Human Molecular Genetics, 2000, Vol. 9, No. 9 1403-1413
© 2000 Oxford University Press
Mutant torsinA, responsible for early-onset torsion dystonia, forms membrane inclusions in cultured neural cells
Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
Received 28 January 2000; Revised and Accepted 22 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Early-onset torsion dystonia is a hereditary movement disorder thought to be caused by decreased release of dopamine into the basal ganglia, without apparent neuronal degeneration. Recent cloning of the gene responsible for this disease, TOR1A (DYT1), identified the encoded protein, torsinA, as a member of the AAA+ superfamily of chaperone proteins and revealed highest levels of expression in dopaminergic neurons in human brain. Most cases of this disease are caused by a deletion of one glutamic acid residue in the C-terminal region of the protein. Antibodies generated against torsinA revealed expression of a predominant immunoreactive protein species similar to the predicted size of 37.8 kDa in neural, glial and fibroblastic lines by western blot analysis. This protein is N-glycosylated with high mannose content and not, apparently, phosphorylated. Overexpression of torsinA in mouse neural CAD cells followed by immunocytochemistry, revealed a dramatically different pattern of distribution for wild-type and mutant forms of the protein. The wild-type protein was found throughout the cytoplasm and neurites with a high degree of co-localization with the endoplasmic reticulum (ER) marker, protein disulfide isomerase. In contrast, the mutant protein accumulated in multiple, large inclusions in the cytoplasm around the nucleus. These inclusions were composed of membrane whorls, apparently derived from the ER. If disrupted processing of the mutant protein leads to its accumulation in multilayer membranous structures in vivo, these may interfere with membrane trafficking in neurons.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TorsinA is a novel protein discovered through identification of the gene responsible for early-onset torsion dystonia (1), an autosomal-dominant movement disorder beginning in childhood and manifesting as contracted, twisted postures throughout life (2,3). About 60% of cases of typical early-onset torsion dystonia have the same mutation in one allele of the TOR1A (DYT1) gene, an in-frame GAG deletion that results in loss of a glutamic acid residue in the C-terminus of the predicted protein (1,4). This, and related forms of dystonia are believed to result from defects in dopamine neurotransmission in the basal ganglia (for review see refs 5,6). In situ hybridization analysis of control human brains revealed highest levels of torsinA message in dopaminergic neurons of the substantia nigra, with selective expression in a number of other brain regions including the cerebellum, hippocampus and locus coerulus (7,8).
The deduced sequence of torsinA provides some clues to its biochemical properties. It predicts a 332 amino acid protein of 37.8 kDa with potential sites for glycosylation and phosphorylation, as well as a predicted N-terminal hydrophobic leader sequence consistent with membrane translocation/targeting, followed by a hydrophobic membrane-spanning domain (1,5). Within the human genome, TOR1A is a member of a gene family comprising at least three other highly homologous genes, TOR1B, which has 70% homology and is located adjacent to TOR1A on chromosome 9q34, and TOR2A and TOR2B with ~50% homology (1,9). Genes encoding proteins highly similar to torsinA occur in many other species, including mouse, rat, nematode, fruitfly, pig and zebrafish, all as yet uncharacterized (1,9).
Several features of torsinA suggest that it is a member of the large AAA+ superfamily of proteins (10), which includes the ATPases associated with a variety of cellular activities (AAA) (11) and the heat-shock proteins (HSP100)/Clp ATPases (12). These features include a Walker ATP/Mg2+ binding site, 11 conserved domains and similar predicted secondary configuration (1,10,13,14). The deleted glutamic acid in mutant torsinA falls within the Box VIII (Sensor 2) region. This family of proteins is critical to the assembly, disassembly and operation of many types of protein complexes, but the functions are so diverse that they provide little clue as to how a new member, like torsinA, might act.
In the present study, antibodies were generated against predicted peptides and an expressed exon of human torsinA and characterized for their specificity and reactivity to proteins in different cell types. These antibodies were used to evaluate the size and subcellular localization of wild-type human torsinA and mutant torsinA by western blot analysis, immunocytochemistry and immunogold electron microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Specificity of antibodies
Rabbit antisera were generated against three synthetic peptides representing different regions of the human torsinA protein: 13 amino acids from the N-terminal region, corresponding to a region with low homology (38%) to torsinB (TA1); and 17 and 14 amino acids from the C-terminal region (TAB3 and TAB1, respectively), both of which have relatively high homology to torsinB (79 and 76%, respectively). The TAB1 peptide spans the region of the GAG deletion. A mouse monoclonal antibody (D-MG10) was generated against a glutathione S-transferase (GST) fusion protein consisting of 42 amino acids in the C-terminal region of torsinA [corresponding to exon 4, which also contains the TAB3 peptide region (9)].
