The sequence of cystatin C was first established more than twenty years ago and can be seen in figure 1.4. The protein is manufactured by the cell as pro-cystatin C with a mature 120 amino acid cystatin C chain linked to a 26 amino acid targeting sequence at the N- terminus which is essential for targeting to the endoplasmic reticulum (Paraoan et al., 2003).
In the normal course of processing this targeting sequence allows the protein to be
processed through the secretory pathway and is cleaved to leave just the mature protein to be secreted from the cell. Consequently the protein only forms its final shape upon the severing of this extra length of amino acids.
In humans it is translated from the CST3 gene which is located on chromosome 20
(Abrahamson et al., 1989; Saitoh et al., 1989) and consists of a series of five β-strands with a large α-helix lying across the surface of the sheet and a short α-helix (Janowski et al. 2001) (figure 1.5).
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Figure 1.4 DNA sequence and primary protein structure of cystatin C. Red text is the precursor sequence which is cleaved during processing to produce the mature protein, highlighted yellow and pink are link regions, highlighted cyan is the alpha chain, highlighted green are the beta chains. The point mutation in the precursor leading to variant B form cystatin C is indicated. Sequence information from Abrahamson et al. (1986) and primary protein structure from Janowski et al. (2001).
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Cystatin C has been demonstrated to be capable of dimerisation. More specifically it dimerises due to the process of three-dimensional domain swapping (Janowski et al., 2001) in which a hinge region allows for partial unfolding of the protein and combining with similarly unfolded proteins (Jaskólski, 2001). It has been argued that domain swapping in itself is enough for the formation of oligomers (Liu et al., 1998) however this process is known to be effected by other factors. In the case of the amyloid-associated L68Q mutant cystatin C (highlighted in Figure 1.4) the substitution results in a form of cystatin C that has a much greater tendency to dimerise and form aggregates in a manner that is temperature dependent (Abrahamson & Grubb, 1994). Further, it has been demonstrated that the tendency of this disease-causing form of the protein to form amyloid fibrils can be inhibited by preventing domain swapping (Nilsson et al., 2004). In short this provides strong evidence that the structure of cystatin C is crucial to its capacity for aggregation and ability to cause aggregation-related disease.
Figure 1.5 Secondary and tertiary structure of cystatin C protein (reproduced from Janowski et al. 2001). It consists of a series of antiparallel β-sheets (blue) twisted around the large α-helix (red/yellow). The active site of the mature protein is circled in red.
38 1.4.2 Mechanism of action and function of cystatin C
Cystatin C is an active cysteine protease inhibitor found extensively throughout the body. Abrahamson et al. (1990) found it to be expressed in every human tissue examined ranging from the kidney to the lung to the placenta. Further it is found in almost all bodily fluids with the highest level being seen in seminal plasma but high levels also in the cerebrospinal fluid and blood plasma (Abrhamson et al., 1986).
As a member of the cystatin type 2 gene family, cystatin C is well established as an extracellular protease inhibitor. Consequently the protein is typically produced within the cell and then specifically passed to the secretory pathway (as directed by the signal targeting sequence) where it is processed, packaged and secreted to the extracellular environment. The essential role of the signal sequence in targeting cystatin C to the secretory pathway was demonstrated experimentally by Paraoan et al. (2003); loss of this signal sequence led to the protein no longer being targeted to the secretory pathway and instead presenting a uniform fluorescence throughout the cell, indicating it is diffused throughout the cytoplasm and nucleus. This clearly shows that the loss of the signal sequence results in the loss of protein targeting. The combination of its ubiquitous expression and its secretion means it is thus a very important regulator of extracellular protease activity.
Cystatin C is believed to have a variety of functions related to the regulation of levels of a number of different proteins in various bodily fluids and is implicated in several diseases and processes. Furthermore it has been used extensively as a marker for kidney dysfunction and there is a great deal of literature available about the use of it as a biomarker. Given its extracellular nature and abundance, cystatin C is likely to be significantly involved in
homeostasis and balance of proteins in extracellular fluid and the modelling of extracellular membranes and structures.
In particular Cystatin C is a potent inhibitor of a number of lysosomal cathepsins (B, H, L and S) and papain (Turk & Bode, 1991; Barrett et al., 1984). Especially cathepsins B, which cystatin C inhibits far more strongly than other cystatins (Barrett et al., 1984); despite the similarities in sequence between the proteins.
Of further note is that cystatin C is a tight-binding but reversible inhibitory protein. It acts through the two hairpin loops and N terminus as seen in figure 1.5 and fits into the active
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site of papain (for example) (Bode et al., 1988). The binding mechanism is functionally similar to how small serine inhibitors work, specifically by binding at the protein active site thus blocking it from use. However it is worth noting that the mechanism itself is quite different to the mechanism typically observed for small serine inhibitors.
Specifically in small serine inhibitors the inhibitor binds to the active site of the enzyme in a manner much like that of a substrate, with the blocking effect being a direct blockage of the active site in a “lock and key” type way (Laskowski Jr. & Kato, 1980). For cystatin C,
however, the binding sites are dissimilar to a bound substrate, yet it still binds to the active site (Bode et al., 1988). Note that this original model was based upon chicken cystatin, however later experiments demonstrated that this model for binding was also applicable to human cystatin C (Lindahl et al., 1992).
Therefore, to inhibit cysteine proteases cystatin C does not impersonate a substrate but does block the active site itself, preventing the binding of a substrate and therefore
blocking the activity of the protease. Specifically the N-terminal region binds to the protein.