©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CuZn Superoxide Dismutase (SOD-1):Tetanus Toxin Fragment C Hybrid Protein for Targeted Delivery of SOD-1 to Neuronal Cells (*)

Jonathan W. Francis (1)(§), Betsy A. Hosler (2)(¶), Robert H. BrownJr. (2)(**), Paul S. Fishman (§) (3)(§§)

From the (1)Department of Anatomy, University of Maryland School of Medicine, Baltimore, Maryland 21201, the (2)Cecil B. Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, and the (3)Neurology Service, Baltimore Veterans Administration Medical Center, and the Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Increased levels of CuZn superoxide dismutase (SOD-1) are cytoprotective in experimental models of neurological disorders associated with free radical toxicity (e.g. stroke, trauma). Targeted delivery of SOD-1 to central nervous system neurons may therefore be therapeutic in such diseases. The nontoxic C-fragment of tetanus toxin (TTC) possesses the nerve cell binding/transport properties of tetanus holotoxin and has been used as a vector to enhance the neuronal uptake of proteins including enzymes. We have now produced a recombinant, hybrid protein in Escherichia coli tandemly joining human SOD-1 to TTC. The expressed hybrid protein (SOD:Tet451) has a subunit molecular mass of 68 kDa and is recognized by both anti-SOD-1 and anti-TTC antibodies. Calculated per mol, SOD:Tet451 has approximately 60% of the expected SOD-1 enzymatic activity. Analysis of the hybrid protein's interaction with the neuron-like cell line, N18-RE-105, and cultured hippocampal neurons by enzyme immunoassay for human SOD-1 revealed that SOD:Tet451 association with cells was neuron-specific and dose-dependent. The hybrid protein was also internalized, but there was substantial loss of internalized hybrid protein over the first 24 h. Hybrid protein associated with cells remained enzymatically active. These results suggest that human SOD-1 and TTC retain their respective functional properties when expressed together as a single peptide. SOD:Tet451 may prove to be a useful agent for the targeted delivery of SOD-1 to neurons.


INTRODUCTION

Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders(1, 2) . CuZn superoxide dismutase (SOD-1), ()a 32-kDa homodimeric enzyme involved in the scavenging of superoxide free radicals, has been widely studied as a neuroprotectant to ameliorate oxidative injury(3, 4, 5, 6, 7, 8) . However, at least two pharmacodynamic factors constrain the use of SOD-1 as a medical treatment. First, human SOD-1 is an intracellular, cytosolic enzyme with an isoelectric point of 4.5 and as such does not readily cross cell membranes(9) . Second, the molecular weight of SOD-1 is well below the renal glomerular filtration cutoff resulting in a rapid clearance of exogenous SOD-1 from the circulation(10) . To overcome these limitations, several modified forms of SOD-1 have been developed. These include SOD-1 entrapped in liposomes, conjugated to albumin or polyethylene glycol, or expressed as a fusion protein with heparin-binding peptide(7, 11, 12, 13) .

Mutations in human SOD-1 have recently been associated with the inherited form of amyotrophic lateral sclerosis(14) . Most of the mutations reduce enzyme activity and may also confer a novel, toxic function upon the enzyme(15, 16, 17, 18) . The possible benefit of supplemental SOD-1 in retarding the characteristic motor neuron degeneration and death in ALS led us to investigate vectors for enzyme delivery which are not only nervous system-specific, but which could target motor neurons in particular. Such a vector might also be beneficial in other diseases which may involve oxidative cellular injury including ischemia, trauma, sustained epileptic activity, Parkinson's disease, and Huntington's disease.

Tetanus toxin has a well-documented capacity for neuronal binding and internalization(19, 20, 21) . In particular, when administered systemically or intramuscularly to animals, the toxin is taken up selectively by motor neurons in the brainstem and spinal cord(22) . The clostridial neurotoxins, tetanus and botulinum toxin, have a common binary structure in which the heavy chain appears to mediate binding and the light chain is responsible for most of the direct toxicity. The carboxyl 451-amino-acid fragment of the heavy chain (tetanus toxin fragment C or TTC) retains the neuronal binding and uptake properties of the holotoxin without the toxic domains(23, 24, 25) . These characteristics led Bizzini et al.(23) to propose that TTC could be used to target and deliver proteins to motor neurons. Chemical conjugation of TTC to large proteins including enzymes has been shown to enhance their uptake by neurons in tissue culture (26) and by motor neurons in animal models(27, 28, 29) . We hypothesized that fusion of SOD-1 to TTC could increase delivery of SOD-1 to the central nervous system in general and motor neurons in particular, potentially providing effective enzyme therapy to neurons.

We therefore joined cDNAs encoding human SOD-1 and TTC in a vector for expression of protein in Escherichia coli. The synthesized hybrid protein, SOD:Tet451 (M = 68,000), contains TTC attached to the carboxyl terminus of the human SOD-1 subunit monomer. We have investigated both the biochemical and biological properties of this hybrid protein as an initial exploration of its potential to deliver SOD-1 to the nervous system.


EXPERIMENTAL PROCEDURES

Plasmid Construction

The TTC cDNA from plasmid SS1261 (from Dr. J. Halpern, FDA)(30) , encoding amino acids 856-1315 of tetanus holotoxin, was cloned into pBluescript KS (Stratagene) as a BamHI/HindIII fragment, producing plasmid pTTC-1. The ribosome binding site and translation initiation signals (XbaI/BamHI fragment) from pAR3040 (31, 32, available from Novagen, Madison, WI) were transferred to pBluescript (which contains a T7 promoter), constructing vector pBex, and to pTTC-1, constructing plasmid pTex (Fig. 1). High-copy number vectors such as pBluescript are not customarily used for protein expression in bacteria, in part because the encoded proteins may be expressed constitutively. While our encoded proteins were expressed to varying extents prior to induction, this expression did not appear to interfere with bacterial growth or the stability of the product.


