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A new enabling proteomics methodology to investigate membrane associated proteins from
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parasitic nematodes: case study using ivermectin resistant and ivermectin susceptible isolates of
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Caenorhabditis elegans and Haemonchus contortus.
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Elizabeth H. Harta*, Peter M. Brophya , Mark Prescottb , David J. Bartleyc, Basil T. Wolfa and Joanne
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V. Hamiltona.
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a
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Aberystwyth University, SY23 3DA, UK
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b
Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building,
Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown Street, Liverpool,
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L69 7ZB, UK
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c
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EH26 OPZ, UK,
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*Corresponding author address Institute of Biological, Environmental and Rural Sciences (IBERS),
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Edward Llwyd Building, Aberystwyth University, SY23 3DA, UK. Tel: +44 (0)1970 622373 Fax: +44
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(0)1970 622350 email: elh18@aber.ac.uk
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Correspondence address for proof as above
Moredun Research Institute, Pentlands science park, Bush Loan, Penicuik, Midlothian, Scotland,
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Abstract
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The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of
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AR management is essential for effective and sustainable control of parasitic helminth worms in
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livestock. Current tests to measure AR are time consuming and can be technically problematic, gold
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standard diagnostics are therefore urgently required to assist in combatting the threat from drug
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resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in
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the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop
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and be measurable in the target associated proteins present in the parasite membrane. The model
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nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein
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expression differences, between IVM resistant and IVM susceptible isolates in the presence and
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absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton x100
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and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane
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proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were
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then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a
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pathogenic blood feeding parasitic nematode which is of global importance in livestock health,
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welfare and productivity. We have demonstrated the successful resolution of membrane associated
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proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the
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zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.
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Key Words
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Membrane associated proteins, Caenorhabditis elegans, 2DE optimisation, sub-proteome analysis,
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Haemonchus contortus.
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1. Introduction
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Anthelmintics have provided effective control against parasitic nematodes in the farming
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industry for over fifty years. However, these gains are compromised by resistance to the traditional
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broad-spectrum anthelmintics; benzimidazoles (BZs), levamisoles (LEV), and macrocyclic lactones.
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More recently two new classes of anthelmintics have been introduced; the amino-acetonitrile
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derivatives (AADs) in 2010 (Kaminsky et al., 2008) and spiroindoles (SI) in 2012 (Little et al., 2010).
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The lack of reversion to susceptibility following long-term drug use by the biological process of co-
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adaptation makes the development of early detection tests increasingly important (Borgsteede and
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Duyn, 1989). The development of molecular based tests is on-going and predominantly focused on
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identifying mutations in anthelmintic membrane protein primary targets (Driscoll et al., 1989, Beech
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et al., 1994, Cleland 1996, Sangster 1996, Dent et al., 1997, Flemming et al., 1997, Xu, et al., 1998,
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Dent, et al., 2000, Köhler, 2001, Riou, et al., 2005, von Samson-Himmelstjerna and Blackhall 2005,
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Lespine et al., 2006). However, as proteins act in complexes and not in isolation (Reid et al., 2010),
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resistance might be occurring and be measurable in target associated proteins in the parasite
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membrane. Global proteomics on anthelmintic sensitive and resistant parasite isolates has the
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potential to systemically reveal resistant sequences (Morgan et al., 2006). In addition, protein level
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defence systems such as phase III toxin sequestration and toxin pumping processes that are also
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potentially involved in the development of anthelmintic resistance (Brophy et al., 2012) are present
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in the membrane fraction of cellular components (Xu et al 1998; Bourguinat et al., 2007; Pritchard
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and Roulett 2007).
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We hypothesise that understanding potential sources of resistance (from primary target via
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associated targets to detoxification pathways) is key to developing a field based test and to sustain
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the working life of new anthelmintic classes, such as the AADs (Kaminsky et al., 2008). This study is
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focused on ivermectin (IVM), as it remains a major global anthelmintic (Crump and Omura, 2011).