The specificity of torsinA antibodies was evaluated initially using PAGE and western blot analysis to determine the size of immunoreactive proteins and their cross-reactivity with different torsinA antibodies. (In all cases preimmune and secondary antibody-only staining revealed no notable immunoreactive bands.) Wild-type and mutant torsinA, overexpressed in CAD cells by transient transfection, yielded a predominant band of 37.1 kDa, similar to the predicted size, in total cell lysates using all antibodies, TAB1, D-MG10, TAB3 and TA1 (Fig. 1). Antibodies TAB1 and D-MG10 gave a stronger signal against this protein species, as compared with antibodies TAB3 and TA1 (rabbit antisera were used at comparable dilutions). The mutant protein yielded somewhat less apparent immunoreactive protein than the wild-type protein for all antibodies, possibly due to reduced transfectability of the mutant versus wild-type construct, relative instability of the mutant protein or toxicity of the mutant protein. This reduced reactivity for the mutant protein was most pronounced with antiserum to the TAB1 peptide, which overlaps the region of the deleted glutamic acid, indicating reduced affinity.
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Expression of immunoreactive protein in different cell types
A number of different cell lines were evaluated for endogenous expression of torsinA-immunoreactive protein, including: monkey kidney fibroblast line, COS-7; mouse neural line, CAD; mouse fibroblast line, NIH 3T3; mouse glioma line, GL261; mouse neuroblastoma line, N1E-115; human kidney fibroblast line, 293T/17; human glioma line, Gli238; and human neuroblastoma line, SH-SY5Y. Using TAB1 antibody, the major immunoreactive band in most cell types was 37.1 kDa and co-migrated with overexpressed torsinA in CAD cells (Fig. 2). In SH-SY5Y and NIH 3T3 cells, the major immunoreactive band was 38.8 kDa. The torsinA–green fluorescent protein (GFP) fusion protein yielded an immunoreactive band of the predicted size, ~60 kDa (Fig. 2, lane 1). In other experiments, the relative abundance of the endogenous immunoreactive protein did not increase in CAD or SH-SY5Y cells in the differentiated versus undifferentiated state (data not shown). These two major immunoreactive species, 37.1 and 38.8 kDa, may represent torsinA and/or torsinB or post-translational modifications of them.
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The identity of immunoreactive bands was confirmed by peptide blocking and immunoprecipitation/western blotting using antibodies against different domains of torsinA. For example, TAB1 immunostaining of the 38.8 kDa endogenous protein in SH-SY5Y cells and the 37.1 kDa protein in CAD cells overexpressing wild-type torsinA were completely blocked by preincubation of immune serum with the corresponding peptide (Fig. 3), whereas a non-specific peptide did not block binding (data not shown). Antibody combinations tested against overexpressed wild-type and mutant torsinA by immunoprecipitation/western blot analysis were: TAB3/TAB1, D-MG10/TAB1, TAB1/D-MG10, TA1/TAB1 and non-specific antibody, 1C4/D-MG10; and for endogenous protein in SH-SY5Y cells were: TA1/TAB1, TAB1/TA1 and TAB1/D-MG10. Similar results were obtained for all immunospecific combinations and no reactive bands were observed when non-specific antibody or pre-immune sera were used for the immunoprecipitation. Representative blots are shown in Figure 4. Note that endogenous torsin in SH-SY5Y cells was enriched by immunoprecipitation (Fig. 4C).