Figure 1: Diagram of steps in construction of plasmid pSOD:Tet451. The TTC cDNA from plasmid SS1261 (30) was cloned into pBluescript KS as a BamHI/HindIII fragment, producing plasmid pTTC-1. The ribosome binding site (RBS) and translation initiation signals (XbaI/BamHI fragment; hatch marks) from pAR3040 were transferred to pTTC-1, constructing plasmid pTex. A cDNA clone for wild type SOD-1 was modified by PCR (described under ``Experimental Procedures''; solid bar) and cloned into pTex as an NdeI/BamHI fragment to generate the expression plasmid pSOD:Tet451. Restriction enzyme sites are indicated as follows: B = BamHI; H = HindIII; N = NdeI; X = XbaI.



A cDNA clone for wild type human SOD-1 was isolated from a lymphoblastoid cell line using reverse transcriptase-polymerase chain reaction, and cloned into pBluescript KS. The reverse transcriptase-PCR primers (upstream: 5`-GGA-ATT-CGT-TTG-CGT-CGT-AGT-CTC-CTG-CA-3`; downstream: 5`-GGA-ATT-CTT-CTG-ACA-AGT-TTA-ATA-CCC-AT-3`) incorporated EcoRI restriction sites for cloning. Subsequently, PCR was used to prepare two modified SOD-1 cDNAs for subcloning. Both reactions used an upstream primer (5`-GGG-AAT-TCC-ATA-TGG-CGA-CGA-AGG-3`) to convert the ATG translation start codon for SOD-1 into an NdeI restriction site. One PCR reaction used a downstream primer homologous to the T3 promoter in Bluescript. This modified cDNA was cloned into pBex as an NdeI/HindIII fragment for bacterial expression of wild-type recombinant human SOD-1 (pBex:SOD-1, for production of rhSOD-1). In a separate PCR reaction, the downstream primer (5`-CGC-GGA-TCC-TTG-GGC-GAT-CCC-AAT-3`) substituted a BamHI restriction site for the SOD-1 translation stop. The product NdeI/BamHI fragment was cloned into the corresponding sites in pTex to obtain plasmid pSOD:Tet451 (Fig. 1). pSOD:Tet451 encodes the desired hybrid protein: full-length human SOD-1 followed by 4 amino acids encoded by restriction enzyme cloning sites (BamHI and SalI), then amino acids 856-1315 of tetanus holotoxin (the carboxyl-terminal 451 amino acids of tetanus toxin heavy chain which constitute TTC plus the 9 amino acids of heavy chain sequence that immediately precede the amino terminus of TTC). The sequence of all PCR products was confirmed.

The two plasmids, pBex:SOD-1 and pSOD:Tet451, were each transferred to E. coli strain BL21DE3, which carries the phage gene for T7 polymerase (Novagen, Madison, WI), for protein expression. Overnight cultures were diluted 1:50 in NZCYM media plus 0.1 mM CuSO, 0.1 mM ZnCl, and 100 µg/ml ampicillin, and grown for several hours at 37 °C. Isopropyl-1-thio--D-galactoside (1 mM) was then added to induce the expression of T7 polymerase and, indirectly, the desired recombinant proteins.

Purification of Expressed Protein

Induced bacterial cultures were pelleted by centrifugation for 10 min at 4000 g. The pellets were resuspended in 50 mM potassium phosphate buffer, pH 7.8, containing 0.1 mM EDTA, 0.1% Triton X-100, 0.1% -mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.2 mg/ml lysozyme. Following sonication, total bacterial lysates were centrifuged at 30,000 g for 20 min at 4 °C. The resulting supernatants (soluble fractions) and pellets (insoluble fractions) were stored frozen at -20 °C until further use. Protein concentrations were determined using a Pierce Coomassie Blue protein assay (for lysates) or a bicinchoninic acid protein assay (for immunoaffinity-purified samples) (Pierce).

Recombinant protein expression and purification were initially evaluated by SDS-PAGE(33) . For immunoblotting, proteins were separated on 4-15% gradient mini-gels (Bio-Rad) and electrophoretically transferred onto nitrocellulose (0.2 µm porosity, Bio-Rad)(34) . Antibody detection of blotted proteins was carried out using a Vectastain ABC-alkaline phosphatase kit (Vector Laboratories). Primary antibody against human SOD-1 (sheep anti-SOD-1 total IgG fraction from Binding Site, Inc., San Diego, CA) was used at a concentration of 1.5 µg/ml. Primary antibody against TTC (rabbit anti-TTC whole antisera, Calbiochem) was used at a dilution of 1:20,000.

