Biotechnol. J. 2010, 5
DOI 10.1002/biot.200900106
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Technical Report
An antibody-based affinity chromatography tool to assess Cu, Zn superoxide dismutase (SOD) G93A structural complexity in vivo Florencia Palacios1, Germán Cota1, Sofía Horjales1, Analía Lima2, Julio Battistoni3, José Sotelo-Silveira4 and Mónica Marín1 1 Sección
Bioquímica-Biología Molecular, Facultad de Ciencias, UdelaR, Montevideo, Uruguay Pasteur de Montevideo, Montevideo, Uruguay 3 Laboratorio de Inmunotecnología, Facultad de Química, Facultad de Ciencias, UdelaR, Montevideo, Uruguay 4 Sección Biología Celular, Facultad de Ciencias, UdelaR and IIBCE, Montevideo, Uruguay 2 Institut
‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSODG93A rats provided evidence about heterodimers rSOD–hSODG93A formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSODG93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.
Received 25 April 2009 Revised 11 November 2009 Accepted 13 November 2009
Keywords: Cu,Zn superoxide dismutase · Human SODG93A · In vivo protein folding · Post-translational modifications
1 Introduction Investigating protein conformation in vivo is a theoretical and experimental challenge. While in vitro experiments have contributed much to our understanding of protein folding, we know much less about how proteins fold in the more complex environment of the cell [1]. Over the last few decades,
Correspondence: Dr. Mónica Marín. Sección Bioquímica-Biología Molecular, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, Uruguay E-mail:
[email protected] or
[email protected] Fax: +598-2-525-8617 Abbreviations: ALS, amyotrophic lateral sclerosis; AP, alkaline phosphatase; EF, eluted protein fractions; SOD, superoxide dismutase; SOD1, Cu,Zn SOD; wt, wild type
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important contributions have shown that protein folding in the cell is affected by a number of factors such as physicochemical conditions of the cellular environment, interactions with chaperones and folding catalysts [2]. Also, cellular growth and translation rate, and local translational kinetics were shown to affect the correct folding of some proteins [3] (for a review see [4]). Despite the activity of specialized systems in assisting protein folding and avoidance of aggregation [5, 6], misfolded proteins are known to accumulate in specific tissues in a growing group of heterogeneous disorders [7, 8]. Some evidence suggests that proteins could adopt different conformations related to the cellular milieu of biosynthesis. By limited proteolysis, previous studies have shown that Ure2 protein
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Biotechnology Journal
from the yeast Saccharomyces cerevisiae, a protein with prion properties, could adopt differentiated conformations when translated in vitro in rabbit or wheat germ-derived translation systems [3]. More recently, using a similar limited proteolysis approach and a sterol ligand-binding assay, we showed that the human estrogen receptor alpha synthesized in these translation systems could adopt at least three distinct conformations [9]. These studies performed in vitro suggested that components of the cellular translation machinery itself might influence the protein-folding pathways and the relative abundance of different conformers when synthesized in different tissues [9]. To further characterize protein properties related to the cellular milieu of biosynthesis, our aim was to compare protein variants synthesized in different tissues. We explored the profile of the human Cu,Zn superoxide dismutase 1 (hSOD1), which is involved in amyotrophic lateral sclerosis (ALS) [10–12]. This neurodegenerative disease is characterized by features indicative of both upper and lower motor neuron lesion [13]. More than 120 mutations in hSOD1 have been identified in approximately 20% of individuals with familial ALS, and patients with these mutations present intracellular inclusions containing insoluble aggregated proteins [14–16]. Despite the fact that this enzyme is widely expressed, at high level in liver and kidney, aggregation has only been described in neural tissues [17, 18].Although the mechanisms by which SOD1 mutations cause degeneration of motor neurons have remained elusive, misfolding of the enzyme and lack of metals are emerging as mechanism underlying motor neuron degeneration [10, 11]. A common property of essentially all ALS SOD1 mutants is their propensity to aggregate [19, 20]. The G93A mutation among others has been shown to weaken the structure, and thus could mean an increased risk of aggregation [21]. Mice and rats expressing human mutation-linked SOD1 transgenes develop an ALS-like disorder characterized by degeneration of spinal motor neurons and by the presence of neurofilament-rich cytoplasmic inclusions in surviving motor neurons [18]. Using an hSODG93A transgenic rat model, our aim was to isolate and characterize the enzyme synthesized in different tissues. We developed a polyclonal antibody-affinity chromatography procedure with which different variants of the hSOD1 produced in vivo could be recovered. Here we describe the characterization of this chromatographic approach and the comparative electrophoretic profiles of hSODG93A extracted from the liver and brain of transgenic rats.
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Biotechnol. J. 2010, 5
2 2.1
Materials and methods Animals
Sprague-Dawley SOD1 G93A L26H rats were kindly provided by Dr. D. S. Howland (Wyeth Research, Princeton, NJ) [18]. Animals were treated in accordance with the guidelines for Care and Use of Laboratory Animals established by the National Institutes of Health, and all protocols for work conducted with mice and rats were previously submitted to and approved by the National Committee for Animal Experimentation (CHEA, Uruguay). Asymptomatic animals of 120 days of age were euthanized using sodium pentobarbital (intraperitoneal injections at 200 mg/kg) and when unresponsive decapitation was performed.