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Clearly, as IVM is hydrophobic the binding site on the parasite receptor will be close to the lipid
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membrane bilayer (Pritchard and Roulett, 2007), resulting in conformational changes of the
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glutamate gated chloride channels (GluCl) (Hibbs and Gouaux, 2011). Therefore, IVM resistance may
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indeed be primarily selected for by mutations in hydrophobic regions such as potential target GluCl
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and gamma-aminobutyric acid (GABA) receptors (Sangster, 1996). Polymorphisms in such channel
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subunits have been shown to be analogous between H. contortus drug resistant isolates and C.
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elegans (McCavera et al., 2007). Despite differences in ecological niches between species (e.g. free-
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living compared to parasitic life cycle stages) there appears very little divergence with respect to
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morphology, genome structure and developmental life cycle (Wood, 1988).
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Proteins from these membrane preparations are challenging to separate due to the high
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lipid content, therefore extraction and fractionation steps to reduce sample complexity are
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important (Ephritikhine et al., 2004, Kota and Goshe, 2011). Membrane proteins are notoriously
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difficult to represent in Two Dimensional Gel Electrophoresis (2DE) due to their hydrophobic nature
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and the extent of solubilisation is highly dependent on the detergent used and the intrinsic
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properties of the proteins of interest (Shaw and Riederer, 2003, Di Ciero et al., 2004). Additional
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challenges for membrane and membrane-associated proteomics are posed by low protein
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abundance, protein degradation, detergent incompatibility and protein precipitation (Ephritikine et
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al., 2004; Komatsu, 2007). For optimal representation and resolution of protein spots the
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solubilisation of the hydrophobic region of membrane associated proteins is essential, making the
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protein extraction step the most important part of the process (Martins et al., 2007). Despite these
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various obstacles 2DE protein arrays remain an effective tool (Santoni et al., 2000, Shaw and
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Riederer, 2003). Although much simpler, the use of 1 Dimensional Sodium Dodecyl Sulphate–
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Polyacrylamide Gel Electrophoresis (SDS-PAGE or 1DE) cannot account for dynamic changes in
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proteins or post-translational modifications (PTMs) (Pionneau et al., 2005). Also, 1DE alone is
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insufficient for comprehensive membrane-associated proteomics, as visual resolution is limited
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(Burre, et al., 2009) and identification of all proteins present in a band using mass spectrometry (MS)
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is difficult as soluble proteins often mask the membrane proteins (Kota and Goshe, 2011). 2DE
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therefore provides a useful tool for both inter-and intra-species proteome analysis via changes in
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protein expression or PTMs (Thelen and Peck, 2007) and the species need not be fully genome
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sequenced.
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The use of surfactants to re-solubilise membrane related proteins has been investigated by
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various authors (Di Ciero et al., 2004, Zhang et al., 2005, Zoubi-Hasona et al., 2005, Layton et al.,
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2012). SDS is traditionally used for protein solubilisation, but produces poor solubilisation for
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membrane associated proteins and results in a loss of electrostatic repulsive effect brought about by
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the polar head group (Luche et al., 2003). The incorporation of a washing procedure prior to
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solubilisation with the addition of either salts or sodium carbonate or detergent partitioning has
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been shown to reduce contamination from soluble proteins (Pionneau et al., 2005). Common
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surfactants used include non-ionic and zwitterionic surfactants, which in the correct concentration
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solubilise the proteins without leading to isoelectric focusing (IEF) interference. For example,3-[(3-
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Cholamidopropyl)dimethylammonio]-1 propanesulfonate (CHAPS is a commonly used detergent
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and in combination with compounds consisting of zwitterionic amphiphilic surfactants such as
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sulfobetaine 3-10 (SB 3-10) and amidosulfobetaine-14 (ASB-14) has shown to lead to a better
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representation of proteins (Shaw and Riederer, 2003; Martins et al., 2007).