SOD:Tet451 was precipitated from the bacterial lysis supernatant by addition of ammonium sulfate to 45% saturation. Wild type recombinant human SOD-1 was obtained from the soluble lysate fraction as a 60-90% ammonium sulfate fraction. The precipitates were resolubilized in 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin and dialyzed to remove the ammonium sulfate. Immunoaffinity columns were prepared by immobilizing sheep anti-human-SOD-1 total IgG or rabbit anti-TTC total IgG to a hydrazide support following manufacturer's instructions (Pierce). For a typical affinity chromatography run, 8-10 mg (1 ml) of solubilized protein from the indicated ammonium sulfate fraction was adjusted to 150 mM NaCl and incubated on the column overnight at 4 °C. The column was then washed extensively with PBS containing antiproteases prior to elution of bound material with 50 mM sodium bicarbonate (pH 11.0). The eluted column fractions were immediately neutralized with 3 M sodium acetate (pH 5.2). Column fractions containing significant amounts of protein as assessed by their optical density at 280 nm were pooled and concentrated using a Centricon-50 (for SOD:Tet451) or Centricon-10 (for recombinant SOD-1) (Amicon).

SOD-1 Enzyme Activity Assays

Superoxide dismutase activity of recombinant proteins and commercial human SOD-1 prepared from erythrocytes (Sigma) was determined using slight modifications of previously described methods(35, 36, 37) . Briefly, this colorimetric assay is based on the ability of SOD to inhibit the superoxide-mediated reduction of nitro blue tetrazolium to formazan. For each protein tested, a concentration-response curve was constructed using a linear transformation of SOD-1 dose-response data as previously published (38). Transformed data were subjected to least squares linear regression, and specific activities (units of SOD/mg of protein) of the various proteins were then determined by deriving the mass of sample protein which produced 50% inhibition of the SOD-inhibitable nitro blue tetrazolium reduction.

Immunocytochemistry

N18-RE-105 cells were plated on 4-well Lab-Tek chambered slides (Nunc; pretreated overnight with 20 µg/ml poly-L-lysine) at 1 10 cells/0.5 ml/well and cultured as described previously with minor modifications (39). Cells were cultured for 3 days prior to use in indirect immunofluorescence experiments, when the cells were fixed in 2% formalin/PBS for 10 min at room temperature. After extensive washing, commercial recombinant TTC (Boehringer Mannheim) or experimental protein preparations were diluted in PBS/0.1% bovine serum albumin and added at room temperature for 2 h. The cells were then incubated for 1 h in primary antibody (rabbit anti-TTC antisera, 1:1000 dilution; Calbiochem), followed by a 1-h incubation with fluorescein-conjugated goat anti-rabbit IgG (1:100 dilution, Boehringer Mannheim). Cells were mounted in PBS:glycerol (1:1) containing 1 mg/ml propyl gallate as an antifading agent and photographed at an objective magnification of 40 using a Zeiss axiophot epifluorescent microscope with a fluorescein filter set.

E18 rat hippocampal cells were cultured in serum-containing media using previously described methods(40, 41) . Bilateral hippocampi were dissected in Ca/Mg-free Hanks' balanced salt solution (Life Technologies, Inc.) and dissociated by brief trypsin digestion and trituration. The final cell suspension was plated at 7 10/0.3 ml/well on 4-well Lab-Tek chambered slides (Nunc) which were pretreated for 3 h with 10 µg/ml poly-L-lysine in 100 mM borate buffer, pH 8.4. Cells were maintained at 37 °C under humidified conditions of 5% carbon dioxide, 95% air. The cultures were used for immunocytochemical experiments after 6 days in vitro. These studies were carried out as described above for the N18-RE-105 cells with the following additions: (i) the incubation of the cells with primary antibody following exposure to SOD:Tet451 included both rabbit anti-TTC antisera and mouse monoclonal antibody against glial fibrillary acidic protein (Sigma, 1:500 final dilution), and (ii) accordingly, the secondary antibody incubation included both fluorescein-conjugated goat anti-rabbit IgG as well as rhodamine-conjugated goat anti-mouse IgG (Sigma, 1:100 final dilution).

Binding/Internalization of SOD:Tet451 in Cultured Neuronal Cells

N18-RE-105 cells were plated onto Costar 12-well culture clusters (Costar) at 1 10 cells/ml/well and were used for experiments after 3 days in vitro. Alternatively, hippocampal cells were plated at 3.5 10 cells/0.5 ml/well and were used after 8-10 days in vitro. These primary cultures were treated with 5 µM 5-fluoro-2`-deoxyuridine (Sigma) after 3 days in vitro to inhibit the proliferation of non-neuronal cells. Hippocampal cultures grown under these conditions typically resulted in a cell population which was 90% neurofilament-positive after 2 weeks in vitro (42).

The effect of dose on the association of SOD:Tet451 with either N18-RE-105 cells or hippocampal neurons was examined by incubating the cells with various concentrations of hybrid protein in DMEM for 1 h at 37 °C. The relative efficiency of SOD:Tet451 versus native human SOD-1 for raising levels of cell-associated human SOD-1 in hippocampal cultures was studied under the same incubation conditions using a fixed concentration of ligand.

Hybrid protein internalization was assessed using a protocol modified from previous studies of tetanus toxin internalization into N18-RE-105 cells (43) or PC12 cells(44) . Like these earlier studies, our experiments used a specialized binding buffer (0.25% sucrose, 20 mM Tris acetate, 30 mM NaCl, 1 mM CaCl, 1 mM MgCl, 0.25% bovine serum albumin, pH 7.0) for incubation of the cells with SOD:Tet451 in order to optimize levels of cell-associated hybrid protein. Briefly, N18-RE-105 cells were incubated with 2 µg/ml SOD:Tet451 for 1 h at either 0 °C or 37 °C. After this time, half of the cells at each temperature were incubated with 0.1 µg/ml Pronase E (Sigma) for 10 min at 37 °C, followed by washing with a mixture of anti-proteases (5 mM aminocaproic acid, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride). This Pronase digestion paradigm was also used to look at the cellular persistence of hybrid protein after pulse treatment of cells with SOD:Tet451. Cells were incubated in DMEM with 2 µg/ml SOD:Tet451 for 1 h at 37 °C. Following removal of the hybrid protein, the cells were washed and incubated at 37 °C in DMEM for various amounts of time prior to the Pronase step. For all experiments dealing with the interaction of SOD:Tet451 with living N18-RE-105 cells or hippocampal cultures, cells were pelleted and lysed using previously described methods(45, 46) . Lysates were clarified by centrifugation for 5 min at 1000 g.

