2015 Group 4 Project
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- 1 Fibronectin
- 1.1 Introduction
- 1.2 History
- 1.3 Structure
- 1.4 Assembly
- 1.5 Function
- 1.6 Abnormalities/defects
- 1.7 Therapeutics
- 1.8 Current Research
- 1.9 References
Fibronectin is an essential extracellular matrix (ECM) glycoprotein, present in all tissues throughout the body. It is especially profuse around lymphatic tissue, connective tissue and muscle fibres. These soluble fibronectin molecules are composed of two monomers that link together via a disulfide bond, forming a dimer. The repeating fragments that make up the molecules serve as the functional domains, enabling the binding of different molecules including heparin, fibrin, as well as binding to itself.
The dimer form of fibronectin is secreted by fibroblasts and is present in the blood, while plasma fibronectin is commonly secreted by hepatocytes. This cellular fibronectin accounts for around 1% of the total concentration of fibronectin in plasma, which is 0.3 mg/ml. It is the insoluble fibrillar network resulting from the association of these dimers, that ultimately dictates the various functions fibronectin plays, including its role in development, disease, and repair. Similarly, its various binding domains enable it to associate with other molecules within the ECM, as seen through the multiple heparin-binding domains present in the fibronectin molecules. Hence, the remainder of this page aims to provide further detail on the structure and function of this component of the extracellular matrix, as well as the abnormalities that can arise when there are mutations.
- 1940: Plasma protein is first described as cold insoluble globulin [Clg] by Harvard's Plasma Protein Program. 
- 1957: Korff notes a protein in a heparin-induced precipitate of plasma with similar properties to "cold-insoluble globulin"
- 1967: found as a plasma antigelatin factor, characterised by its interference in the determination of collagen antibodies. discovered by Wolff et al
- 1973: Ekki Ruoslahti and his team discover Fibronectin on surface of fibroblasts whilst searching for cell attachment molecules 
- 1978: Hynes RO show that fibronectin bind to platelets and promoted their spreading 
The dimer state of fibronectin is formed by a covalent bond joining the tails (C-terminus) of the two monomers. These 250 kDa monomers each comprise of three distinct protein fragments, each known as type I, II, and III repeats. More specifically, cellular fibronectin is a miscellany of heterodimers of various subunits, which differ in their ED-A and ED-B segments. For example, plasma fibronectin is often deficient in extra domain (ED) segments.
Fibronectin (FN) consists of repeated units of the domains FNI, FNII and FNIII. More specifically, the structure of the type III repeating fragment within the fibronectin monomers, has a hydrophobic core and electrostatic networks through an increased number of salt bridges. These forces in combination with the removal of non-conserved interactions, increases the stability of this fragment's structure. Fibronectin is a glycoprotein of around 440000 - 500000 kilo Daltons. It is a heterodimer, and its monomers are linked by disulfide bridges. Fibronectin is abundant around blood vessels, in lymphatic tissue stroma and in loose connective tissue. It circulates in the blood stream in a soluble form, but it is constantly assembled into insoluble extracellular matrix fibrils by a cell-dependant process. There is the cellular and the plasma fibronectin. Despite sharing many similarities, these two molecules are not equal regarding its structure and function. Most of the differences are due to primary structure differences (the primary structure of a protein is the linear sequence of its amino acids) of each one, which are a result of alternative splicing in, at least, but not limited to, two distinct regions (ED and IIICS) of the pre-RNAm. .
The cell attachment site of fibronectin is RGDS, and is postulated to mediate the concentration of fibronectin in cells.
Regarding the secondary structure (the secondary structure of a protein is the 3D forms os segments of the molecule) of the Fibronectin molecule, it is formed mainly by anti-parallel β-forms. This protein has a very compact structure, and it has a length that ranges between 14.2 and 16.8 nm, a width that ranges between 7.1 and 10.5 nm. The axial ratio is around 2:1. There is also a complex tertiary structural (the tertiary structure is the way that the protein molecule folds itself, it is the description of the 3D geometry of the molecule) organization (domains) in the Fn molecule, and the domains are interconnected by flexible non-structured segments of the polypeptide chain. These domains are not globular: they form long, thin and flexible strands instead.
