A proteomics study of the response of North Ronaldsay sheep to copper challenge
© Simpson et al; licensee BioMed Central Ltd. 2006
Received: 13 October 2006
Accepted: 27 December 2006
Published: 27 December 2006
The objective of this proteomics study was to identify proteins that changed expression as a result of copper challenge in the uniquely copper sensitive North Ronaldsay sheep and further, to compare those changes in expression with the more copper tolerant Cambridge breed. Such data gives us a proteome-centered perspective of the pathogenesis of copper-induced oxidative stress in this breed.
Many proteins respond to copper challenge, but this study focuses on those exhibiting a differential response between the two breeds, related to liver copper content. As copper accumulated in the tissue, the pattern of expression of several proteins was markedly different, in North Ronaldsay sheep as compared to the Cambridge breed.
The pattern of changes was consistent with the greatly enhanced susceptibility of North Ronaldsay sheep to copper-induced oxidative stress, focused on mitochondrial disturbance with consequent activation of hepatic stellate cells. The expression profiles were sufficiently complex that the response could not simply be explained as a hypersensitivity to copper in North Ronaldsay sheep.
Copper is an essential trace element and the cofactor of several enzymes involved in a wide variety of physiological processes. However by virtue of its ability to participate in single-electron transfer reactions, copper can also generate reactive oxygen species which can be highly damaging to cell membranes and biomolecules. For this reason copper homeostasis has evolved as a tightly regulated process with an array of transporter and chaperone proteins regulating the uptake and excretion of copper, preventing its free accumulation within cells. Interruption or modification of this homeostasis can cause disease. A group of clinically and pathologically indistinguishable copper-associated diseases occur in infancy and childhood and are named Indian childhood cirrhosis (ICC), idiopathic copper toxicosis (ICT) and endemic Tyrolean infantile cirrhosis (ETIC). These life-threatening disorders, characterised by a florid pericellular fibrosis and cirrhosis have been linked with exogenous copper and also a genetic predisposition in the Tyrol  and in N. Germany . ICC and ETIC have been attributed to excess copper intake from the use of brass or copper vessels to prepare infant feed  or well water with low pH . The aetiology of sporadic cases of ICT in Germany, implicated the absence or short duration of breast feeding and the substitution of formula milk made with water at pH < 6.5 contaminated with copper 3–26 mg/L (WHO requirements <2 mg/L).
The North Ronaldsay (NR), a primitive breed of sheep, has been identified as a possible model of these non-Wilsonian infant copper toxicoses . Sheep generally are intolerant of dietary copper excess due to their impaired ability to excrete copper in the bile, and liver copper accumulation with ensuing toxicity is well recognised. The NR breed manifest this species propensity in extreme form and are the most copper-sensitive mammals known. This ancient and isolated breed of sheep occupy an ecological niche on the foreshore of North Ronaldsay island in the Orkney archipelago, where they have been exposed to a copper-impoverished diet, mainly seaweed [Cu < 5 ppm], for so long that they have evolved mechanisms to maintain adequate copper reserves . In so doing they appear to have compromised their homeostatic mechanism, such that under copper-replete conditions they accumulate copper to excess with ensuing toxicity. NR sheep and their cross-breeds absorb more dietary copper than other breeds of domesticated sheep, proving that this trait is heritable . In addition to an ecogenetic aetiology, Ronaldsay copper toxicosis (RCT) has a pathomorphology distinct from copper poisoning in domesticated sheep , but similar to the infant copper hepatic toxicoses . Recent studies with artificially fed copper-supplemented North Ronaldsay lambs confirmed the unique status of this animal model with regard to the childhood disorders .
Genetic factors must ultimately underlie these differences in response to copper between different breeds of sheep, but at the metabolic level it is proteins that are responsible for its expression. An exploration of the effect of copper challenge on protein expression in the copper sensitive North Ronaldsay sheep compared with a more copper-tolerant breed, the Cambridge, would aid understanding of their respective pathophysiologies and contribute to understanding of copper toxicosis in humans. The study of Cambridge sheep has been completed and the pattern of biochemical changes was consistent with an early adaptive response to copper challenge, followed by an impaired ability to compensate for an increasing copper burden, initiating oxidative stress-induced injury in the prehaemolytic phase. These findings are reported in full elsewhere  and provide the basis from which to compare the response of North Ronaldsay sheep which are the main subject of this investigation. An evaluation of the pathological changes at the ultrastructural level in the two breeds of sheep has formed a parallel study .
