ENTREVISTA A DIFERENTES MIEMBROS DE LA COMUNIDAD EDUCATIVA

Fibronectins are a class o f high molecular weight glycoproteins found in two distinct forms. Plasma fibronectin is secreted into the blood stream by hepatocytes whilst the cellular or insoluble form is produced by fibroblasts and remains localized on the cell surface. The soluble form of fibronectin is also found in most body fluids.

1.3.1. History and Isolation

In 1948, Morrison et al. observed, during further purification o f a fibrinogen rich plasma fraction, an unrecognised component which had a lower solubility than fibrinogen at low temperatures. Accordingly this component was named ‘cold-insoluble globulin’.

A pure fraction o f cold-insoluble globulin was not made until 1970, when Mosesson and Umfleet (1970) used diethylaminoethyl cellulose chromatography to remove fibrinogen and other plasma proteins from cryoprecipitate. Confirmation that the cold-insoluble globulin had an electrophoretic migration rate similar to a fast (3 globulin and that no change was noted after heating to 56°C or treatment with thrombin distinguished it from fibrinogen and it was then regarded as a separate plasma protein.

Other research during the 1970s concentrated upon the isolation o f a cell surface protein from fibroblasts (Hynes 1990). Yamada and Weston (1974) isolated a protein, present on the surface o f normal cells but absent after neoplastic transformation. This cell surface protein (CSP) was seen to increase with cell density and deemed important in growth control. In the mid-1970s it was concluded, by a number o f groups, that the cell surface protein and cold insoluble globulin were related with properties including cell adhesion and morphology. Antibodies were used both to confirm similarities between the two proteins (Hynes 1990) and some slight structural differences (Atherton and Hynes, 1981). Gelatin afiBnity chromatography was described as a rapid and simple method to purify proteins from both sources (EngvaU and Ruoslahti, 1977).

Research into both soluble and insoluble forms o f the protein and their properties had been carried out independently resulting in a number o f different names for the protein each describing its identified functions. The protein had been described as cold-^^

insoluble globulin, antigelatin factor, ceU attachment protein, cell spreading factor and opsonic surface binding glycoprotein whilst the cell surface forms had been known as large, external, transformation-sensitive (LETS) protein, surface fibroblast antigen, galactoprotein a, cell surface protein and zeta protein. To avoid further confusion it was decided that the proteins should be described as cellular and plasma forms o f fibronectin, derived firom Latin fibra - fibre and nectere - to connect or link, referring to the adhesion properties o f the protein (Hynes, 1990).

By 1982 a fairly clear picture o f the basic structure and functions o f the molecule was established and with the advent of molecular biology, by 1988, complete amino acid sequences o f a number o f mammalian fibronectins were known as well as a more detailed knowledge o f the protein structure (Hynes, 1990).

1.3.2. Primary structure and conformation

Fibronectin is a dimer, o f molecular weight approximately 440 kDa, consisting o f two almost identical chains which are discretely separable on an electrophoretic gel. Estimates o f the molecular weight o f each chain have been given as 220 and 215 kDa and this difference is attributed to the release o f a small C terminal peptide post- translationally rather than the synthesis o f two types o f chain (Mosesson et al., 1975). The chains are covalently joined by disulphide bonds at their C termini, as shown in Figure 1.2. At the N terminus there are a number o f intrachain disulphide bonds and in the central portion o f each chain 1 or 2 fi*ee sulfhydryl groups are found as well as the carbohydrate residues, which are believed to stabilise the protein against proteolysis (Mosesson and Amrani, 1980).

p i 8.2 4.9-S.3

Domain binding functions Fibrin Heparin Collagen Bacteria + 8.7 4.2-4.8 _{4.6- 8.3- 5.0-} 4.9 8.5 5.8 DNA dNA

Heparin Heparin Cells Heparin Fibrin

30kDa

N 29kDa 45kDa 30kDa 30kDa 23kDa

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1 and 2

Figure 1.2. Schematic diagram o f the fibronectin molecule. Fibronectin consists o f two almost identical chains, one shown here completely and one partly shown, joined at their C termini by disulphide bonds to give a flexible structure. Each chain is divided into a number o f domains, each with particular binding functions, shown above the diagram. Approximate molecular weights fo r each domain are given, with the rest o f the molecular weight o f the chain (total 215 - 220 kDa) being made up by connecting sequences. 90 % o f the fibronectin molecule is made up o f 1 o f 3 repeat amino acid sequences and the type o f repeat predominant in each binding domain is shown below the chain. The p i fo r individual sections o f the chain was found to differ and these are also shown in approximate positions along the chain as well as the ionic charge fo r that region at neutral p H (adapted from Markovic et al, 1983b and Petersen et al., 1989).