There are multiple isoforms of fibronectin due to the variations in splicing during its formation. These isoforms vary in their binding domains, hence influencing their interactions with integrins, with some being identified as having a higher affinity for certain interactions than others. Subsequently, it is acknowledged that changes to the structure of the fibronectin molecule, ultimately influence the functional role it plays in that particular cell.
- module is 43 residues in length each forming a β-hairpin
- 3 stranded anti-parallel β-sheet
- stabilised by pair of interchain disulphide bonds (absolutely conserved).
- mediates human protein-to-protein and protein-to-oligosacchiride interactions.
- Tandem modules (FnI2-FnI5) interact with FnIII modules and binding of heparin.
- FNI4-FNI5 have capacity to bind to fibrin. FnI mudules near C-terminus also bind to fibrin
- FNI modules only found in vertebrates
- 49 residues in length
- Module is 5 stranded
- stabilised by 2 disulphide bonds
- only conserved structural element is small 2-stranded β-sheet.
- only 2 modules occur as single tandem repeat.
- FnII2 forms tight complex with FnII6
- FNII confined to metazoans
- 90-100 residues contained within an all-β structure
- module fold contains 7 strands. 2 anti-parallel β-sheets of 3 or 4 stands forming hydrophobic core
- does not require stabilising intrachain disulphide bonds.
- structurally plastic when under strain (disulphide bonds enforce rigidity)
- found mainly in metazoans (>80%), but also in plants/bacteria/archaea
- metazoan module occurs commonly as tandem repeats in other cell surface proteins and in EC regions of some cell surface receptors.
The assembly of fibronectin molecules into a fibrillar network is cell mediated. Furthermore, this assembly is a result of electrostatic forces that enable the joining of the head and tail of two dimers. It is suggested that the association of these dimers to form larger complexes is essential for providing enough binding sites in order to create a stable attachment with the cytoskeleton, that the dimer is otherwise unable to achieve. The beginning steps of this assembly includes the binding of these sites to surface receptors and ECM proteins, and is essential in forming this stable complex.
Moreover, the state of the fibroblasts influences the structure of the fibronectin secreted. More specifically, if the tissue-associated fibroblasts are unstretched, the resulting fibronectin will assemble in a mesh-like arrangement. This contrasts to the well-organised, linear placement of fibronectin secreted from fibroblasts that have been stretched. Such a difference in fibronectin deposition suggests a possible association to the role of mechanical stresses in cancer-associated fibroblasts.
Fibronectin plays a pivotal role in the process of wound healing. In particular, it enables the migration of fibroblasts, which is vital for the formation of granulation tissue and hence healing. Fibronectin is present in the collagenous matrix as well as the fibrin-rich matrix of the wound, and it is suggested that fibronectin ultimately is essential for enabling the fibroblasts to interact with the fibrin. Fibronectin plays a particularly important role in the early phases of wound healing. The release of platelets in the first step of haemostasis when a wound occurs causes a platelet plug to form. the platelet to platelet bind is formed by integrins via fibrinogen. This fibrin clot is then stabilised further by factor XIII, where it binds the fibrin to fibronectin, forming the fibrin-fibronectin clot.
During inflammatory phase within the ECM, fibronectin is able to opsonise ECM debris. It acts as a nonimmune opsonin for the debris' phagocytosis by fibroblasts, keratinocytes and even macrophages under some conditions. The fibronectin also acts as an enhancement for the phagocytosis of immune-opsonised particles by monocytes. 