Results and discussion
Liver copper and tissue microarrays
Differential protein expression
Identification of copper-responsive proteins in North Ronaldsay sheep. 2-D gels for each animal were run in triplicate and placed in analysis sets. Statistical analysis sets were created from two copper-challenged Cambridge (n = 6 gels) and North Ronaldsay (n = 6 gels) animals and statistically significant changes in protein expression were identified by Student's t-test at p ≤ 0.05.
1. Cathepsin D
2b Epoxide hydrolase
3. Ferritin light chain
7. Peroxiredoxin 3 (SP-22)
8. Plasma retinol binding protein
Identification of copper-responsive proteins in Cambridge sheep. 2-D gels for each animal were run in triplicate and placed in analysis sets. Statistical analysis sets were created from two copper-challenged Cambridge (n = 6 gels) and North Ronaldsay (n = 6 gels) animals and statistically significant changes in protein expression were identified by Student's t-test at p ≤ 0.05.
Fold change (%)
2a. Epoxide hydrolase
4. Heat shock protein Hsp 27
5. Cytosolic NADP+-dependent isocitrate dehydrogenase
6. Methionine adenosyl transferase
Methionine adenosyl transferase, identified by MALDI-ToF MS and peptide mass fingerprinting was more than 2-fold higher in abundance (p < 0.05) in the gels prepared from the Cambridge versus NR liver tissue however, spot quantitation in control versus copper challenged Cambridge sheep showed no copper-responsive increase in the expression of this protein, indicative of a breed difference in the expression of this protein. Conversely, in a comparison of control versus copper-challenged NR gels there was a 2-fold decrease in expression of the protein at high copper challenge. Methionine adenosyl transferase catalyses the formation of S-adenosylmethionine as part of the activated methyl cycle. Supplementation of ethanol fed rats with S-adenosylmethionine resulted in a 40–50% increase in the mitochondrial glutathione pool accompanied by mitochondrial protection . A decrease in the expression of methionine adenosyl transferase in NR sheep liver as a result of copper challenge may result in a decrease in the availability of glutathione to the mitochondria and thus contribute to the oxidative stress-induced mitochondrial damage observed in NR livers .
Several copper toxicoses in humans and animals are mutations of a specific carrier protein . This occurs in Wilson disease with WND (ATP7B) expressed in the liver, the related MNK (ATP7A) in Menkes disease, expressed in the intestine and the MURR1 (COMMD1) deletion expressed in the liver of Bedlington terriers affected with copper toxicosis .
In humans, endemic Tyrolean infantile cirrhosis is genetically distinct from Wilson disease  and Bedlington terrier copper toxicosis . Moreover, NR sheep do not to share the MURR1 deletion of the Bedlington terrier (Wijmenga, C. – unpublished data).
In humans it is increasingly recognized that differential expression of copper transporter proteins is time-dependent during the neonatal period. Copper absorption of humans at birth is high and reflects copper intake but declines soon after, in concert with the modulated expression of transporters Ctr1 and ATP7A . Also liver copper concentrations are considerably higher in the foetus and neonate compared with adults and plasma caeruloplasmin is subnormal . In the post natal period (3–6 m) there is an induction of biliary copper excretion and caeruloplasmin synthesis, presumably due to activation of the ATP7B (WND) gene. By comparison, in sheep foetal copper levels are lower than in adult sheep  and whilst there is no evidence of defective expression of WND (ATP7B), biliary copper excretion, but not caeruloplasmin synthesis, is impaired . Recent work has identified different isoforms of WND protein in sheep which may explain the anomaly and by extension the sensitivity of sheep as a species to copper poisoning .
North Ronaldsay sheep accumulate copper in their livers to ten times the extent of Cambridge sheep . This suggests that infantile copper importer mechanisms may be still operable having been selected by evolutionary pressure for a uniquely copper deficient environment. A similar maturational dysregulation of homeostatic mechanisms may also operate in the childhood condition although this must remain speculative.