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This study demonstrates the development and optimisation of sub-proteomic methods for
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the solubilisation and representation of membrane associated proteins via 2DE in IVM-resistant and
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IVM-susceptible isolates of the model nematode, C. elegans, and a parasitic counterpart H.
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contortus.
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2. Materials and Methods
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2.1 General
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Chemicals used were of analytical grade from either Fisher (Loughborough, Leicestershire, UK) or
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Sigma-Aldrich (Gillingham, Dorset, UK) unless otherwise stated and all solvents used were of high
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performance liquid chromatography (HPLC) grade for mass spectrometry analysis. Where
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appropriate, solutions were autoclaved for 15 min at 15 KPa at 121oC.
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2.2 Culture of Caenorhabditis elegans
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Isolates of C. elegans (Caenorhabditis Genetic centre, USA) were maintained on nutrient growth
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medium agar 9cm petri dishes at 20oC according to the methods by Brenner (1974). Strains used
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were the wild-type, IVM susceptible (Bristol N2 strain, Brenner 1974), nematodes with moderate
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resistance to IVM (DA1302; avr-14 (ad1302) I; avr-15 (ad1051)V, Dent et al., 2000) and nematodes
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highly resistant to IVM (DA1316; avr-14 (ad1302) I; avr-15 (ad1051)glc-1 (pk54), Dent et al., 2000).
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Plated C. elegans stocks (5-8 plates) were transferred for monoxenic liquid culture of mixed and L4
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life stages (Brenner, 1974).
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2.3 Exposure of C.elegans to drug challenge conditions
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All three strains of C. elegans were cultured to the L4 developmental stage for 2DE analysis. Median
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lethal concentration assays (LC50 assays) were conducted on the harvested L4 stage to determine
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the minimal exposure level of each strain to IVM. Ivermectin standards were prepared as described
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in Prieto et al., (2003). The resulting dose of IVM added to the culture media for each strain was
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calculated as 20% of the LC50 value for each strain, to expose the worms to substantial drug
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pressure without causing mortality.
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2.4. Parasite collection
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All animals in this experiment were used under license in accordance with local legislation (Animals
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Scientific Procedures Act (ASPA) 1986. HMSO, London). Six, parasite free wether lambs in total were
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randomly selected. Three lambs were randomly allocated to experimental treatment 1, where lambs
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were infected with 10,000 H. contortus L3. These were designated ISE isolates (MHco3 isolate
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susceptible to all main classes of anthelmintic, originally derived from the MHco1 (MOSI) isolate
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maintained at the Moredun Research Institute) (Roos et al., 1990, Redman et al., 2008). The
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remaining three randomly selected lambs were allocated to experimental treatment 2, where lambs
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were infected with 10,000 CAVR H. contortus L3 (Chiswick avermectin resistant strain (MHco10),
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originally isolated from the field in Australia, Le jambre, 1993, Redman et al., 2008). To prevent cross
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infection lambs were individually housed with spatial separation between treatments. After 28 days
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post infection worms were harvested following euthanasia from the abomasum by washing with
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approximately 500 ml warm (39oC) physiological saline solution (0.85% NaCl w/v) and transferred
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into a 50 ml centrifuge tube containing warm physiological saline solution. Approximately 100 adult
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female worms of each isolate were selected and transferred into cryovials containing 500 l of
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potassium phosphate buffer (pH 7.4), 0.1% Triton X-100 and protease inhibitor cocktail (Roche).
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Worms were snap frozen in liquid nitrogen and stored at -80oC prior to analysis.