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Potential post-translation processing of immunoreactive torsinA was also evaluated. To determine if torsinA is glycosylated, total cell lysates from non-transfected SH-SY5Y cells and CAD cells transfected with wild-type or mutant forms of torsinA were treated with endoglycosidase H, which cleaves high mannose and hybrid carbohydrates present in the endoplasmic reticulum (ER), and with an N-glycosidase, PNGase F, which cleaves these as well as complex oligosaccharides present in the Golgi and subsequent cellular compartments. Both deglycosidases completely reduced the size of the endogenous immunoreactive band from 38.8 to 33 kDa in SH-SY5Y cell lysates, and the overexpressed wild-type and mutant torsinA band from 37.1 to 31.1 kDa in CAD cell lysates (Fig. 5), consistent with high mannose glycosylation characteristic of proteins in the ER. Treatment of SH-SY5Y and CAD cell lysates with calf intestine phosphatase (CIP) did not result in a shift in apparent molecular weight of the immunoreactive torsinA, indicating that the protein is apparently not phosphorylated (data not shown).
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Cellular localization of torsinA-like proteins
The intracellular localization of overexpressed wild-type and mutant forms of torsinA was investigated by immunocytochemistry in CAD cells using TAB1 antibody. Endogenous immunoreactive torsinA-like protein was not apparent in untransfected CAD cells, presumably due to relatively low levels of expression (see western blot, Fig. 2). When wild-type torsinA was transiently expressed in either undifferentiated or differentiated CAD cells, immunoreactivity was seen throughout the cytoplasm, excluding the nuclei, and extending into neurites (Figs 6A and D, and 7A). Cells overexpressing wild-type torsinA retained their neuronal-like morphology. In contrast, transient expression of mutant torsinA in both undifferentiated and differentiated CAD cells, yielded multiple, large inclusions containing immunoreactive protein, which were especially abundant around the cell nucleus (Figs 6G and J, and 7C and E). Many cells transfected with the mutant protein showed a dramatically altered morphology, appearing flattened with retracted neurites, suggesting cell toxicity. Similar staining patterns for overexpressed wild-type and mutant torsinA were observed using torsinA antibody, D-MG10. SH-SY5Y cells transfected with wild-type and mutant proteins and stained with TAB1 and D-MG10 antibodies showed similar morphologies to that observed for CAD cells, with the exception that mutant-induced membrane inclusions tended to be smaller (data not shown). Membrane compartmentalization of wild-type and mutant torsinA in transfected CAD cells was supported by fractionation of cells by homogenization and differential centrifugation, followed by western blot analysis, which revealed highest levels of torsin-immunoreactivity in the 17 000 g pellet, which contains large membrane organelles, including Golgi, mitochondria, vesicles and ER membranes (data not shown).
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Double-label immunocytochemistry was carried out to identify cellular compartments using TAB1 antibodies for torsinA and antibodies/lectin for a number of cellular markers, including: Golgi proteins (GM130 and VVL), vesicles (SV2, nSec1, synaptophysin and synaptobrevin/VAMP-2), ER [protein disulfide isomerase (PDI)], endosomes (transferrin receptor), lysosomes (LAMP-1), microtubules (ß-tubulin) and neurite growth cones (13H9). The greatest extent of co-localization for overexpressed wild-type torsinA was seen with the ER marker, PDI (Fig. 6A–F); both extended in an overlapping punctate pattern throughout the cell body and processes, albeit with the edges of some cell bodies positive for PDI only (Fig. 6F). In contrast, the large intracellular inclusions seen in cells overexpressing mutant torsinA did not co-localize with any of the markers tested, except PDI, which in some cells was distributed throughout the cytoplasm but also in the inclusions (Fig. 6G–I; note greenish-yellow inclusions in I), and in other cells showed concentration in the torsinA inclusions (Fig. 7J–L). The presence of PDI in mutant inclusions was confirmed by electron microscopy (see below). The coalescence of PDI into inclusion bodies in cells overexpressing the mutant protein may reflect a progressive disruption of the ER triggered by the mutant protein. In CAD cells overexpressing wild-type torsinA, partial co-localization was found with all the vesicular markers tested, e.g. VAMP-2 (Fig. 7A and B) with high density at neurite varicosities, but no co-localization of this or other vesicular markers was observed for mutant inclusions (Fig. 7C and D). Double-label immunocytochemistry with Golgi markers (shown for GM-130) and torsinA antibodies showed that mutant torsinA inclusions clustered around, but were distinct from, the Golgi (Fig. 7E and F).