The two chains, each approximately 2000 amino acids long, are separated at physiological pH by a 70° angle (Mosher, 1984). The position o f the chains under various environmental conditions is described more fully is section 1.3.3.

The fibronectin molecule was found to consist o f a number o f homologous repeat sequences, listed below:

Type 1: 45-50 amino acids long and containing disulphide bonded loops Type 2: as type 1

The central 150-170 kDa portion contains only type 3 repeats and thus no disulphide bonds, although the two free sulfhydryl groups are found here. Each repeat exists as a basic binding module and so repeat sequences increase the specific binding potential o f each domain. The cell binding domain was seen to contain a number o f repeats o f the four amino acids, L-arginine, glycine, aspartate and serine or closely related variants and these are known to promote the cell attachment and cell spreading functions o f fibronectin (Hynes, 1985).

Boughton and Simpson (1984) examined afiBnity chromatography purified fibronectin which appeared homogeneous by SDS gel electrophoresis but could be split into five fractions by isoelectric focusing, each with a different isoelectric point (pi) ranging from 5.6- 6.1. The fraction with pi = 6.1 was the only fraction found to promote the ingestion o f Sacchromyces cerevisiae and constituted only 15 % o f the total fibronectin. They also found that fibronectin in plasma had a higher pi than purified fibronectin although the explanation for this was unclear. Markovic et al. (1983b) foimd that different regions o f the chain had varying values o f pi and this may play an important role m the conformation o f the molecule and its binding functions described later. Tooney et al. (1983) have measured the pi o f a 6.8 mg/mL solution as 5.2 ± 0.2.

It is known that there is only one gene for fibronectin (Petersen et al., 1989) and that different types of fibronectin are formed after being transcribed in a different way. It is as yet unknown whether different fibronectins originate from different tissues or whether they interact with different molecules preferentially (Boughton and Simpson, 1984).

1.3.3. Effect of environmental change on the fibronectin molecule

The effect o f various environmental conditions such as pH, temperature, ionic strength and chemical agents has been examined thoroughly to determine whether they effect the conformation o f the molecule. Changes in molecule shape may affect the binding properties o f the protein.

1,3,3,1, Effect o f p H and ionic strength

Electron microscopy has been used to measure the dimensions o f the fibronectin molecule. Most accurate results are believed to be achieved when fibronectin is freeze

dried on a carbon film at pH 7.0. Under these conditions, Tooney et al. (1983) measured the molecule as 24 nm x 16 nm.

There have been a number o f reports suggesting that the fibronectin molecule changes fi’om a compact to an elongated conformation under extremes o f pH and ionic strength. Tooney et a l (1983) noted that the protein unfolds at pH 2.8 or 9.3 or when the ionic strength is raised at pH 7.0. Similar results were seen by Williams et a l (1982) who described an increase in chain flexibility and Sjoberg et al. (1989) who stated that fibronectin molecules in solutions with sodium chloride concentrations <0.3 M are disc­ shaped, whilst in 0.3 M solutions they take on a reversible transition to a more open structure.

A number o f studies have been made to investigate changes in electrostatic bonding in the molecule with changes in the environment. A study by Markovic et al. (1983b) indicated, using sedimentation velocities, that fibronectin molecules formed elongated structures with ‘outstretched arms’ at pH 3.0 and 11.0. To test whether the effect was due to intra or inter chain effects a 140 kDa fi*agment fi-om the central portion o f one chain was also tested. A similar pH dependent effect was seen. Small segments taken fi-om the N terminal end o f the chain showed no pH dependence on sedimentation velocity. These may have been stabilised by disulphide bonds in the region. The group also noted that the sedimentation velocity for pH 6-10 alluded to a compact structure and a rod-like form for pH < 6.0, concentrations 0.15 mg/mL. This is represented diagrammatically in the upper part o f Figure 1.3. The pH-dependent reaction is believed to be brought about by interactions between the domains o f the molecule. Electrostatic interactions between domains o f different net charges (due to differing pi in adjacent segments along the chain. Figure 1.2) were believed to be important for intramolecular association. At neutral pH the opposing charges interact to produce a fairly symmetrical compact conformation whereas at extremes o f pH a more extended, thread-like conformation forms due to repulsive interactions.

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