Research Opportunities

The Biological Mass Spectrometry Center currently has 2 PhD projects available through the Child Health Research Appeal Trust (CHRAT) Studentship scheme.

If you are interested in this project  project please apply through the CHRAT website.

Project 1.

'The search for the elusive deacylase'

The Determination of the Anabolism & Catabolism of Ceramide Trihexoside in Fabry Disease

Supervisors: Dr Wendy Heywood, Prof Simon Heales, Dr Simon Eaton & Dr Kevin Mills

Fabry disease is an X-linked lysosomal storage disorder caused by abnormalities in the GLA gene which results in a deficiency of α-galactosidase A [1]. This leads to a progressive intracellular accumulation of neutral glycosphingolipids primarily ceramide trihexoside (CTH) in the lysosome of the cell. Organs that are primarily affected by Fabry disease include the kidney and heart [2].  CTH and more recently lyso-CTH, a deacylated form of CTH are the only available biomarkers. However; each has its own limitations and the identification of new and improved biomarkers are essential to improve diagnosis, monitor treatment and disease progression in Fabry disease. The CTH molecule is comprised of three distinct parts, a tri-saccharide moiety, a sphingosine backbone and a fatty acid portion (see figure 1).  It is the fatty acid moiety that causes the heterogeneity of the CTH molecule observed in human urine.

Figure 1.

Figure 1 Chemical structure of CTH showing the three distinct portions of the molecule, the hexose portion (grey), the sphingosine backbone (red) and the variable fatty acid moiety (blue). 

Figure 2 shows a typical mass spectrum of the CTH isoforms seen in human urine; the most abundant species being the C24 and C24:1 isoforms followed by the C22, C20 and smaller amounts of C16-C18-CTH molecules

Figure 2

Figure 2 Mass spectrum of CTH isoforms in a Fabry patient urine

The reason for why there is heterogeneity in the fatty acid side chain, how they originate, the role and the toxicity of these differing versions of the same molecule is unknown. Lyso-CTH, the deacylated version of CTH, is almost undetectable using conventional analyses because it is present at a concentration 1000 x less that CTH levels [3]. Although there are 3 known deacylase enzymes present in the body, none of them are known to be able to deacylate CTH, as they can only act on the ceramide portion of the molecule after sugar moieties have been removed [4]. Using patient fibroblasts, we would like to search the subcellular components of the cell to find this 'elusive' enzyme.  The discovery of this enzyme would allow us to test drug targets and reduce the amounts of this extremely toxic chemical being produced and hence reduce the damage seen in Fabry disease.

To go about answering these questions, the biochemistry research group at ICH have developed novel methods for the synthesis of deuterated CTH analogues by chemical modification of the fatty acid side chain present on CTH [5]. Using these methods we have synthesized fully deuterated C6-C26 homologues of CTH that can only be distinguished from native CTH molecules using mass spectrometry as they are chemically and structurally identical. Therefore, these molecules, which the body cannot distinguish from native CTH, can be used as tracer molecules in cellular systems to monitor their transport, incorporation into particular subcellular organelles and more importantly, how they are metabolised. The experimental outline of the proposed project is illustrated in figure 3. 

Figure 4

Figure 3. Schematic outlining the proposed experimental workflow to investigate the catabolism and anabolism of CTH. Catabolism will be studied by adding deuterated CTH analogues to cells this will be compared with a fabry model with added alpha-galactosidase inhibitors. Cells will then be fractionated and analysed using MS based methods. To identify if and where they may a deacylase the cells will be fractioned first and CTH analogues added to the fractions. Anabolism of CTH will be studied by the addition of deuterated fatty acids.

By monitoring the intact deuterated CTH molecule using UPLC-MS/MS and the liberation of the deuterated fatty acids by GC-MS, we will be able to either confirm the fatty acid oxidation breakdown hypothesis or be able to identify the deacylase.

The project will involve using cardiac, kidney and endothelial cell lines to establish the normal metabolism of CTH in these cell types. In a dual approach we would like to create an artificial Fabry model of these cell lines using the alpha-galactosidase inhibitor 1-deoxynojirimycin, which would then be tested with deuterated CTH analogues. Cells will be sub-cellular fractionated (to yield nuclear, mitochondrial fraction, endoplasmic reticulum-Golgi and cytosolic fractions) by homogenisation, differential centrifugation and ultra-centrifugation to elucidate the catabolic pathway for CTH.  Fractionation will help identify the organelle/fraction that contains the potential un-identified deacylase By isolating the cellular compartment of the labelled CTH/lyso-CTH molecules we will be able to focus on the organelle that contains the elusive deacylase. In a reverse approach, we would also like to subcellular fractionate these cells into their respective organelles prior to incubation and then incubate them with our deuterated analogues of CTH/ lyso CTH.  Although these experiments will monitor the catabolism of CTH there is increasing evidence that the salvage mechanism of sphingolipid metabolism may also be involved in the production of lyso-CTH [4].  Therefore, we will also incubate cell lines with deuterated fatty acid molecules of various lengths and monitor their incorporation into CTH, thus monitoring both the anabolism and catabolism of CTH in the cell.

These targeted and hypothesis driven analyses can also be augmented with hypothesis generating omic analyses carried out in the same experiment and even on the same sample.  The purification of the sub-cellular organelles from the kidney, cardiomyocyte and endothelium cell culture / deuterated CTH incubation experiments will not only allow us to monitor the metabolism of CTH, but also allow us to monitor which of these organelles are more susceptible to CTH incorporation / uptake.  In addition, these metabolomic analyses will require precipitation of the protein content of the organelles. These protein fractions can then be used for proteomic analyses such as the mass spectrometry based method of label- free quantitation to study what effect that high levels of CTH/lyso-CTH have on protein expression and hence biological mechanisms in the cells of these different tissues.  In effect, giving insights into the disease mechanisms of Fabry disease which are still not fully understood.

By understanding the mechanism of the breakdown of CTH to lyso-CTH we will gain greater insight into the disease mechanisms involved in Fabry disease. The identification of the elusive deacylase that creates the highly toxic lyso-CTH, will allow the development of chemical inhibitors to target the enzyme. Finally, this work is extremely important from a perspective that enzyme replacement therapy does not cross the blood brain barrier and new treatments will be required.

Reference List

(1) Zarate YA, Hopkin RJ. Fabry's disease. Lancet 2008 Oct 18;372(9647):1427-35.

(2) Mehta A, West ML, Pintos-Morell G, Reisin R, Nicholls K, Figuera LE, et al. Therapeutic goals in the treatment of Fabry disease. Genet Med 2010 Nov;12(11):713-20.

(3) Auray-Blais C, Ntwari A, Clarke JT, Warnock DG, Oliveira JP, Young SP, et al. How well does urinary lyso-Gb3 function as a biomarker in Fabry disease? Clin Chim Acta 2010 Dec 14;411(23-24):1906-14.

(4) Mullen TD, Hannun YA, Obeid LM. Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 2012 Feb 1;441(3):789-802.

(5) Mills K, Johnson A, Winchester B. Synthesis of novel internal standards for the quantitative determination of plasma ceramide trihexoside in Fabry disease by tandem mass spectrometry. FEBS Lett 2002 Mar 27;515(1-3):171-6.

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