Immunogold ultrastructural visualization of overexpressed wild-type torsinA in CAD cells revealed association with double, undulating membranes throughout the cell body and in proximity to the nuclear membrane, consistent with an ER location (Fig. 8A). Mutant torsinA overexpressed in CAD cells was primarily concentrated in multilayer, concentric membranous structures ranging in size from 0.5 to 2.5 µM in diameter (Fig. 8B) and to a lesser extent in ER-like structures, as seen for the wild-type protein (data not shown). The size of the membrane whorls was in the same range as the inclusions seen by immunocytochemistry. The double membrane structure of layers in whorls and their proximity to the nuclear membrane, suggests that they are derived from the ER. Nuclei and mitochondria appeared intact in all cells. Staining with the lumenal ER marker, PDI, showed marked staining of the mutant-derived, whorled inclusions (Fig. 8C), as well as staining of ER-like structures throughout the cytoplasm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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This study demonstrates that torsinA, the protein responsible for early-onset dystonia, localizes primarily to a membranous compartment in neural cells. Overexpressed wild-type protein exists in a high mannose, N-glycosylated state, characteristic of ER proteins, and is located in a punctate pattern throughout the cell body and neurites, with high density at neurite varicosities. Wild-type torsinA shows a high degree of co-localization with the ER marker, PDI, and partial co-localization with vesicular markers e.g. VAMP-2 (synaptobrevin). In contrast, overexpressed mutant torsinA, bearing the loss of a single glutamic acid residue in the C-terminus (as in dystonia patients), accumulates in multilayered, concentric membrane whorls in the cytoplasm, frequently accompanied by flattening of the cell body and retraction of neurites. The whorls stained for both torsinA and PDI and appear to derive from the ER. These findings, and the characteristics of early-onset dystonia, are consistent with the mutant protein interfering with ER integrity, membrane trafficking and downstream vesicular release from neurons.
Several interesting aspects of torsinA are revealed by these studies. First of all, the overexpressed wild-type and mutant forms seem to be processed and retained in the ER, consistent with the deduced signal sequence (1) and the observed high mannose content of both overexpressed and endogenous torsin. Both forms of the protein also appear to be associated with a membrane compartment, as predicted from the hydrophobic, putative membrane-spanning amino acid sequence at the N-terminal region of the protein (downstream from the putative signal sequence). Particularly intriguing with regard to the present study are the double phenylalanine residues, which are characteristic of ER proteins that cycle between the ER and Golgi (15,16), that lie just downstream of the deleted glutamic acid in mutant torsinA (1).
In the present study, it is difficult to determine whether endogenous immunoreactive species in different cell types represent torsinA or torsinB, given the high homology (70%) of these proteins (1). Still, in the case of wild-type and mutant forms of the protein transiently expressed in cells at high levels, it is safe to assume that the excess protein, over that seen in non-transfected cells, represents torsinA. The presence of overexpressed wild-type torsinA in a punctate pattern throughout the cytoplasm, high extent of co-localization with an ER marker protein, ultrastructural association with ER membranes and high level of mannose residues indicate that it is localized primarily in the ER. Partial co-localization of wild-type torsinA with the vesicular proteins, e.g. VAMP-2 (synaptobrevin), may be an artifact of the close proximity of ER and vesicles in these cells or may implicate transit of torsinA between these compartments. The co-localization of PDI and overexpressed mutant torsinA in membrane whorls (seen by immunoelectron microscopy) and disruption of normal ER morphology seen in some cells suggest that the mutant form can interfere with normal ER membrane trafficking. Furthermore, the altered morphology of neural cells transfected with mutant torsinA, and not with wild-type torsinA, suggests that high levels of the mutant protein, or the whorls derived from it, are toxic to or can otherwise disrupt the structural integrity of these cells.
The torsinA message is expressed in most tissues in high abundance and, thus, this protein appears to be a functional component of many cell types (1). Its initial identification as the cause of a neuronal disease may result from the selective expression of torsinA, with little torsinB message, in human brain and its high regional localization there, especially in the dopaminergic neurons of the substantia nigra (7,8). The mutation responsible for early-onset torsion dystonia is unique on several counts. First, it confers loss of a single, specific amino acid in the protein, and no other mutations have been found in the coding region of this gene in a screen of 17 early-onset dystonia cases, who do not have the GAG deletion (9). Second, the GAG deletion produces disease only in ~40% of heterozygous carriers (17) with onset of symptoms restricted to a period of postnatal development between 1 and 28 years of age (3). No individuals homozygous for mutations in TOR1A have been found, and such a genotype might, given the widespread tissue distribution of torsinA, affect multiple tissues and cause systemic disease.