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2.5. Solubilisation of membrane associated proteins
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Whole worm samples for each biological replicate (N=3) were homogenised by bead beating in
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potassium phosphate buffer (KHPO4) buffer (pH 7.4) in 30s bursts followed by 1 min on ice, which
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was repeated three times. The membrane fractions were then separated from the samples by ultra-
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centrifugation at 100,000 x g for 30 mins. Membrane fractions were enriched in sodium carbonate
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(NaCO3 )buffer (0.1 M NaCO3 pH 11, 10 mM EDTA, 20 mM DTT plus protease inhibitor cocktail) for 1
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h at 4oC (Everburg et al., 2006). Following re-solubilisation samples were centrifuged at 13,000 x g
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for 30 mins at 4oC and the supernatant removed. The remaining pellet was re-suspended again in
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NaCO3 buffer and incubated at 4oC for 30 mins. This process was repeated for a total of three washes
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before re-suspension of the pellet in solubilisation buffer (4% SDS in 20 mM potassium phosphate
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(pH 7.4) plus protease inhibitor cocktail and heated to 95oC for 5 mins.
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Following initial solubilisation samples were centrifuged for 15 mins at 13,000 x g at 4oC
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before precipitation in 20% trichloroacetic acid (TCA) in acetone. The resulting pellet was re-
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suspended in one of the isoelectric focusing (IEF) buffers listed in Table 1 to assess optimum
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conditions for the solubilisation of membrane associated proteins in the second dimension (Fig. 1).
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Protein concentrations were determined using the reagent compatible detergent compatible (RCDC)
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protein assay (Biorad, UK).
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Final membrane associated samples were solubilised at room temperature in IEF buffer 4
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(Table 1) for 1 h before a total of 125 µl containing 50 µg of protein were actively rehydrated and
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focused on 7 cm linear pH 5-8 IPG strips at 20oC and then focussed between 8 -15 Vh using the
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Protean IEF cell (BioRad, UK). Equilibration of each IPG strip was conducted for 12 mins in 2.5 ml
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equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M Urea, 30 % Glycerol v/v and 2 % SDS w/v, with the
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presence of Dithiothreitol (DTT) (Melford, U.K.) at 10 mg/ml followed by a second equilibration with
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Iodoacetamide (IAA) (Sigma, UK) at 25 mg/ml). Proteins were separated in the second dimension
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using the Mini protean system (BioRad, UK) for 25 mins at 200V using 12.5% T, 3.3% C
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polyacrylamide gels. Gels were visualised via Coomassie blue staining (PhastGel Blue R, Amersham
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Biosciences, U.K.) and images captured using a GS-800 calibrated densitometer (Biorad, UK) and
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analysed using Progenesis PG220 v.2006 (Nonlinear dynamics, UK). Analysis was performed on 3
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biological replicates using normalised spot volumes to distinguish between a +/- 2 fold change in
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protein regulation. Protein expression changes were further analysed using a two way ANOVA (SAS,
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version 9.1.3). Spots were excised followed by trypsin digest (modified trypsin sequencing grade,
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Roche, UK).
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2.6 Analysis of peptides
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Gel spots were manually excised. Coomassie stained gel plugs were destained in 50% (v/v)
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Acetonitrile (ACN) and 50 mM (NH4)HCO3 at 4oC overnight until completely de-stained. Gel plugs
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were dehydrated in 100% ACN until opaque by incubating at 37oC for approximately 45 mins and
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rehydrated with 50 mM (NH4)HCO3 and 10 ng/µl trypsin (Roche, UK; prepared according to
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manufacturer’s instructions) for 45 mins at 4oC. These were incubated at 37oC overnight in order to
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digest the proteins. Digested proteins were eluted by the addition of 60% (v/v) (NH4)HCO3 and 1%
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Trifluoroacetic acid (TFA) and placed in a sonicator bath for 5 mins in 30 sec bursts. The supernatants
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were collected by centrifugation at 7,000 x g for 1 min. This was repeated for a second time and the
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supernatants pooled and vacuum dehydrated using a Maxi dry plus vacuum centrifuge (Hete-Holten
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A/S, Allerød, Denmark). Peptides were re-suspended prior to being sent for mass spectrometry
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analysis in 10 µl 1% (v/v) formic acid.
