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Fibronectin

History
Brief history of Fibronectin.jpg
Introduction
Fibronectin is an essential extracellular matrix (ECM) glycoprotein,[1] present in all tissues throughout the body. It is especially profuse around lymphatic tissue, connective tissue and muscle fibres.[2] 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 and fibrin.[3]

The dimer form of fibronectin is secreted by fibroblasts and is present in the blood,[4] while plasma fibronectin is commonly secreted by hepatocytes.[5] This cellular form accounts for approximately 1% of the glycoprotein's total plasma concentration,[5] which is 0.3 mg/ml.[6] 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.[7] Similarly, its various binding domains enable it to associate with other molecules within the ECM,[8] as seen through the multiple heparin-binding domains present in the fibronectin molecules.[9] 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.


Structure


Structure of Fibronectin Protein.
<mediaplayer>https://www.youtube.com/watch?v=B6cWSEQx934</mediaplayer>

Fibronectin is a glycoprotein of around 440000 - 500000 kilodaltons (kDa). It is a heterodimer with its monomers linked by disulfide bridges.[10] These molecules circulate in the blood stream in a soluble form, but constantly assemble into insoluble ECM fibrils by a cell-dependant process.[1] Despite plasma and cellular fibronectin sharing many similarities, they are not identical in regards to their structure and function. Most of these differences result from variations in their primary structures, which is caused by alternative splicing in at least but not limited to, two distinct regions (ED and IIICS) of the pre-mRNA.[11]

The dimer state of fibronectin is formed by a covalent bond joining the tails (C-terminus) of the two monomers.[12] These 250 kDa monomers[13] comprise of three distinct protein fragments, each known as type I, II, and III repeats.[14] More specifically, cellular fibronectin is a miscellany of heterodimers of various subunits, which differ in their ED-A and ED-B segments.[3] For example, plasma fibronectin is often deficient in these ED segments.[5] Also, the cell attachment site of fibronectin is RGDS and is postulated to mediate the concentration of fibronectin in cells.[15]

As mentioned above, fibronectin consists of repeated units of the domains FNI, FNII and FNIII.[16] In particular, 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.[17]



Secondary and Tertiary Structure
Complex of ROBO1 Fn3-B2212A Fab and its interactions.[18]

The secondary structure of fibronectin is formed mainly by anti-parallel β-sheets. It is a very compact arrangement with a length that ranges between 14.2 and 16.8 nm and a width of 7.1 to 10.5 nm. The axial ratio is around 2:1. In particular, the secondary structure of fibronectin Type-III domain-containing protein (ROBO1) can be seen in the adjacent figure.


Fibronectin also has a complex tertiary structure whereby the domains are interconnected by flexible non-structured segments of the polypeptide chain.[10] These domains are not globular, but rather form long, thin and flexible strands.[19]

Isoforms

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.[20] Subsequently, it is acknowledged that changes to the structure of the fibronectin molecule ultimately influence the functional role it plays in that particular cell.[21]



Repeating Units
A three stranded anti-parallel β-sheet stabilised by a pair of interchain disulphide bonds.

FnI[22]

  • Module is 43 residues in length, each forming a β-hairpin.
  • Three stranded anti-parallel β-sheet.
  • Stabilised by a pair of interchain disulphide bonds (absolutely conserved).
  • Mediates human protein-to-protein and protein-to-oligosaccharide interactions.
  • Tandem modules (FnI2-FnI5) interact with FnIII modules and binding of heparin.
  • FNI4-FNI5 have the capacity to bind to fibrin. FnI modules near C-terminus also bind to fibrin.
  • FNI modules only found in vertebrates.


A five stranded module that is 49 residues in length, stabilised by 2 disulphide bonds.


FnII[22]

  • Module is 49 residues in length.
  • Five stranded molecule.
  • Stabilised by two disulphide bonds.
  • Only conserved structural element is the small 2-stranded β-sheet.
  • Only two modules occur as single tandem repeat.
  • FnII2 forms tight complex with FnII6.
  • FNII is confined to metazoans.




A seven stranded module with 90-100 residues contained within an all-β structure; two anti-parallel β-sheets of three or four strands form a hydrophobic core and does not require stabilising intrachain disulphide bonds.


FnIII[22]

  • Module with 90-100 residues contained within an all-β structure.
  • Module fold contains seven strands; two anti-parallel β-sheets of three or four strands form a hydrophobic core.
  • Does not require stabilising intrachain disulphide bonds.
  • Structurally plastic when under strain as the 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.










Assembly

Steps of fibronectin matrix assembly: initiation, unfolding and fibrillar assembly.[23]

The assembly of fibronectin molecules into a fibrillar network is cell mediated[4] and is a result of electrostatic forces that enable the joining of the head and tail of two dimers.[12] 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.[24] The beginning steps of this assembly includes the binding of these sites to surface receptors and ECM proteins, and is fundamental in forming this stable complex.[25] The three step process of this matrix assembly consists of initiation, unfolding, and lastly fibrillar assembly, which are illustrated in the neighbouring figure.[23]

Moreover, the state of the fibroblasts influences the structure of the fibronectin secreted. 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.[26]

Function

Fibronectin is an important molecule within cells due to the numerous roles that it adopts. Such roles are listed and explained below.