Fibronectin Wound healing 
Role in Tumours
Plasma fibronectin is found to be upregulated in tumour cells, ultimately supporting tumor maintenance. Tumor cell adhesion to and invasion in fibrin when paired with fibronectin (fibFN) is mediated mainly by integrin avB3 and activated by the former. pFN, in association with fibrin, is shown to assist in the cell adhesion of clotted plasma, however it does not assist in the initial tumor cell arrest. Moreover, fibronectin plays more of a function in organising the arrangement of other elements during tumour angiogenesis, compared to its structural role in embryo angiogenesis.
Plasma fibronectin is found to help as well as hinder atherosclerosis. It increases the number and size of Atherosclerosis plaques that form with it's deposition within lesions, but also helps in the formation of a protective fibrous cap, helping prevent rupture of said athersclerotic plaque. Also indicated that plasma fibronectin, rather than the hemopoietic cell-driven fibronectin, is deposited at atherosclerosis-prone sites prior to the development of atherosclerotic lesions.
Role in Blood Vessels
Plasma fibronectin is found to deposit in vessel injury sites (independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets) before accumulation of platelets, which was previously understood to be the first wave of hemostasis. In particular, it promotes platelet aggregation when in conjunction with fibrin but acts conversely when fibrin is absent. Working off the fibrin gradient, fibronectin is a regulator of thrombosis
Role in Promoting and Inhibiting Cell Adhesion
Fibronectin within sheep carotid arteries was seen to increase cell attachment of proteins with the vessel walls, in comparison to other methods. However, it has also been observed that cell adhesion mediated by fibronectin can be inhibited by fibronectin when a high concentration of fibronectin is reached, or there is a long incubation period. This inhibition is observed to be associated with the 75kDa cell-binding component of the fibronectin molecule.
Fibronectin is seen to play a role in the maturation of the ovarian follicle. During the first and second trimesters of gestation, the mesenchymal compartment cells of the ovarian cortex and the tunica albuginea express fibronectin. This fibronectin is observed throughout the basement membrane of the developing blood vessels. Hence, fibronectin is seen to play a role in the assembly of the primordial follicles, as well as assisting in the embedding of blood vessels. More specifically, fibronectin influences the defining of the mesenchymal compartment during fetal development.
Furthermore, fetal fibronectin can be used to predict preterm birth through observing the presence of this fibronectin in secretions of the cervical posterior fornix. Whilst the presence of fibronectin is expected in the blood and amniotic fluid during pregnancy, high levels of it in secretions indicate a significant increase in the chance of preterm birth.
Fibronectin is one specific extracellular matrix protein that most effectively enables differentiation of human embryonic stem cells to definitive endoderm. Subsequently, it can be identified as having a significant role in early embryogenesis. This differentiation of cells is facilitated by integrin receptors, with the expression of integrin subunits increasing as this differentiation is occurring. In particular, a5 integrin is required for human embryonic stem cells to bind to fibronectin. Moreover, the cells that are exposed to fibronectin, and other extracellular proteins, begin to secrete extracellular proteins, encouraging further differentiation.
Fibronectin is also seen to play a pivotal role in the left-right axis determination that takes place during embryogenesis. This is important in ensuring correct development of organs distributed asymmetrically throughout the body. It is suggested that fibronectin performs this function by giving nodal cells polarity, which is necessary for their formation of the nodal pit. 
Furthermore, it is suggested that fibronectin plays a role in cranial neural crest cell migration, as well as neural fold and neural crest formation. This is proposed through the study as a5 integrin was observed in the anterior neural ridge, the cranial neural fold, and in the neural crest cells. Moreover, fibronectin was identified as increasing the activity of a specific protein-coding gene known as BMP5. This gene is associated with chicken cranial neural crest formation. Subsequently, fibronectin is noted as influencing the expression of a growth factor. This can also be seen through the role fibronectin plays in cardiac development of the embryo. For example, the transcriptional repressor gene snail1 assists in the migration of cardiac precursors by influencing the assembly of fibronectin in the embryo. Abnormalities in fibronectin function result in cardiac defects. In particular, the ordered deposition of fibronectin in control embryos contrasts to the reduced and disorganised arrangement in modified embryos with inactivated snai1b. Hence, snai1b is needed for effective fibronectin assembly, which enables cardiac precursors to migrate where they are needed for successful cardiac development. Moreover, integrins are crucial for effective fibronectin function. In cases were snai1b is inactivated, addition of integrins salvages fibronectin assembly and minimises cardiac defects. Thus, it can be seen that snai1b modulates the expression of a5 integrin, which ultimately modulates fibronectin assembly essential for cardiac development.