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Samples were analysed using Liquid Chromatography tandem mass spectrometry (MS/MS) using a
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LC Packings Ultimate nano-HPLC System. Sample injection was conducted with an LC Packings Famos
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auto-sampler and the loading solvent was 0.1% formic acid. The pre-column used was a LC Packings
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C18 PepMap 100, 5 mm, 100 A and the nano HPLC column was a LC Packings PepMap C18, 3 mm,
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100 A. The solvent system was: solvent A (2% ACN with 0.1% formic acid), and solvent B (80% ACN
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with 0.1% formic acid). The LC flow rate was 0.2 µl/min with a gradient using 5% solvent A to 100%
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solvent B in 1 h. The HPLC eluent was sprayed into the nano-ES source of a Waters Q-TOFµMS via a
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New Objective Pico-Tip emitter. The MS was operated in positive ion mode and multiply charged
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ions were detected using a data–directed MS/MS experiment. Collision induced dissociation (CID)
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MS/MS mass spectra were recorded over the mass range m/z 80-1400 Da with scan time 1 s.
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MassLynx v 3.5 (Waters, UK) Fragmentation spectra was combined and smoothed (using the Savitzky
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Golay method), background subtracted and deconvoluted using Max Ent 3 software (Mass Lynx v
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3.5). Peptide sequencing was conducted automatically using peptide sequencing (Mass Lynx v 3.5).
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An intensity threshold was set as 1 Da and fragment mass tolerance of 0.1 Da. Protein modifications
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considered were carboxyamidomethylcysteine, cysteine acrylamide and methionine sulphoxide.
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Trypsin was specified as the enzyme choice for protein digestion. Peptide sequences generated were
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used to search against the metazoans of the NCBI non-redundant database (www.ncbi.nlm.nih.gov/)
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using Protein BLAST (Altshul et al., 1990). Significant matches were determined for BLAST matches
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with an e value <1.00.
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3. Results
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3.1 Protein enrichment and solubilisation of membrane associated proteins of C. elegans.
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Sodium carbonate based washing of the associated membrane fraction of C. elegans reduced the
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diversity within the protein profile as evident from the 1D SDS-PAGE (Fig. 1, supplementary data).
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Average gels were produced for each of the IEF buffers listed in table 1 (Fig. 1). IEF buffers 3 and 4
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(Table 1, Fig. 1) were shown to produce the most abundant array of proteins, with 57 and 66 spots
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respectively compared to the remaining buffers averaging 10, 32, 31 and 39 spots for buffers 1, 2, 5,
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and 6. Therefore IEF buffers 3 and 4 were investigated further. Membrane associated proteins were
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initially separated across a pH of 3-10, however this pH range focused C. elegans proteins centrally
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on the broad gel format therefore, a pH range 5-8 was used to further resolve this subset of proteins
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(Fig. 2, supplementary data). Although similar good resolution and abundance of proteins were
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achieved using both IEF buffer 3 and 4; IEF buffer 4 was selected for the remainder of the study, as
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approximately 10 more protein spots were visualised using the ASB-14 and CHAPS combination (Fig.
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1, Table 1).
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3.2. Hydrophobicity plots for putatively identified associated membrane proteins.
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To increase confidence that proteins putatively identified by ESI MS/MS were membrane associated
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proteins, sequences were analysed for the presence of hydrophobic regions (Kyte and Doolittle,
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1982) using the bioinformatic Mobyle portal (Expasy). Proteins examined demonstrated regions of
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their amino acid sequence > a value of 0 and other regions where this value exceeded 1 (data not
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shown). This suggests the presence of hydrophobic regions within these proteins and their potential
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to interact with the cellular membrane.
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3.3. Comparison of membrane associated proteins for C. elegans isolates.
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C. elegans isolates were cultured separately with and without the presence of sub-lethal
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concentrations of IVM in the culture media (section 2.3). Three biological replicate gels were
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produced for each C. elegans isolate (section 2.2) in the presence and absence of drug (Fig. 2 and 3).