It is tempting to evoke toxicity of mutant torsinA inclusions as the cause of early-onset dystonia, as, in a number of neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease and Alzheimer’s disease, inclusions have been associated with neuronal death (for review see refs 18–20). However, to date, pathological analysis of autopsy brain tissue from early-onset dystonia patients has revealed no specific histopathological changes or neuronal degeneration (21,22). Furthermore, the cytoplasmic inclusions seen in neural cells transfected with the mutant torsinA expression construct are morphologically distinct from the dense aggregates found in these neurodegenerative diseases. The concentric whorls of membrane formed by mutant torsinA described here appear similar to the rough ER/nuclear membrane whorls described in Xenopus laevis hepatocytes (23), and the ER stacks noted in fibroblasts overexpressing the inositol 1,4,5-triphosphate receptor (24) and in Purkinje cells in culture (25). The double-membrane appearance and apparent ER derivation of mutant torsinA whorls distinguish them from the plasma membrane-derived whorls seen in dying neurons expressing mutant degenerins (26). Two possible derivations of these mutant torsinA whorls can be considered. First, the mutant protein could trigger an ER stress response due to protein misfolding or inability to oligomerize, resulting in upregulation of ER stress proteins, like PDI (27). Mutations in other proteins that affect protein processing and cause an ER stress response include the prion protein in hereditary Creutzfeld–Jakob disease, peripheral myelin protein 22 in Charcot–Marie Tooth Syndrome and 
1-antitrypsin (PiZ variant) in hereditary emphysema (for review see ref. 28). Second, mutant torsinA could interfere in some way with the normal membrane dynamics, such as budding or transport of ER vesicles to the Golgi or of other types of vesicles along neurites (29). Other members of the AAA+ family that have a role in membrane trafficking include: NSF, which mediates vesicle fusion between the ER and the Golgi (30) and between synaptic vesicles and presynaptic nerve terminals (31,32), and the dynein-associated complex, which serves as the motor to move membranous organelles along microtubules in neurites (33).
Although in the early stages of elucidation, torsinA appears to be processed through the ER compartment. The wild-type protein may be involved in membrane fusion, vesicle movement or other events of membrane trafficking, and/or serve as a molecular chaperone to mediate proper folding of proteins in the ER. The mutant form of torsinA may compromise neuronal function by disrupting processing of normal torsinA or other proteins, or by physical accumulation in inclusions, which may be toxic to cells at high levels and/or may interfere with generation, transport and targeting of membrane organelles. Dopaminergic neurons, which express high levels of torsinA, may be especially sensitive to the disruptive influence of the mutant form during the postnatal period of development when restructuring of synaptic terminals in the striatum is taking place. This study provides the basis for investigation of the function of torsinA and the aberrant function of mutant torsinA with a focus on ER and chaperone functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cells
The following cell lines were used: human neuroblastoma SH-SY5Y [American Type Culture Collection (ATCC) (34)], human glioma Gli238 (provided by Dr D. Louis, Massachusetts General Hospital), human embryonic kidney fibroblasts 293T/17 [Dr D. Baltimore, MIT (35)], mouse neuroblastoma, N1E-115 [Dr M. Nirenberg, NIH (36)], mouse fibroblasts NIH 3T3 (ATCC), mouse glioma GL261 [Dr Saris, New England Medical Center (37)], monkey kidney fibroblasts COS-7 (ATCC) and mouse neural cells CAD [Dr J. Wang, Tufts University (38)]. All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Rockville, MD) supplemented with 4.5 g/l glucose, 2 mM glutamine, except CAD cells, which were grown in DMEM/F12 (1:1) (Gibco BRL). All media were supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco BRL) and cultures were incubated at 37°C in 5% CO2/95% air. SH-SY5Y and CAD cultures were differentiated at ~20% confluency by the addition of 20 µM retinoic acid for 4 days, and 20 µg/ml transferrin (Sigma, St Louis, MO), 50 ng/ml sodium selenite (Sigma) in serum-free DMEM/F12 for 3 days, respectively.