The abnormalities presented by Fibronectin (FN) are very vast and also have a huge range of clinical and pathological presentations, being caused by alterations on its production, which might be due to gene expression mutations or even over-production without any abnormalities in the structure.
Similarly, human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organise fibronectin and similar proteins in the extracellular matrix, as well as down-regulate alpha2beta1 integrin, whilst up-regulating alphavbeta3 instead of alpha5beta1 integrin. Such mutations are seen in patients with Ehlers-Danlos Syndrome (EDS) types I and IV, who are ultimately identified as having a diminished fibrillar network. Providing such patients with pure fibronectin encourages binding between FN and integrin alphavbeta3.
Glomerulopathy with Fibronectin Deposits (GFND)
Glomerulopathy with Fibronectin (GFND) deposits is a dominant inherited disease that affects the kidney through deposition of fibronectin in the glomeruli. This disease results in symptoms such as the loss of protein, blood in the urine, and hypertension. It is postulated that GFND is caused by mutations in FN1, an encoder of fibronectin, with forty per cent of GFND cases in a particular study showing these mutations.
A Study  compared the levels of fibronectin in normal tissue against wounds that developed keloid, a process which consists on an over-production of collagen and other extracellular matrix components. The keloid tissue presented a huge production of both intracellular and extracellular fibronectin (due to its mRNA increment), that had no abnormalities in the structure, process and degradation. The fibronectin receptors were also raised in the abnormal tissue. When glucocorticoids were administered, fibronectin production was generally stimulated in both normal and keloid fibroblasts.
Metastatic Breast Cancer
Fibronectin consists of two alternatively spliced extra domains, ED-A and ED-B, which are expressed during embryonic angiogenesis, but ultimately decrease in normal adults. Nonetheless, the ED-A and ED-B are detectable in the angiogenesis of tumours and matrix remodelling. A study  showed that mice who were injected with anti-ED-A/B tend to have less metastasis, as well as immunisation against ED-B diminished angiogenesis and tumor growth. ED-A is known to be expressed at high levels in human breast carcinomas and general metastasis, whereas ED-B shows very low levels in these types of tumours. The immunisation of the mice with anti-ED-A reduced tumour burden and the metastasis were suppressed. This subsequently leads to the consideration of targeting vaccines as part of the usual therapy, or in replacement of monoclonal antibodies.
Type 1 Diabetes
Elevated levels of fibronectin can also serve as a possible biomarker for damage to blood vessels in diabetics due to the manner in which increased fibronectin concentrations have been observed in diabetic patients with cardiovascular impediments. Moreover, fibronectin has been observed to play a role in a positive feedback mechanisms in Type 1 diabetics. In particular, monocytes of diabetics secrete greater amounts of myeloid-related protein MRP8/14 than controls. This in turn promotes cells to adhere to fibronectin, in turn causing further production of this serum. Hence, it is suggested that this influences the maintenance of the immune response.
Fibronectin can function as a molecular target for therapeutics of hepatocellular carcinoma. In particular, ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a target for specific therapeutics. A monoclonal antibody, called B2212A, binds the third fibronectin domain (Fn3) of ROBO1. The results of the study, according to the article´s text, promises to lead future researches towards a new antibody drug for the treatment of hepatocellular carcinoma.
Recent PubMed Articles
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