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The total number of protein spots present on each average sub-proteome gel for C. elegans isolates
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were 18, 18, 20 respectively for gels A-C (Fig. 2) and 23, 23, 25 respectively for gels A-C (Fig. 5).
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Despite various problems in the production of membrane associated gels, these methods produced
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similar profiles of replicate gels with a mean of over 70% matching between biological replicates.
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Putative identifications of all but two excised spots, demonstrated the presence of membrane
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associated proteins on the 2D gels, validating the potential of this methodology to purify and resolve
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relatively hydrophobic proteins for further analysis (Fig. 4, Table 3).
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3.4. H. contortus membrane associated proteins profiles.
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H. contortus samples were prepared following the novel associated membrane protocol described
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above which was optimised using C. elegans samples (Fig. 2 and 3, sections 2.5 and 3.3). However,
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minor modifications were required for representation of the parasitic samples. This included an
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extra centrifugation step before ultracentrifugation, where after bead beating, samples were
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centrifuged at 13, 000 x g for 30 mins in order to remove excess insoluble material such as residual
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abomasal debris. H. contortus membrane gels exhibited profiles similar to that of C. elegans
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membrane protein arrays (Fig. 5); for example, spot number 2 (Fig 2B) and spot number 11 (Fig. 2C)
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has approximately the same location (isoelectric point (pI) and molecular weight (MW)) on the gel as
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spots 2 and 8 (Fig. 5). Protein spots were resolved using this novel 2DE protocol and differences in
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average expression levels were observed between resistant and susceptible H. contortus isolates
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(Fig. 5). Putative protein identifications were determined for H. contortus resistant and susceptible
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proteomes (only spots with hits to the database are shown, Table 4) with proteins confirmed as
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potential membrane associated proteins (Fig.6 , Table 4), endorsing the new membrane associated
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assay protocol.
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4. Discussion
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2DE methodologies are well established for the separation of soluble nematode proteins. In this
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study we report for the first time the optimisation of the separation and visualisation of membrane
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associated proteins for 2DE from both a model (C. elegans) and parasitic (H. contortus) nematode
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species. In this new approach we have established a protocol for the successful solubilisation and
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resolution of associated membrane proteins from a parasitic nematode of global veterinary
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importance. Subsequently, a combination of ASB-14 and CHAPS solutions was shown to be the most
277
successful combination to resolve membrane associated proteins in this study (Fig. 1). ASB-14 was
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previously used for membrane solubilisation in several other studies such as Chevallet et. al., (1998),
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Martins et. al., (2007), D’Andrea et al., (2011) and Jagannadham and Chowdhury, (2012), where the
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long ASB carbon tail effectively coats the hydrophobic proteins improving efficiency. However it is
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worth noting, as highlighted by Luche et al., (2003), that the efficiency of a particular detergent to
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solubilise proteins largely depends on the sample under test.
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A total of 12 spots were chosen for identification in order to give a good representation of
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spots present on the gels and to determine whether the protocols optimised the recovery of
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associated membrane proteins. The majority of the putative protein identifications for C. elegans
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were membrane associated proteins. In total, 70% of proteins represented on gels were found to be
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membrane associated. Of the 70% of membrane associated proteins, 50% were located in the inner
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mitochondrial membrane, 10% in the outer mitochondrial membrane and 10% located in the outer
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cellular membrane (Fig. 4B). Of the remaining proteins 20% were related to cell structure and 10%
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from the cytoplasm (Fig. 4A).
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From this study we were able to putatively identify some proteins which may provide a
292
potential biomarker role for ivermectin resistance. Amongst the C. elegans isolates, spot 5 was
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putatively identified as a cytochrome C oxidase family member. These proteins are associated with
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the mitochondrial membrane and are involved in energy transduction across the membrane (Richter
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and Ludwig, 2003).
