that electrostatic forces play a major role in the aggregation of fibronectin. Calcium already prevented precipitation by heparin at low concentrations. If precipitation was performed on hydrophobized glass cover slides, the formation of fibrils visible by phase-contrast microscopy was observed. On hydrophilic surfaces amorphous precipitates were generally obtained, most likely due to trapping of aggregates by adsorption prior to their arrangement to fibrils. The results are discussed on the basis of a model assuming that heparin induces a conformational rearrangement of plasma fibronectin so that masked binding sites responsible for self-association become exposed.
Aggregation und Fibrillenbildung von Plasmafibronektin durch HeparinZusammenfassung: Die Ausfällung von Plasmafibronektin durch Heparin wurde in Abhängig-keit von verschiedenen Parametern untersucht. Bei zunehmender Heparin-Konzentration stieg die Niederschlagsmenge bis zu einem Maximum an und fiel dann wieder ab. Ausbeuten um 80% wurden bei 4 °C erzielt, aber auch bei 37 °C wurden Niederschläge beobachtet. Die Niederschlagsbildung war sehr stark abhängig von der Ionenstarke, was auf die Mitwirkung elektrostatischer Kräfte bei der Aggregation von Fibronektin schließen läßt. Calcium verhinderte die Fällung bereits bei niedrigen Konzentrationen. Wurde Plasmafibronektin auf hydrophobierten Glasoberflächen ausgefällt, dann entstanden Fibrillen, die im Phasenkontrastmikroskop sichtbar waren. An hydrophilen Glasoberflächen erhielt man dagegen amorphe Niederschläge, was damit gedeutet wurde, daß die gebildeten Aggregate an die Oberfläche adsorbiert wurden, bevor sie sich zu Fibrillen ordnen konnten. Die Ergebnisse werden auf der Basis eines Modelies diskutiert, das eine Konformationsumwandlung von Plasmafibronektin unter dem Einfluß von Heparin vorschlägt, wobei maskierte Bindungsstellen für eine Eigenassoziation freigelegt werden.
A mild cathepsin D digest of fibronectin only contained single-chain peptides of 200, 140 and 70 kDa and doubIe-chain fragments of about 300 and 140 kDa containing the C-terming d~sulfide link. Among the single-chain fragments the 200 kDa peptide was a precursor of the 140 kDa and 70 kDa peptides. The latter was correlated to the N-terminal and the former to the central region of the fibronectin subunit chains.
Fibronectin Carhepsin D
Gela fin-SepharoseHeparin-Sepharose
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