2.2
Antibodies against hSOD1
Recombinant wild-type (wt) hSOD1 was kindly provided by Dr. Beatriz Alvarez, (Facultad de Ciencias, UdelaR). The protein was produced in E. coli strain BL21 (DE3) pLysS, metallated and purified as described [22]. Rabbit antibodies were raised against this recombinant hSOD1 at the Immunotechnology Laboratory, Polo Tecnológico, UdelaR. A gamma globulin fraction was obtained from rabbit sera by ammonium sulfate precipitation. Specific antibodies were purified by affinity chromatography to the immobilized recombinant enzyme on Sepharose 4B (Pierce). To recover antiSOD antibodies, two elution conditions were employed: (i) elution with 0.1 M glycine pH 2.5 (fraction called anti-hSOD); and (ii) elution with 40% DMSO in 0.1 M glycine pH 2.5 to recover antibodies strongly bound to the stationary phase (fraction called anti-SOD-DMSO). In both cases, eluted antibodies were immediately neutralized with 1.5 M Tris pH 9.
2.3
Western and dot blots
Proteins were transferred onto Hybond C membrane (GE Healthcare) and incubated overnight at 4°C with blocking buffer (5% milk, 2% glycine in phosphate buffer containing 0.1% Tween 20). The membrane was incubated for 1 h with rabbit polyclonal anti-hSOD (or anti-SOD-DMSO) antibody in blocking buffer, then washed and incubated for 1 h at room temperature with horseradish peroxidaseconjugated goat anti-IgG rabbit (Promega) or alkaline phosphatase (AP)-conjugated goat anti-IgG rabbit antibody. The blots were developed with a chemiluminescent substrate kit (Super Signal, Pierce) or for AP-labeled anti-IgG rabbit antibody
Biotechnol. J. 2010, 5
with 4-nitro blue tetrazolium (NBT)-5-bromo-4chloro-3 vidolyl phosphate BCIP.
2.4
hSOD1 extraction by affinity chromatography
Anti-hSOD antibodies (from the fraction antihSOD only) were immobilized to activated Sepharose 4B (Pierce) as described. Rat brain and liver tissues were cut into pieces and homogenized in 10% lysis buffer containing 15 mM Tris pH 7.4; 25 mM EDTA and other protease inhibitors (Roche inhibitor cocktail). Potter homogenization was carried out on ice with five 1-min embolic incursions. Homogenates were centrifuged at 1000 × g at 4°C for 10 min, and supernatants were further analyzed.
2.5
SOD1 activity assay in gel
Native PAGE was stained with NBT in the presence of riboflavin and TEMED, as previously described [23]. When exposed to light, active SOD in gels was detected as colorless bands.
2.6
2-DE
First-dimensional separation was performed with commercially available IPG strips (7 cm, non-linear 3–11 pH gradient, GE Healthcare). Samples containing 70 µg proteins, previously purified using the 2-D Clean-Up kit (GE Healthcare) and dissolved in 125 µL rehydration solution [7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer 3–11 NL (GE Healthcare), 0.002% bromophenol blue], were loaded onto IPG strips by passive rehydration over 12 h at room temperature. The isoelectric focusing was done in an IPGphor Unit (Pharmacia Biotech) employing the following voltage profile: constant phase of 300 V for 30 min; linear increase to 1000 V in 30 min; linear increase to 5000 V in 1 h 20 min and a final constant phase of 5000 V for 15 min. Prior to running the second dimension, IPG strips were reduced for 15 min in equilibration buffer (6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromophenol blue) supplemented with DTT (10 mg/mL) and subsequently alkylated for 15 min in equilibration buffer supplemented with iodoacetamide (25 mg/mL). The second-dimensional separation was performed by SDSPAGE in 15% gels using an SE 260 mini-vertical gel electrophoresis unit (GE Healthcare). Electrophoresis was carried out for 15 min at 10 mA per gel, and then at 20 mA per gel until the bromophenol blue dye front reached the bottom of gels. The size markers used were Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis
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(GE Healthcare). The gels were silver stained according to the protocol described by Shevchenko et al. [24]. Images were digitalized using a UMAX PowerLook 1120 scanner and LabScan 5.0 software (GE Healthcare).
2.7 MS sample preparation and MALDI-TOF MS analysis Peptide mass fingerprinting of protein spots selected for identification was carried out by in-gel trypsin treatment (Sequencing-grade Promega). Peptides were extracted from the gels using 60% acetonitrile in 0.2% TFA, concentrated by vacuum drying and desalted using C18 reverse phase micro-columns (OMIX Pippete tips, Varian). Mass spectra of digestion mixtures were acquired in a 4800 MALDI-TOF/TOF instrument (Applied Biosystems) in reflector mode using a matrix solution (α−cyano-4-hydroxycinnamic acid in 60% aqueous acetonitrile containing 0.2% TFA) and were externally calibrated using a mixture of peptide standards (Applied Biosystems). Collision-induced dissociation MS/MS experiments of selected peptides were performed. Proteins were identified by database searching with peptide masses using the MASCOT program (Matrix Science http://www.matrixscience.com/ search_form_select.html), and based on the following search parameters: all taxonomic entries; monoisotopic mass tolerance, 0.08 Da; fragment mass tolerance, 0.6 Da; partial methionine oxidation, cysteine carbamidomethylation and one missed tryptic cleavage allowed. Significant Mowse scores (p