Furthermore NR sheep are uniquely susceptible to copper resulting in a lower threshold of sensitivity and hence increased susceptibility to oxidative stress in which the mitochondria play a key role. Mitochondrial changes in the NR sheep have been contrasted with those in Wilson disease, Bedlington Terrier copper toxicosis and LEC rats in a companion paper . However, some superficial similarities exist between the pathology of the mitochondria in NR hepatocytes and those of the toxic milk mouse, a murine model of Wilson disease [32, 33] although the primary genetic defect in NR sheep has not been elucidated.
Interestingly, the yeast mitochondrion contains a dynamic pool of copper localised in the matrix as part of a low molecular weight anionic complex . Expansion of this copper pool by the addition of copper salts to the growth medium did not result in any respiratory defects . In contrast to the copper-challenged Cambridge sheep which showed minimal evidence of mitochondrial damage, ballooning and rupture of this organelle occurred in the NR sheep with an equivalent liver copper overload, which may suggest a failure to regulate this expandable copper pool resulting in the generation of reactive oxygen species and the pathological changes noted .
A comparison of differential protein expression in response to copper challenge between the two breeds of sheep, complemented by ultrastructural studies, has affirmed the exquisite sensitivity of NR sheep to Cu-induced oxidative stress and suggest that this may arise from within mitochondria. Secondary consequences derived from the oxidative stress contribute to the overall pathology. The NR sheep is a unique model of altered or arrested copper homeostasis that will be of considerable value in the ontology of copper metabolism and its pathology in human infants and animals.
Fourteen NR ewes aged 10 months were used in this study, of which six received a diet of hay (Cu 5 mg/kg dry weight) and 250 g/head/day of beet (Cu 5.4 mg/kg dry weight) over a six month period. The remaining animals were given the same diet except that the beet contained 15 mg/kg copper, a supplement of CuSO4 having been added. The copper-supplemented animals were killed in pairs at interval of 1, 2, 4 and 6 months after the commencement of the experiment, to achieve liver copper concentrations similar to those produced in Cambridge sheep . The non-supplemented animals were killed in pairs at 1, 2 and 6 months.
Nine Cambridge ewes were housed and fed for a period as described above except that six animals received copper-supplemented pellets containing (Cu 155 mg/kg) and three animals received the same copper un-supplemented diet containing less than 50 mg/kg copper. With the basal diet of hay each animal received 500 g/day pelleted feed.
Ethical statement: The animal experiments were carried out under licence granted by the ANIMAL (SCIENTIFIC PROCEDURES ACT) 1986.
Sample preparation and analysis of liver tissue by inductively-coupled plasma mass spectrometry (ICP-MS) has been described previously .
2-DE of soluble liver proteins
Preparation of liver supernatants and their resolution and visualisation on 2-D gels has been described previously .
Image analysis of 2-DE gels
Gels were scanned using an Epson 1680 pro flat bed scanner (266 dpi, 24-bit colour) and images were saved in an uncompressed TIFF file format. The gels were compared using the PD Quest 7.1 gel analysis software (Bio-Rad). Gels were normalised by reference to total intensity on the gel and replicate groups created from replicate gels (n = 3). Statistical analysis sets were created between two copper-challenged Cambridge and North Ronaldsay animals and statistically significant changes in proteins were identified by Student's t-test with a confidence level of 95%. Proteins were excised from the gels by the ProteomeWorks spot cutter (Bio-Rad). Excised gel plugs were robotically de-stained, digested with trypsin and spotted onto a MALDI target plate by a MassPREP Workstation (Waters, Manchester, UK).
Characterisation of protein spots by MALDI-MS
Peptide mass fingerprinting of protein digestion products was performed on a M@LDI R mass spectrometer (Waters, Manchester, UK). Protein databases were searched using the Mascot search engine . The monoisotopic masses of the tryptic peptides were compared to the Swiss-Prot and NCBI mammalian databases [36, 37] using a mass tolerance of 150–200 ppm, allowing for up to one missed tryptic cleavage, and with carbamidomethylation of cysteine residues as a fixed modification and oxidation of methionine as a variable modification.