Forms of fibronectin playing different roles in wound healing processes.[23]

Wound Healing

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.[27] 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.[28]

This ECM glycoprotein is also observed in the early phases of wound healing. In particular, platelets are released in the first step of haemostasis when a wound occurs, causing a platelet plug to form. The resulting platelet to platelet bond 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.[29]

Furthermore, during the inflammatory phase, fibronectin is able to opsonise ECM debris. It acts as a non-immune opsonin for the debris' phagocytosis by fibroblasts, keratinocytes, and even macrophages under certain conditions. The fibronectin also acts as an enhancement for the phagocytosis of immune-opsonised particles by monocytes.[30]

Role in Tumours

Plasma fibronectin (pFn) is found to be upregulated in tumour cells, ultimately supporting tumor maintenance. Tumor cell adhesion to and invasion in fibrin when paired with fibronectin 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.[29] 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.[31]

Atherosclerosis

Plasma fibronectin is found to help as well as hinder atherosclerosis. It increases the number and size of atherosclerotic plaques that form with its deposition within lesions, however it also helps in the formation of a protective fibrous cap, which assists in preventing rupture of these plaques. In addition, it is important to note that plasma fibronectin, rather than the haemopoietic cell-driven fibronectin, is identified as being deposited at atherosclerosis-prone sites prior to the development of atherosclerotic lesions.[32]

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 haemostasis. 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.[33]

Examining Plasma Fibronectin in Haemostasis.
<mediaplayer>https://www.youtube.com/watch?v=3avSmLwgOp4</mediaplayer>

Role in Promoting and Inhibiting Cell Adhesion

Fibronectin is observed to increase the cell attachment of proteins to the walls of blood vessels.[34] 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 when there is a long incubation period. This inhibition is observed to be associated with the 75kDa cell-binding component of the fibronectin molecule.[6]

Fetal Fibronectin

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.[35]

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.[36]

Embryogenesis

Fibronectin is one specific ECM 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.[37]

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 the formation of the nodal pit.[38]

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 as a5 integrin is observed in the anterior neural ridge, the cranial neural fold, and in the neural crest cells. Moreover, fibronectin is 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.[39] 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 required for successful cardiac development. Moreover, integrins are crucial for effective fibronectin function. In cases where 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.[40]

Abnormalities/defects

The abnormalities presented by fibronectin are very vast and have a huge range of clinical and pathological presentations. These are caused by alterations in its production, which can result from excessive production of the glycoprotein as well as mutations in its gene expression.

Ehlers-Danlos Syndrome:
Ehlers-Danlos Syndrome causing Hyperelastic skin and increased flexibility.
Ehlers-Danlos Syndrome causing hypermobility of joint movement.

Similarly, human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organise fibronectin and similar proteins in the extracellular matrix. These mutated fibroblasts also 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 this glycoprotein and the integrin alpha5beta3.[41] The disease clinically manifests as fragility and hyperelasticity of the skin and joint laxity.[42]


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.[43]
Keloid:
A rare keloid on the third and fourth toes.

A study [44] compared the levels of fibronectin in normal tissue against wounds that developed keloid, an abnormality that results from an over-production of collagen and other ECM components. The keloid tissue presented a huge production of both intracellular and extracellular fibronectin (due to its mRNA increment), that had no abnormalities in its structure, process or 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. Overall, it can be seen that fibronectin plays a pivotal role in the development of keloids.

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, ED-A and ED-B are detectable in the angiogenesis of tumours and matrix remodelling. A study[45] showed that mice who were injected with anti-ED-A/B tend to have less metastasis, and that immunisation against ED-B diminished angiogenesis and tumour 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 suppressed metastasis. Subsequently the use of targeting vaccines as part of the usual therapy, or in replacement of monoclonal antibodies, is being considered.
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.[5] 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.[46]

Current Research

Therapeutics

Fibronectin can function as a molecular target for therapeutics of hepatocellular carcinoma. In particular, ROBO1, fibronectin Type-III domain-containing protein, is a target for specific therapeutics. A monoclonal antibody, called B2212A, binds to the third fibronectin domain of ROBO1, hence the results of the study suggest a possible direction towards the development of a new antibody drug for the treatment of hepatocellular carcinoma.[18]

Recent PubMed Articles

<pubmed limit=5>Fibronectin</pubmed>

Glossary

Term Meaning
Angiogenesis The growth of new blood vessels
Atherosclerosis The deposition of fats, cholesterol, and other fatty materials in and on the artery walls
ED-A Extra domain - A
ED-B Extra domain - B
Extracellular matrix A meshwork of materials that surrounds the outside of cells
Glycoprotein A specific class of proteins that consist of a carbohydrate attached to them
IIICS A domain of the primary gene transcript for fibronectin that is targeted for splicing
Opsonise Modifying a cell as to make it liable for phagocytosis
Primary Structure The linear sequence of a protein’s amino acids
RGDS Arg–Gly–Asp sequence of a fibronectin molecule
ROBO1 Fibronectin Type-III domain-containing protein
Secondary structure The three-dimensional arrangement of the segments of a protein
Tertiary structure The resulting three-dimensional geometry of a molecule due to folding and covalent interactions.

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