RNA interference (RNAi) studies in C. elegans have suggested that these
12
296
proteins are important for reproduction and development (Braekman et al., 2009). Although, little is
297
known about these proteins in parasitic nematodes, it is suggested that cytochrome c oxidases are
298
involved in gametogenesis and are required for survival in the anaerobic environment of the host
299
(Braekman et al., 2009).
300
consequences for nematode survival (Campbell et al., 2008). Spot 9 was putatively identified as a
301
mitochondrial prohibitin (PHB) complex family member. A depletion of PHB has been correlated to
302
an increased production of reactive oxygen species (ROS) (Merkwirth and Langer, 2009), which may
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be important for cellular defence. Prohibitins have been associated with chaperone activity and for
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the maintenance of mitochondrial integrity by functioning as protein or lipid scaffolds for the inner
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membrane (Merkwith and Langer, 2009). Spot 11 was putatively identified as a voltage dependent
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anion channel (VDAC family), which have been suggested to be important for the excretion of
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organic acids (Blair et al., 2003). VDACs are perforin proteins (porins) in the mitochondrial outer
308
membrane and are important for the transport of purine nucleotides. It has been suggested that
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they could conduct ATP in response to physiological stress (Okada et al., 2004), which may account
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for an up-regulation in protein expression observed when subjected to drug challenge conditions.
Therefore the interference of these processes could have severe
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From the H. contortus isolates spots 2, 3 and 4 appeared to dominate the H. contortus
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profiles corresponding to putatively identified actin proteins, which is not entirely unexpected as
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these proteins are amongst the most abundant proteins in the cell (Dominguez and Holmes, 2011).
314
Thus explaining the presence of this protein in both C. elegans and H. contortus associated
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membrane protein preparations. These proteins correlated with spot 2 on the C. elegans profiles
316
and as with H. contortus demonstrated up-regulation in the resistant isolate. Actin is an important
317
component of the cytoskeleton which interacts with the cell membrane (Cowin and Burke, 1996)
318
and the effect of Benzimidazoles (BZs) on these proteins have been shown to compromise the
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formation of the cytoskeleton (Lacey, 1990). An up-regulation of these putative actin proteins in the
320
resistant isolate may relate to differences in functional development between isolates. Spot 1,
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putatively identified as glutamate dehydrogenase (GDH) was present on the susceptible profile and
13
322
has been previously involved in vaccine trials. GDH was established as an important vaccine
323
candidate antigen (Skuce et al., 1999), although
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component during vaccine trials (Knox et al., 2005). GDH has also been shown to be present in C.
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elegans and it is thought that it may form a complex with intergral membrane proteins (Skuce et al.,
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1999). Spot 19, putatively identified as an apical gut membrane polyprotein was present on the
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resistant profile. These proteins have previously shown to induce protective immunity to challenge
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infection in goats and have also been considered for the design of recombinant antigens for vaccine
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trials as a potential target for nematode control (Jasmer, 1996). From studies by Jasmer (1996), it
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was shown that P46 and P52 proteins are derived from P100 (gut membrane protein), which may
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have involvement with integral membrane proteins. The antigens derived from these nematode gut
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proteins to produce vaccines have shown some protective immunity against parasitic nematodes
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(Miller and Horohov, 2006).
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Conclusion
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Key aspects of nematode biochemistry and physiology are associated with events in their
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membranes. To date, membrane based proteomics has been hampered by problematic sample
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preparation of the more hydrophobic cellular proteins. We have put the C. elegans model (Gilleard,
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2006) to work in order to devise new enabling methodology to study proteins associated with
339
membranes in parasitic nematodes. We demonstrated that a new membrane protein enrichment
340
process that encompasses chaotrophic agents plus detergents and a suitable IEF compatible buffer
341
facilitated the isolation of membrane associated proteins from C. elegans and the methodology
342
could be translated to the parasitic nematode H. contortus.