EIA for Human SOD-1

Hybrid protein binding and internalization into intact neuronal cells was analyzed through the use of a two-antibody sandwich EIA specific for human SOD-1. Corning 96-well polystyrene enzyme-linked immunosorbent assay plates (Fisher Scientific) were coated with 10 µg/0.1 ml/well of sheep anti-human SOD-1 total IgG (Binding Site, Inc.) in 100 mM sodium carbonate buffer, pH 9.6. The plates were incubated for 4 h at 37 °C and then stored overnight at 4 °C. The next day, the wells were washed and blocked for 15 min with 1% bovine serum albumin in wash buffer (PBS, pH 7.4, containing 0.02% thimerosal and 0.05% Tween 20). The blocking solution was aspirated and 50 µl of mouse anti-human SOD-1 (Sigma, 1:5000 final dilution) was added along with either 50 µl of SOD:Tet451 (standard curve) or 50 µl of cell lysate. The antibody/antigen mixture was incubated at room temperature for 3 h, the wells were washed, and bound mouse antibody was detected with 0.1 ml/well of alkaline phosphatase-conjugated goat anti-mouse IgG (Boehringer Mannheim; 1:2000 final dilution). The plate was incubated at room temperature for 1 h, and the wells were washed 4 times prior to the addition of alkaline phosphatase substrate. The enzyme reaction was initiated by adding 0.2 ml/well of 4-nitrophenyl phosphate (Boehringer Mannheim, 1 mg/ml in 10 mM diethanolamine, pH 9.5). The rate of change in absorbance at 405 nm was measured at ambient temperature with a Thermomax microplate reader (Molecular Devices, Menlo Park, CA) in conjunction with Softmax for Windows software (Molecular Devices). The concentration of SOD:Tet451 in N18-RE-105 cell lysates was derived from a hybrid protein standard curve with a linear concentration range of 1.6-25 ng/ml.


RESULTS

SDS-PAGE and Western Blot Analysis

Soluble protein fractions from bacterial lysates containing the SOD:Tet451 hybrid protein or recombinant human SOD-1 were subjected to SDS-PAGE analysis performed under reducing conditions (Fig. 2A). Compared to the soluble protein fraction obtained from bacteria containing the expression plasmid without an insert, SOD:Tet451 extracts showed a novel, minor band at the predicted subunit molecular mass of 68 kDa. The soluble extract from bacteria expressing wild-type human SOD-1 also revealed a novel band at the subunit molecular mass of approximately 19 kDa. The identities of these bands were confirmed by Western blots using antibodies against human SOD-1 and TTC (Fig. 2, B and C, lanes 3 and 5). As evidenced by the presence of a single major immunoreactive band in each recombinant protein extract, the proteins were obtained from the soluble lysate fraction predominantly as full-length products (Fig. 2, B and C, lanes 3 and 5). Although recombinant human SOD-1 represents a substantial portion of the total bacterial protein in some preparations (estimated on Coomassie Blue gels at 20-30%), SOD:Tet451 is uniformly expressed at a lower level. The lower expression level for the hybrid protein incorporating TTC is consistent with previous reports that the unmodified TTC coding sequence is not expressed well in E. coli(47, 48, 49) . The bacterially produced human SOD-1 protein showed a reduced mobility on SDS-PAGE gels relative to that predicted by its amino acid composition, as has been reported previously(50, 51) . The higher molecular mass band detected in the recombinant human SOD-1 sample by the anti-SOD-1 antibodies (Fig. 2B, lane 6) is likely to be incompletely reduced/denatured human SOD-1 dimers(52) .


Figure 2: SDS-PAGE and Western blot analysis of SOD:Tet451, TTC, and recombinant human SOD-1. A, SDS-PAGE Coomassie Blue-stained gel. Amounts of protein loaded in the lane are given in parentheses. Lane 1, protein molecular mass standards; lane 2, commercial human SOD-1 (3 µg); lane 3, commercial TTC (4 µg); lane 4, Bex vector without cDNA insert, total protein, soluble fraction (10 µg); lane 5, SOD:Tet451, total protein, soluble fraction (10 µg); lane 6, SOD:Tet451, anti-TTC-column purified (4 µg); lane 7, rhSOD-1, total protein, soluble fraction (10 µg); lane 8, recombinant human SOD-1, anti-SOD-1-column purified (3 µg). B, Western blot of SDS-PAGE gel, with anti-SOD-1 antibody. Amounts of protein loaded in the lane are given in parentheses. Lane 1, commercial human SOD-1 (500 ng); lane 2, Bex vector without cDNA insert, total protein, soluble fraction (5 µg); lane 3, SOD:Tet451, total protein, soluble fraction (5 µg); lane 4, SOD:Tet451, anti-TTC-column purified (1 µg); lane 5, recombinant human SOD-1, total protein, soluble fraction (5 µg); lane 6, recombinant human SOD-1, anti-SOD-1-column purified (250 ng). C, Western blot of SDS-PAGE gel, with anti-TTC antibody. Amounts of protein loaded in the lane are given in parentheses. Lane 1, TTC (50 ng); lane 2, Bex vector without cDNA insert, total protein, soluble fraction (5 µg); lane 3, SOD:Tet451, total protein, soluble fraction (5 µg); lane 4, SOD:Tet451, anti-TTC-column purified (1 µg); lane 5, recombinant human SOD-1, total protein, soluble fraction (5 µg); lane 6, recombinant human SOD-1, anti-SOD-1-column purified (250 ng).