Transfection of CAD and SH-SY5Y cells was carried out using the Lipofectamine Plus method (Gibco BRL), according to manufacturer’s instructions, using 10 µg DNA of pcDNA3 constructs per culture (10 cm dishes). Cultures were analyzed for 72 h post-transfection; transfection efficiency was ~30%, as assessed by parallel transfections with lacZ. Transfection of 293T/17 cells was carried out essentially as described (35), using 30 µg DNA/calcium phosphate co-precipitates and chloroquine (Sigma). Cells were analyzed 72 h post-transfection; transfection efficiency was ~80%.
Expression vectors
For bacterial expression, a region of torsinA was expressed as a GST fusion protein. This fusion protein, GST–C6/C7, which includes torsinA residues 208–249, was amplified by RT–PCR using normal adult human fibroblast RNA. The oligonucleotide primers had an EcoRI site at the 5" end and a NotI site at the 3" end. PCR amplifications were performed with Pfu polymerase (Stratagene, La Jolla, CA), the amplified fragments were digested with EcoRI and NotI and subcloned into the pGEX4T-1 vector (Amersham Pharmacia, Piscataway, NJ). The integrity of the insert was confirmed by sequencing. Expression and purification of the GST fusion protein were performed essentially as described (39), with minor modifications, using Escherichia coli BL21 cells transformed with the pGEX4T-1 recombinant and induced with 0.05 mM isopropyl-ß-D-thiogalactopyranoside overnight at room temperature. Following cell lysis and removal of debris by centrifugation, the fusion protein was collected by binding to glutathione–Sepharose 4B (Amersham Pharmacia), and then eluted from Sepharose beads using 100 mM glutathione.
For mammalian expression, the entire coding sequences of human torsinA (1–332), both wild-type and mutant (GAG deleted), were cloned into the mammalian expression vector, pcDNA3 (Clontech, Palo Alto, CA). The full-length coding sequence of wild-type torsinA was also expressed as a fusion protein with GFP (torsinA 5" and GFP 3") employing the vector pEGFP-N3 (Clontech) including 331 amino acids of torsinA, three ‘stuffer’ amino acids and eGFP. Expression constructs were transfected into 293T/17 cells, CAD cells and SH-SY5Y cells (see above).
Generation of polyclonal and monoclonal antibodies for torsinA
Three anti-peptide antisera were produced against the following regions of torsinA: TA1, amino acids 51–63 (GQKRSLSREALQK); TAB3, amino acids 222–239 (WRSGKQREDIKLKDIEH); and TAB1, amino acids 299–312 (RVAEEMTFFPKEER). TA1 and TAB1 peptides were synthesized onto a lysine core (40); TAB3 peptide was conjugated to KLH (41). Peptide synthesis, immunization of rabbits and bleeding were performed by Research Genetics (Huntsville, AL). Peptide antibodies were purified by affinity elution from overexpressed torsinA–GFP following resolution by SDS–PAGE and transfer onto nitrocellulose, employing the method of Olmsted (42).
For monoclonal antibody production, four mice were injected with 50 µg purified GST–C6/C7 fusion protein. After the final boost, the best responders were killed, and spleens were removed and fused to SP2 myeloma cells (obtained from Dr E. Harlow, Massachusetts General Hospital). Fusions and selection were carried out as described (43). The antibody, D-MG10 was determined to be an IgG1, IgL
using an isotyping kit (Amersham Pharmacia).
For preabsorption (blocking) experiments, the rabbit antibody, TAB1, was incubated with 100 µg TAB1 peptide for 2 h at room temperature, and the mouse monoclonal D-MG10 was incubated with 100 µg GST–C6/C7 for the same period.
Antibodies/lectin
Other antibodies used in this study included rabbit antisera (at 1:1000 for western blots, unless otherwise indicated) to: nSec1 (Stressgen Biotech., Victoria, BC, Canada), a protein associated with neuronal vesicles; VAMP-2 (synaptobrevin), an integral membrane protein in synaptic vesicles (Stressgen); and synaptophysin (Stressgen), also an integral membrane protein of synaptic vesicles; and mouse monoclonal antibodies to: SV2, a synaptic vesicle membrane protein [NICHD Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA (44)]; Grp75 to mitochondrial Hsp70 (Stressgen); GM130 to a cis-Golgi matrix protein (Transduction Labs, Lexington, KY); 1C4 to merlin (41); 13H9 to filopodia of growth cones (Dr Frank Solomon, MIT); SPA-891 to the ER protein, PDI (45) (Stressgen); LAMP-1 to a lysosomal membrane glycoprotein (NICHD Hybridoma Bank); to anti-ß-tubulin (NICHD Hybridoma Bank); and H68.4 to the transferrin receptor in endosomes (Zymed, San Francisco, CA). Fluorescein-labeled Vicia Villosa lectin (VVL, Vector Laboratories, Burlingame, CA) was used as a marker for the trans-Golgi (46).
