Dialysis Related Amyloidosis (DRA) is a serious complication of long term haemodialysis. Amyloid deposits accumulate in the joints causing great pain & restricting mobility of sufferers. The main constituent of these amyloid deposits is fibrillar β2-microglobulin (β2m), although additional components are found which are thought to affect the formation and stability of the β2m fibrils. β2m fibrils formed in vitro under acidic conditions appear to have the same morphology as fibrils formed in vivo under pathological conditions when studied using electron microscopy and atomic force microscopy. However, the in vitro formed fibrils are not stable at neutral pH and quickly dissociate into monomeric and low oligomeric species. This raises the question as to why fibrils do not dissociate in vivo at physiological pH. In vivo serum amyloid P component (SAP) is always found associated with β2m fibrils and thought to stabilise the fibrils by preventing dissociation. Here we present evidence from pull-down assays that SAP binds tightly to acid produced β2m fibrils. The behaviour of the acid produced fibrils with and in the absence of SAP at neutral pH has being characterised using Thioflavin T fluorescence studies and has revealed that SAP does have a small stabilising effect on acid produced fibrils at the concentrations tested. The studies also imply that ionic strength as well as free protein concentration are important determining factors into the longevity of the fibrils at neutral pH. Studies of β2m in inclusion bodies prior to refolding demonstrate that they are not identical to β2m fibrils but that NMR studies do show areas of structural homogeneity with fibrils suggesting that the inclusion bodies may have structure and are not amorphous aggregate as previously thought. Soluble β2m has been assigned using solution-state NMR to identify regions of structural transition between soluble and fibrillar forms of β2m. Solid-state NMR spectra of acid produced fibrils have been acquired at both acidic and neutral pH and reveal that at a molecular level the fibrils are structurally homogenous, giving rise to spectra with site specific resolution. Sequential assignment of fibrillar β2m has therefore been possible using specific labelling techniques to overcome spectral crowding. To identify the interaction interface between β2m fibrils and SAP we have undertaken solid-state NMR studies of β2m with and without SAP bound. Comparing the chemical shifts from these studies has allowed us to identify that SAP is interacting with the side chain carboxylates of fibril aspartates and glutamates. Subsequent chemical modification of these carboxylates to remove their charge resulted in complete inhibition of SAP binding; confirming that they are essential for SAP binding to occur. However there is no strong interaction between monomeric β2m and SAP occurring demonstrating that a collective action of these acidic side chains is needed for binding to occur. Fibrils provide this in the form of acidic strips along the fibril axis brought about by the parallel and anti-parallel beta-strand structure of fibrils.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:548274 |
| Date | January 2011 |
| Creators | Taylor, Garrick F. |
| Contributors | Williamson, Philip |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/300060/ |
Page generated in 0.002 seconds