Immunoaffinity-purified samples of SOD:Tet451 showed significant enrichment of the 68-kDa protein as determined by SDS-PAGE analysis (Fig. 2A). This protein was recognized on immunoblots by antibodies against either SOD-1 or TTC (Fig. 2, B and C, respectively), and affinity-purified material appeared identical by SDS-PAGE and Western blotting regardless of whether anti-SOD-1 or anti-TTC antibodies were used in the purification (not shown). For wild-type human SOD-1, affinity-purified samples showed a subunit molecular mass of approximately 19,000 daltons following SDS-PAGE under reducing conditions (Fig. 2A). This protein was recognized on immunoblots by anti-SOD-1 (Fig. 2B) but not anti-TTC antibodies (Fig. 2C).

Enzyme Activity

The enzymatic activities of commercial human SOD-1 isolated from erythrocytes, recombinant human SOD-1, and the SOD:Tet451 hybrid protein are summarized in Fig. 3(inset). The affinity-purified recombinant human SOD-1 possesses greater than 80% of the enzymatic activity present in the commercial preparation of human SOD-1. On a per mg of protein basis, SOD:Tet451 was only 10-13% as active as the commercial enzyme. However, due to the greatly increased molecular mass of the hybrid protein relative to wild-type SOD-1, specific activity calculated on a basis of total protein does not accurately reflect the amount of activity retained by each molecule. When the enzymatic activities of the various proteins were calculated on a molar basis, the specific activities of the recombinant enzyme and the hybrid protein were more comparable (47 units/nmol versus 26-33 units/nmol, respectively). The hybrid protein showed similar specific activity regardless of whether it was affinity-purified using anti-SOD-1 or anti-TTC antibodies. This suggests that the SOD activity present in the hybrid protein preparations is not due to SOD domains which lacked a TTC moiety before immunoaffinity purification. This interpretation is consistent with the results from our immunoblot analysis with anti-SOD-1 antibodies, in that SOD-1 immunoreactivity was associated with full-length hybrid protein and not with any potential contaminating cleavage or degradation products (Fig. 2B). The activities of all samples were inhibited by 1 mM KCN, confirming that the observed activities are due to the human CuZnSOD rather than bacterial SOD, which is not inhibited by KCN(51) .


Figure 3: Concentration-dependent SOD activity of commercial human SOD-1, recombinant human SOD-1, and SOD:Tet451. The commercial enzyme standard and affinity-purified recombinant proteins were analyzed for SOD enzyme activity as defined by inhibition of superoxide-mediated nitro blue tetrazolium reduction (see ``Experimental Procedures'' for details). Data are plotted according to Asada et al. (38). Concentration-response curves were linear for all samples tested (r > 0.995). Inset: SOD specific activity of commercial human SOD-1 and affinity-purified recombinant proteins. Specific activity was determined by estimating the sample protein concentration at which there was 50% inhibition of SOD-inhibitable nitro blue tetrazolium reduction ([V/v] - 1 = 0.73 for this assay; see ``Experimental Procedures'' for details).



Immunocytochemistry

To determine whether the SOD:Tet451 hybrid protein possesses the characteristic nerve cell binding properties of TTC, we first compared the immunocytochemical labeling profile of SOD:Tet451 to commercial recombinant TTC in the neuroblastoma hybrid cell line, N18-RE-105. Unlike most neuroblastoma cell lines, N18-RE-105 cells have a surface ganglioside composition similar to normal brain tissue and thus bind high amounts of tetanus toxin(43) . Both recombinant TTC and the SOD:Tet451 hybrid protein bound to fixed N18-RE-105 cells (Fig. 4). At equal protein concentrations, the labeling intensities for commercial TTC and SOD:Tet451 appeared to be equivalent. However, a 10-fold lower concentration of TTC showed markedly reduced labeling (data not shown). This finding suggests that the avidity of binding of SOD:Tet451 is similar to that of TTC. Labeling of N18-RE-105 cells was similar for SOD:Tet451 affinity-purified on either an anti-SOD-1 or anti-TTC column. Thus, SOD:Tet451 labeling of fixed membranes is also likely to be due to the full-length, hybrid protein rather than smaller degradation products. We verified the neuron-specific binding properties of SOD:Tet451 through similar studies on primary, mixed cultures of embryonic rat hippocampus. The hybrid protein appeared to interact exclusively with neuronal cell bodies and processes (Fig. 5B). As defined by their positive immunoreaction with antibodies against glial fibrillary acidic protein, astrocytes were uniformly unlabeled by anti-TTC antibodies following incubation with SOD:Tet451 (Fig. 5C). These findings are consistent with previous immunocytochemistry studies which have shown the neuron-specific binding of tetanus toxin or TTC(19, 30, 53) .