PAGE and western blot analysis
Total cell lysates were prepared by washing the cells twice with phosphate-buffered saline (PBS), and resuspending the cell pellet in lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40 and protease inhibitors (PI; Complete; Boehringer Mannheim, Indianapolis, IN). Protein concentrations were determined using the Coomassie plus protein assay reagent (Pierce, Rockford, IL) and a bovine serum albumin (BSA) standard (Bio-Rad, Hercules, CA). Lysates were fractionated by electrophoresis in 10% polyacrylamide gels according to the method of Laemmli (47), electrophoretically transferred to nitrocellulose (Bio-Rad) and stained for total protein with 0.2% Ponceau-S (Sigma). After staining, the membranes were blocked overnight in 10% non-fat dry milk powder in TBST (150 mM NaCl, 50 mM Tris pH 7.9, 0.05% Tween). The blots were probed with torsinA antibodies diluted in 2% milk in TBST and the proteins were visualized with horseradish-peroxidase-conjugated secondary antibodies and the ECL Reagent system (Amersham Pharmacia, Piscataway, NJ). Affinity purified polyclonal antibodies were used at dilution of 1:40 or 1:60; the monoclonal antibody culture supernatants were used directly. Secondary antibodies for western blots were sheep anti-mouse IgG–HRP (1:10 000, Amersham Pharmacia) and donkey anti-rabbit IgG–HRP (1:7 500, Amersham Pharmacia). Controls included cells transfected with empty vector (data not shown).
Post-translation modifications
To evaluate glycosylation, endoglycosidase H or PNGase F digestion was performed on cell lysates prior to SDS–PAGE, as recommended by the manufacturer [New England Biolabs (NEB), Beverly, MA]. To evaluate phosphorylation, 20 µg total lysates from SH-SY5Y cells and CAD cells transfected with torsinA wild-type and mutant constructs were treated with 10, 20 and 50 U CIP (NEB) at 30°C for 16 h, prior to SDS–PAGE. Western blot analysis was performed using TAB1 antibody and control monoclonal 1D12 to NHE-RF.
Immunoprecipitation
Transfected CAD cells and non-transfected SH-SY5Y cells were harvested by trypsinization and centrifugation, and washed twice in Ca2+/Mg2+-free PBS by repeated centrifugation (1 000 g for 5 min) and resuspension. Pellets were resuspended in 1.2x pellet volume with lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40), incubated on ice for 30 min and kept at 4°C throughout. Lysates were centrifuged at 17 000 g for 30 min and supernatants were precleared by incubation with normal rabbit serum (10 µl per 500 µl lysate) and protein A/G–agarose beads (1:1, 50 µl; Boehringer Mannheim) for 1 h. Following centrifugation the supernatant was incubated with protein A/G–agarose and 10 µl TAB1 antiserum overnight. Beads were collected by centrifugation (saving first supernatant), washed five times with 500 µl 1% NP-40 lysis buffer with PI by repeated centrifugation, and then resuspended in 60 µl 1x sample buffer for SDS–PAGE. An irrelevant antibody, 1C4, and pre-immune sera (data not shown) were used as controls for immunoprecipitation.
