About Follistatin

About Follistatin

Follistatin as a therapeutic

One of the advantages of follistatin as a therapeutic for inflammatory and fibrotic disorders is that it is a naturally occurring glycoprotein expressed by many cells in the body. Its primary function is to bind to and neutralize activins. Activins are a critical component of the innate immune response and in the development of fibrosis. Numerous studies have shown activin levels increase in the serum and various tissues in acute and chronic inflammatory diseases. Activins are also known to stimulate fibrosis and tissue repair and also mediate the fibrotic actions of other key growth factors. Follistatin therefore offers great potential for modulating inflammatory processes and preventing or resolving accompanying fibrosis.

Structure and biochemistry of follistatin

The follistatin gene is found on the long arm of human chromosome 5 (5q11.2). It is a highly conserved molecule in evolution with a follistatin-like gene sequence found in sponges. In fact, there is 98% identity between human follistatin and mouse follistatin, indicating its fundamental importance in biology.

The follistatin gene consists of six exons (from which mature proteins are built), resulting in a protein that has the following elements as shown diagrammatically below:

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FIGURE 1 The basic structure of follistatin consists of an ‘N-terminal’ domain (ND), followed by three follistatin domains (FSD1, FSD2 and FSD3). Through alternative splicing of Exon 6, the two major forms of follistatin are follistatin 288 and follistatin 315, the latter containing Exon 6. A heparin binding sequence (HBS) is located within FSD1.

Each follistatin molecule has three similar domains, known as follistatin domains (FSD), varying individually at the amino acid level but having the same structural features. Some other proteins that are not closely related to follistatin also have follistatin domains in their structure. Before the first follistatin domain, there is another domain known as the ‘N-terminal’ domain (ND). The follistatin gene undergoes what is known as an ‘alternative splicing’ event, where some follistatins are made with all six exons and some are made with only five, resulting in a shorter protein. These two major forms of follistatins are known by the number of amino acids they have, and are commonly known as FS315 and FS288.

Two additional features of follistatin structure are important for its biochemical properties. In the first FSD is a sequence of amino acids that can bind to (has affinity for) the surfaces of cells – this sequence is known as a heparin binding sequence (HBS). This property is a key element in follistatin’s action and is outlined more fully below. Follistatin is also a glycoprotein and contains a type of amino acid in its structure, called asparagine, where sugar groups called oligosaccharides are linked. Follistatin has a number of these amino acids in its sequence and so has various types of oligosaccharides bound to its structure. This glycoslyated nature of follistatin affords increased stability and circulating half-life of follistatin in vivo.

How does follistatin work as a therapeutic?

Follistatin was originally discovered in the 1980’s and the first scientific paper describing follistatin was published in 1987. It was first known as FSH suppressing protein, as the original description was based on its property of inhibiting release of the reproductive hormone, follicle-stimulating hormone (FSH), from the pituitary gland.

We now know that follistatin’s primary function results from its ability to bind and block the effects of the growth factor, activin. Activin is a key member of the transforming growth factor beta (TGFß) superfamily of proteins.

When a molecule of follistatin meets a molecule of activin, this results in an almost irreversible binding event that prevents activin from binding to its specific receptors and causing gene activation. The activin molecule is a dimer comprising two identical or similar ß subunits. Two molecules of follistatin are required to fully neutralize an activin molecule, as shown in the schematic below:

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FIGURE 2 Free activin molecules are able to bind to their cellular receptors and initiate cell events such as inflammation and fibrosis. In the neutralization pathway, two molecules of follistatin bind to activin with high affinity and prevent activin from binding to its receptors.

In addition to activin, follistatin also binds with lower affinity to some other members of the TGFß superfamily, including BMPs 2, 4, 5, 6, 7, 11 and 15, myostatin (GDF8) and GDF9. Follistatin does not bind to TGFß1 or TGFß2, but can bind to TGFß3. The anti-cachexia property of follistatin conferred through neutralization of myostatin potentially offers significant therapeutic benefits to patients suffering from muscle wastage accompanying chronic diseases.

As previously mentioned, follistatin contains a heparin binding sequence (HBS) that enables follistatin to bind to proteoglycans on cell surfaces. The shorter form of follistatin, FS288, is known for this property of binding to cells and is thought of as a local activin regulator within a particular cell or tissue environment. The longer form of follistatin, FS315, has a tail that masks the HBS when the molecule is in an unbound state. When FS315 is first made, it does not bind to cell surfaces and is free to circulate in the bloodstream. But after binding to activin, the tail region unmasks the HBS and the activin-FS315 complex can then bind to cell surface proteoglycans, as shown in the diagram below:

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FIGURE 3 Free FS288 (left-hand path) is able to bind cell surfaces through its unmasked heparin binding sequence or HBS. It is therefore a ‘local’ regulator of activin and binding of an activin molecule will trigger removal by a lysosomal breakdown pathway. In contrast, FS315 (right-hand path) is the ‘circulating’ form of follistatin. Upon binding to activin, its HBS is unmasked enabling the FS315-activin complex to bind to cell surface proteoglycans triggering removal by a lysosomal breakdown pathway.

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