Electrospray Ionisation Mass Spectrometry
Electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MSMS) were performed on a Waters Q-ToF Micro instrument, fitted with a nanospray source. The electrospray was created from a silver-coated glass capillary with a 10 μm orifice (Presearch, Hitchin, UK), held at a potential of +2000 V relative to the sample cone. Prior to ESI-MSMS peptides were separated by reversed phase HPLC on a Dionex Ultimate system. The system was fitted with a PepMap C18 column (LC Packings, Camberley, Surrey, UK), 15 cm × 75 μm, bead size 3 μm and pore size 100Å. Prior to separation, the sample digest (20–25 μL) was taken up into the injection loop of the Dionex Famos autosampler and desalted in-line using a Dionex Switchos apparatus, fitted with a 5 mm × 300 μm, C18 precolumn. The precolumn was initially equilibrated in 0.2%(v/v) formic acid (solvent A) at 30 μL/min. Peptides were then loaded and washed for 3 min at the same flow rate, after which, the trap and downstream PepMap column were developed with 90% acetonitrile/0.2% formic acid (solvent B) introduced as a linear gradient of 0–50% in 30 min at 0.2 μL/min. The column eluant was monitored for uv absorbance at 214 nm in an in-line flow cell prior to being introduced into the mass spectrometer. The initial quadropole analyser was set to allow the transmission of selected precursors into the gas cell, where they were fragmented by collision with argon. The masses of the resulting fragment ions were then measured by the ToF analyser. Selection of precursors and fragmentation energy were controlled automatically using the data-dependent acquisition facility within the MassLynx software. Precursor spectra were acquired between m/z 400 and 1500 at a scan/interscan speed of 2.4/0.1 s. Product ion spectra were acquired between m/z 100 and 2000 at a scan/interscan speed of 1.0/0.1 s. Raw product ion spectra were deconvoluted using the MaxEnt 3 alogorithm within the MassLynx software. The charge state of the parent peptide was determined from the isotope envelope in the precursor spectrum. Interpretation of product ion spectra and the determination of peptide sequences were facilitated by the PepSeq module within MassLynx. Peptide sequences were searched using Blast (Basic Local Alignment Search Tool) .
Immunohistochemical analysis of tissue microarrays
Tissue microarrays (24 × 2 mm cores) were prepared from formalin-fixed paraffin-embedded liver (dorsal and ventral lobes) using a Tissue Micro Array Builder (Abcam, Cambridge, UK). Ultrathin sections from the donor block were mounted on charged slides permitting the simultaneous screening of tissue from a number of animals with antibodies. The TMAs were de-waxed in xylene and re-hydrated by immersion in a series of alcohol, followed by water baths. Endogenous peroxide activity was blocked using the peroxidase solution provided with the Envision dual labelling kit (DakoCytomation, Cambridge, UK). The TMA slides were washed in Tris-buffered saline (TBS) pH 7.6 0.05% (v/v) Tween 20 before incubation with 1% (w/v) BSA in TBS pH 7.6 for 1 hr at room temperature. After removal of the blocking solution the TMAs were incubated with a 1:250 dilution (~200 μL/slide) of anti-metallothionein antibody (E9 monoclonal, DakoCytomation) in TBS pH 7.6 overnight at 4°C. TMA slides were then developed further using the Envision®+ Dual link system. A control slide was included in the experiment and was incubated in blocking solution overnight. The slides were given 3 × 5 min washes in TBS/0.05%(v/v)Tween then incubated with a peroxidase-labelled polymer conjugated to goat anti-mouse and goat anti-rabbit immunoglobulins included with the kit, for 30 min at room temperature. After a further 3 washes, as above, the TMAs were developed using the diaminobenzidine (DAB) buffered substrate provided with the kit. Colour development was stopped by immersing the slides in TBS-Tween containing 0.05%(w/v) azide, slides were counterstained with haematoxylin.
This work was supported by a research grant from the Wellcome Trust: 063730/Z/01/Z/GM/AC/AF. Grants from the BBSRC supported the purchase of instrumentation. We wish to thank Duncan Robertson for assistance and training on the Q-ToF mass spectrometer. Michael J. Loughran for the metal analysis, Gordon Ross for the electron microscopy and Sean Williams for preparing the donor TMA blocks.
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