Figure 4: Immunocytochemical labeling of N18-RE-105 hybrid neuroblastoma cells following incubation with SOD:Tet451 or commercial TTC. Shown are phase contrast and fluorescence photomicrographs of N18-RE-105 cells stained immunocytochemically with rabbit anti-TTC antisera after exposure to bovine serum albumin (1 mg/ml, A and B), commercial TTC (5 µg/ml, C and D), SOD:Tet451 anti-TTC affinity-purified (5 µg/ml, E and F), or SOD:Tet451 anti-SOD-1 affinity-purified (5 µg/ml, G and H). Ligand binding was visualized indirectly with fluorescein-labeled secondary antibodies. Cell binding of SOD:Tet451 appeared to be equivalent to TTC regardless of whether the hybrid protein was purified using anti-TTC or anti-SOD-1 antibodies (compare D, F, and H). Bar in A = 30 µm.




Figure 5: Immunocytochemical labeling of primary dispersed cultures of embryonic rat hippocampus following incubation with SOD:Tet451. Shown are phase contrast (A) and fluorescence photomicrographs of hippocampal cells stained immunocytochemically with rabbit anti-TTC antisera (B) and mouse monoclonal antibody against glial fibrillary acidic protein (C) after exposure to SOD:Tet451. Antigens were visualized indirectly with the appropriate rhodamine- or fluorescein-labeled secondary antibody. A, B, and C are the same photographic field. Arrows in A and B indicate neuronal cell bodies for orientation. Bar in C = 30 µm.



Binding and Internalization in Cultured Neuronal Cells

The capacity of SOD:Tet451 to deliver human SOD-1 to intact cultured neuronal cells was evaluated using an antibody-sandwich enzyme immunoassay (EIA) specific for human SOD-1. The N18-RE-105 cell line, like mammalian brain tissue, possesses high levels of endogenous SOD-1 (1-2 µg of SOD-1/mg of protein). Determination of cell-associated SOD:Tet451 by EIA allowed us to specifically assay for the hybrid protein human SOD-1 moiety in spite of a high background level of rodent SOD-1. Incubation of cells with increasing amounts of hybrid protein resulted in a dose-dependent increase in cell-associated SOD:Tet451 which was linear over the concentration range of 0.3-10 µg/ml (Fig. 6). N18-RE-105 cells incubated with 0.1 µg/ml SOD:Tet451 failed to show a level of cell-associated hybrid protein which could be reliably detected by our EIA, while limiting amounts of hybrid protein did not permit us to thoroughly examine treatment concentrations above 10 µg/ml.


Figure 6: Dose-response curve for SOD:Tet451 association with N18-RE-105 cells. Cells were incubated at 37 °C for 1 h with various concentrations of SOD:Tet451 in DMEM. Cell lysates were analyzed for cell-associated hybrid protein by EIA for human SOD-1. Data points are means ± S.E. of 4-5 replicates for each dose.



To determine if the hybrid protein is internalized into N18-RE-105 cells, we examined whether cell-associated SOD:Tet451 is resistant to brief Pronase treatment of intact cells. This experimental paradigm has been useful in previous studies of the internalization of tetanus toxin (43, 44) and is based on the observation that binding to a cell-surface membrane (Pronase-sensitive) compartment is not a temperature-dependent process, whereas internalization (movement of the ligand to a Pronase-resistant location) is reduced at low temperature. Similar to these earlier reports, our internalization study used a low ionic strength, neutral pH binding buffer to optimize cellular binding of SOD:Tet451 under the two different incubation temperatures. As expected with the use of a tetanus toxin-like ligand, N18-RE-105 cells not incubated with Pronase had approximately equal amounts of cell-associated SOD:Tet451 regardless of incubation temperature (Fig. 7). However, while less than 15% of cell-associated hybrid protein was resistant to Pronase treatment when cells were incubated at 0 °C, greater than 90% of cell-associated SOD:Tet451 was Pronase-resistant after incubation at 37 °C. When cells were incubated at 37 °C with hybrid protein in DMEM instead of binding buffer, there were considerable reductions in the levels of both total cell-associated and Pronase-resistant SOD:Tet451 (Fig. 8). Under these physiological conditions, only about one-third of cell-associated SOD:Tet451 was seen to be resistant to Pronase digestion. Thus, while the overall efficiency of hybrid protein uptake appears to be diminished under physiological conditions, it is nonetheless apparent that SOD:Tet451 is readily internalized into N18-RE-105 cells.


Figure 7: Effect of Pronase treatment on cell-associated SOD:Tet451 in N18RE105 cells. Cells were incubated for 1 h at either 0 °C or 37 °C with a fixed concentration of SOD:Tet451 in binding buffer. Thereafter, half the cells at each temperature were briefly treated with Pronase to define internalized SOD:Tet451. Cell lysates were analyzed for cell-associated hybrid protein by EIA for human SOD-1. Results are means ± S.E. of 5-6 replicates for each treatment.




Figure 8: Persistence of cell-associated SOD:Tet451 in N18RE105 cells following pulse incubation with hybrid protein. Cells were incubated at 37 °C for various amounts of time in DMEM containing 2 µg/ml SOD:Tet451. For each time point, half the cells were treated with Pronase to define internalized hybrid protein. Results are means ± S.E. of 4-5 replicates for each treatment.