343
demonstrated that membrane associated proteins could be resolved by 2DE. The methodology was
344
supported by identification of predicted membrane associated proteins from both C. elegans and H.
345
contortus using mass spectrometry, many of these proteins are predicted to be part of electron
346
transport and energy reaction pathways. The finding of the different protein profiles in a drug
GDH was confirmed as a non- protective
Moreover, sub-proteomic analysis
14
347
resistant H. contortus isolate compared to a drug sensitive isolate tentatively suggests that
348
differential compound (including drug) response pathways are operating in the resistant phenotype.
349
Acknowledgements.
350
The authors would like to thank Dr Gustavo Chemale for all his help and advice with buffer
351
components for membrane extraction and to Dr Russ Morphew for all of his helpful suggestions and
352
proteomic advice. C. elegans isolates were kindly provided by the CGC (Caenorhabditis elegans
353
genetic centre) and H. contortus isolates were provided by the Moredun Institute, thanks to Dr
354
Phillip Skuce. We would also like to thank Hybu Cig Cymru (HCC) and the European Social Fund for
355
funding.
356
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531
Table 1. IEF solubilisation buffers
22
532
Table 2 .Effect of isolate and challenge on normalised spot volumes of C. elegans.
533
Table 3. Membrane associated protein identifications of C. elegans isolates via ESI MS/MS.
534
Sequence peptides were BLASTed against the NCBI database with significant matches determined
535
at e < 0.05, only spots with significant hits are shown.
536
Table 4. Membrane associated protein identifications of H. contortus isolates via ESI MS/MS.
537
Sequence peptides were BLASTed against the NCBI database with significant matches determined
538
at e<1, only spots with significant hits are shown.
539
540
Figure 1. Gel images (A to F) of corresponding IEF buffers shown in table 1 (1 to 6). Average spot
541
numbers for each IEF buffer for gels (A to F) based on three replicate gels were 10, 32, 57, 66, 31,
542
39 respectively. Proteins were separated on 12.5% T, 3.3% C acrylamide SDS-PAGE gels across a
543
non linear pH range of 3-10. Proteins were stained with Coomassie Blue.
544
545
546
Figure 2. Membrane associated protein arrays for C. elegans. Unchallenged isolates average gels
547
from 3 biological replicates. Proteins were separated across a non linear pH range of 5-8, 12.5% T,
548
3.3%C SDS PAGE and show protein arrays for (A) N2, (B) moderate resistant (DA1302) and (C)
549
highly resistant (DA1316) isolates. Proteins were visualised using Coomassie blue stain. Circled
550
areas indicate spots excised for ESI MS/MS identification.
551
Figure 3. Membrane associated protein arrays for C. elegans challenged isolates. Average gels
552
were created for each isolate from 3 biological replicates cultured with a sub-lethal concentration
553
of ivermectin. Proteins were separated across a non linear pH range of 5-8, 12.5% T, 3.3% C SDS
23
554
PAGE and show protein arrays for (A) N2, (B) moderate resistant (DA1302) and (C) highly resistant
555
(DA1316) isolates. Proteins were visualised using Coomassie Blue stain.
556
Figure 4. Charts to show the distribution of subcellular location of proteins from C. elegans gels.
557
(A) shows the number of membrane associated proteins putatively identified on gels compared to
558
structural proteins and those found on the cytoplasm. (B) shows the breakdown of these proteins
559
into their various subcellular locations.
560
Figure 5. Protein array of membrane associated proteins of H. contortus. Membrane associated
561
protein analysis of three biological replicates of H. contortus female isolates at the adult stage.
562
Protein separation was conducted on 12.5% T, 3.3% C acrylamide SDS-PAGE gel across a 7cm non
563
linear pH range of 5-8 and Coomassie stained. Protein profiles are for A) susceptible (ISE) isolate
564
and B) resistant (CAVR) isolate.
565
Figure 6. Charts to show the distribution of subcellular location of protein putatively identified
566
from H. contortus gels.
567
24