Immunocytochemistry
Cells were transfected with expression constructs for wild-type or mutant torsinA and allowed to recover after transfection for 24 h before being trypsinized and plated on coverslips. Transfected CAD and non-transfected SH-SY5Y cells were differentiated and fixed with 4% paraformaldehyde in PBS at 37°C for 30 min. After thorough rinsing with PBS, cells were incubated with 0.1% NP-40 in PBS for 20 min followed by blocking solution, 10% goat serum (Vector Laboratories) in PBS, for 1 h. Cells were incubated with affinity eluted or monoclonal torsinA antibodies (undiluted) with or without other antibodies or lectins (see above) for 1 h. Fluorescein-conjugated goat anti-rabbit secondary antibody or rhodamine-conjugated goat anti-mouse secondary antibody (Tago Immunologicals, Camarillo, CA) was then used (30 min with 1:200 dilution in PBS plus 1% BSA). Coverslips were mounted onto slides using gelvatol mounting medium containing 15 µg/ml 1,4-diazabicyclo(2.2.2)octaine (DABCO; Aldrich Chemical Co., Milwaukee, WI), an anti-fade agent. Cells were examined on a Nikon fluorescence microscope using a 40x 1.3 N.A. and 60x 1.4 N.A. objectives with images recorded on Kodak Tri-X-Pan 400 (Eastman Kodak Co., Rochester, NY), or using a Zeiss IM35 with a planapo 63x 1.4 N.A. objective and recorded electronically on a Princeton cooled CCD camera using Metamorph. Confocal images were generated with a Nikon TE 300 microscope and a Bio-Rad MRC 100 Laser Confocal Imaging system. Controls included pre-immune sera, preabsorption with TAB1 peptide for TAB1 antiserum, and cells transfected with an empty vector, pcDNA3 (Clontech; data not shown).
Cryo-immunogold electron microscopy
CAD cells transfected with the mutant and wild-type torsinA constructs were grown for 1 and 2 days in DMEM/F12 medium. Cells were collected by rinsing monolayers off dishes with a stream of media and centrifugation. Cell pellets were washed three times with 200 mM HEPES buffer, pH 7.4, layered onto 4% paraformaldehyde, in HEPES buffer and centrifuged for 1 min at 8000 g. Cell pellets were fixed for 1 h at room temperature and then incubated at 4°C for an additional 4 h. The fixative was removed and cells were washed three times with HEPES buffer and then stored overnight at 4°C in the same.
Small pieces (~2 mm3) of cell pellet were infiltrated with 2.1 M sucrose in PBS containing 0.2 M glycine for 30 min and then mounted on an aluminium pin before being plunged into liquid nitrogen. Ultrathin sections (~60 nm thick) were cut at –120°C using a cryo-diamond knife (Reichert Ultracut-S Microtome, Germany) and transferred to formvar/carbon coated copper grids.
For immunogold staining, grids were treated with 0.15 M glycine to quench any residual fixative and then blocked with 1% BSA in PBS. Grids were then incubated with affinity eluted TAB1 antibody (undiluted) or antibodies to PDI (diluted 1/250), washed with PBS, and incubated with protein A conjugated to 10 nm gold particles (Dr Jan Slot, University Utrecht School of Medicine, The Netherlands). Grids were then washed with PBS, fixed with 1% glutaraldehyde in PBS briefly, washed with double-distilled water, then counterstained with 2% uranyl oxalate and completed by incubating in a 1.8% methylcellulose/0.3% uranyl acetate mixture. Grids were then examined on a JEOL 1200EX electron microscope.
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ACKNOWLEDGEMENTS |
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We thank Ms Denise Pinney, Dr Miguel Sena-Esteves, Ms Maryanne Anderson and Ms Barbara Jean Johnson for advice and help with techniques and reagents; Ms Maria Ericsson, Dr Jessie Garcia and Dr Jaime Garcia-Anoveros for advice and assistance with electron microscopy; Dr David Corey for insights into torsinA structure and help with imaging; Drs Alfred Goldberg and Dennis Brown for advice on cell biology; and Ms Suzanne McDavitt for skilled preparation of this manuscript. This work was supported by the Jack Fasciana Fund for Support of Dystonia Research and NINDS grants NS28384 (J.H., D.B., X.O.B.), NS37409 (X.O.B., L.O., V.R., D.J.), NS38372 (X.O.B., D.S., D.B.), NS24279 (V.R., C.G.-A.) and the Deutsche Forschungsgemeinschaft (P.Z.).
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FOOTNOTES |
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+ These authors contributed equally to this work
§ Present address: Department of Psychiatry Research, University of Zürich, 8008 Zürich, Switzerland
¶ Present address: Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
To whom correspondence should be addressed at: Massachusetts General Hospital-East, Department of Molecular Neurogenetics, 13th Street, Building 149, 6th floor, Charlestown, MA 02129, USA. Tel: +1 617 726 5728; Fax: +1 617 724 1537; Email: breakefi@helix.mgh.harvard.edu
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REFERENCES |
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