The cellular uptake and persistence of SOD:Tet451 under physiological conditions was examined in N18-RE-105 cells which were pulse-loaded with hybrid protein and chased in DMEM. The results of this experiment show a relatively rapid loss of total cell-associated SOD:Tet451 over the first 4 h, followed by a much more gradual loss over the next 20 h (Fig. 8). The majority of the early loss of cell-associated hybrid protein appeared to be from the noninternalized (Pronase-sensitive) compartment. During this early phase, levels of Pronase-sensitive SOD:Tet451 declined to 13% of the levels initially present at time zero, whereas levels of internalized hybrid protein were reduced to only 30% of their initial value. The small amount of hybrid protein that remained associated with the cells after 24 h appeared to be only in the internalized compartment.

To ascertain whether levels of cell-associated SOD:Tet451 as determined by EIA represent enzymatically active hybrid protein, we measured cell-associated SOD:Tet451 and total SOD activity in N18-RE-105 cells which had been incubated with a relatively high concentration of hybrid protein. As shown in , under conditions which optimize hybrid protein binding/internalization, cells treated with 25 µg/ml SOD:Tet451 showed a modest elevation of total SOD activity relative to untreated controls. Statistical analysis of the enzyme activity results by one-way analysis of variance was unable to demonstrate a significant treatment effect for these small groups (n = 4) at the level of = 0.05 (df = 2, F = 4.13, p = 0.053). The incremental increase in SOD activity (0.4 µg of SOD/mg of protein) detected following SOD:Tet451 incubation appeared consistent with the amounts of cell-associated hybrid protein as measured by EIA, adjusting for the molar specific activity of the hybrid protein and the partial purity of the affinity-purified sample.

Because the N18-RE-105 cell line has certain characteristics which are not typical of mammalian nerve cells, we confirmed the neurotropic property of SOD:Tet451 by demonstrating its binding to cultured embryonic rat hippocampal neurons. Similar to our findings with the neuronal cell line, the association of hybrid protein with hippocampal cultures appeared to be dose-dependent and linear over the concentration range examined (Fig. 9). The primary neuron cultures also showed higher levels of cell-associated SOD:Tet451 when compared to N18-RE-105 cells. This result is consistent with an earlier study of tetanus toxin binding which showed that the toxin binding capacity of synaptic membrane preparations from rat brain was almost 3-fold greater than microsomal fractions from N18-RE-105 cells (43).


Figure 9: Dose-response curve for SOD:Tet451 association with cultured hippocampal neurons. Hippocampal cultures (10 days in vitro) were incubated with various concentrations of hybrid protein in DMEM for 1 h at 37 °C. Cell lysates were analyzed for cell-associated SOD:Tet451 by EIA for human SOD-1. Results are means ± S.E. of 4-5 replicates for each dose.



Finally, the efficiency of the hybrid protein in raising levels of cell-associated human SOD-1 in hippocampal cultures was directly compared to that of native human SOD-1. The hybrid protein was far superior to the native enzyme in delivering the SOD-1 moiety to cultured neurons. As shown in , the efficacy of SOD:Tet451 for delivering human SOD-1 to hippocampal neurons from the incubation media was at least 1000-fold greater on a molar basis compared to the native form of the enzyme. During this relatively brief (1 h) exposure, a substantial fraction (approximately 6%) of the total hybrid protein initially present in the incubation media was delivered to the hippocampal neurons.


DISCUSSION

In this report, we describe the production and preliminary biological studies of a bifunctional protein, SOD:Tet451, composed of human SOD-1 and TTC. Our results strongly suggest that the individual components of the hybrid protein retain their respective functional properties when expressed together as a single linked peptide. Although previous genetic engineering studies have constructed other hybrid or fusion proteins involving either human SOD-1 (12, 54) or TTC(55) , SOD:Tet451 is the first recombinant hybrid protein designed to specifically exploit the neurotropic property of TTC to deliver another protein into nerve cells. The recombinant hybrid construct may be seen to offer certain advantages over a chemical conjugate of SOD-1 and TTC. The hybrid protein is obtained as a single molecular species compared to the mixture of different conjugate species typically present in chemical conjugates. Furthermore, the trace contamination of chemical conjugates with small amounts of unconjugated reactants could potentially confound the interpretation of studies assessing biological properties of the conjugate. The initial results presented in this study provide strong incentive for further experiments directed at assessing the neuroprotective potential of SOD:Tet451.

We elected to fuse the amino terminus of TTC to the carboxyl terminus of SOD-1 for three reasons. First, mutations at the amino terminus of SOD-1 (Ala-Val) significantly disrupt both the activity and stability of the enzyme(15, 16) . For this reason, we elected to leave the amino terminus of SOD-1 in its native state. Second, the amino terminus of TTC is normally joined by a peptide bond to the rest of the tetanus toxin heavy chain. And third, removal of the carboxyl-terminal 10 amino acids from TTC drastically reduces neuronal and ganglioside binding activity, suggesting that the carboxyl terminus should be left unmodified(56) .

SDS-PAGE and Western blot experiments show that SOD:Tet451 is expressed in E. coli at a subunit molecular mass of 68 kDa, in accord with the size of the predicted 617-amino-acid polypeptide. The hybrid protein is recognized on immunoblots by both anti-SOD-1 and anti-TTC polyclonal antibodies. This antigenic profile confirms the accurate readthrough and translation of the TTC moiety following initial translation of hSOD-1. Calculated per mol of protein, affinity-purified preparations of SOD:Tet451 show between 55 and 70% of the SOD activity present in recombinant human SOD-1 produced in the same bacterial expression system. Improved purification techniques may increase the specific activity of the hybrid protein preparations. It is not known at present whether the activity of SOD:Tet451 is attributable to a monomeric or homodimeric species. Given that monomeric SOD-1 is devoid of enzymatic activity(54) , it may be that the hybrid protein allows for normal dimerization of the SOD-1 subunit moiety.

SOD:Tet451 appears to be a bifunctional protein: in addition to its SOD enzymatic activity, it also binds to neuronal membranes. Our hybrid protein showed binding and internalization properties similar to both tetanus toxin and TTC. Similar to previous binding studies with tetanus toxin and TTC(56, 57) , the binding of SOD:Tet451 to intact neuronal cells under physiological conditions appeared to be nonsaturable over the concentration range tested. A significant portion of the bound hybrid protein was also rapidly internalized, particularly under conditions that favor binding of tetanus toxin(21, 43, 44) . Once internalized, the hybrid protein persists for hours within the cells.

Given the bifunctional properties of SOD:Tet451, does the hybrid protein enhance the cellular uptake of SOD? It is difficult to compare the efficacy of SOD:Tet451 in delivering SOD-1 to neuronal cells in vitro relative to other modified forms of the enzyme developed previously. We elected to use EIA and enzyme activity assay to assess cellular SOD-1 while other studies employed indirect/functional measurements to analyze delivered SOD-1(6, 58) . Although both native and modified forms of SOD-1 were shown to be neuroprotective in these earlier tissue culture models of hypoxia or starvation, high bath concentrations were invariably required (1000 units/ml)(6, 58) . Interestingly, a 5-fold higher uptake of native SOD-1 induced by K depolarization of superior cervical ganglion neurons resulted in cytoprotection from starvation(6) . We have demonstrated using primary hippocampal cultures that SOD:Tet451 is at least 1000-fold more efficient than the native SOD-1 enzyme in delivering hSOD-1 to neurons.

Numerous studies have demonstrated a neuroprotective role for exogenous SOD-1 in vivo as well(3, 4, 5, 7, 59) . In light of the poor pharmacodynamic properties of native SOD-1 previously discussed (8-10), many of these investigations employed modified forms of SOD-1 to enhance its association with cell membranes and/or extend its circulating half-life in vivo. Similarly, SOD:Tet451 potentially offers several advantages over native SOD-1 as a neuroprotectant in vivo. First, consistent with the recombinant SOD-1 polymer study by Hallewell et al.(54) , the larger molecular weight of SOD:Tet451 would be expected to result in an increased retention time in the circulation relative to native SOD-1. Second, while the uptake of SOD:Tet451 into normal central nervous system is likely to be restricted to neurons with peripheral axon terminals, the protein could be a valuable means of targeting SOD-1 to neurons at risk for oxidative damage in certain neurological disorders where the blood-brain barrier is compromised (e.g. stroke, trauma). Even with a normally functioning blood-brain barrier, the selective uptake of SOD:Tet451 by motor neurons in the spinal cord and brainstem could be a potential route for delivering SOD-1 to motor neurons in disorders such as familial ALS. Through this pathway, the hybrid protein could access other central nervous system neurons as well, given the ability of TTC to undergo retrograde trans-synaptic transfer(60) .

Finally, given the long half-life of tetanus toxin in the central nervous system in vivo (6.9 days(22) ) and in cultured neurons in vitro (5.7 days(61) ), one might expect that SOD:Tet451 would also have a long half-life in neurons. The half-life of TTC in nerve cells in vitro or in the central nervous system in vivo is unknown, although TTC immunoreactivity in the spinal cord has been demonstrated 20 days after a single injection (62). By contrast, in this study, we observed that SOD:Tet451 was eliminated from N18-RE-105 cells in about 24 h (Fig. 8), with a biphasic time course. The fairly rapid loss of cell-associated hybrid protein seen over the first 4 h is consistent with the short half-life of exogenous native SOD-1 following endocytosis into cultured endothelial cells, which was approximately 3-6 h(11) . Thus, while we do not yet know the intracellular lifetime of SOD:Tet451 in the central nervous system, it is possible that the SOD-1 enzymatic activity of the hybrid protein may persist for an extended period of time (24 h or more).

Although neuronal uptake of SOD:Tet451 is highly efficient, large quantities of SOD-1 will be required to raise cellular SOD-1 levels by physiologically meaningful amounts. Transgenic mice which overexpress human SOD-1 are resistant to the neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and also show reduced levels of brain damage following temporary cerebral ischemia or methamphetamine treatment(63, 64, 65) . However, the level of SOD-1 in the nervous system of these transgenic animals is over 3-fold greater than that of their nontransgenic littermates(66) . We did not test concentrations of our hybrid protein at levels high enough to expect a similar increment in cellular SOD-1 activity. Physiological solutions of 50-100 µg/ml would be expected to double SOD-1 levels in hippocampal cells if uptake remained nonsaturable at this concentration.

While there is great conceptual appeal for the potential use of SOD:Tet451 to treat various neurological diseases involving free radical damage to neurons, several caveats deserve careful consideration. Of fundamental importance is the question of whether a soluble, cytosolic enzyme such as SOD-1 will retain its enzymatic activity if compartmentalized into intracellular membrane-bound vesicles by the action of the TTC moiety. Even if SOD:Tet451 is enzymatically active in this atypical subcellular compartment, its potential neuroprotective properties may be changed due to the altered distribution of the enzyme in the cell. The antigenic properties of the TTC moiety of SOD:Tet451 are also a concern, as most people in developed countries have been immunized with tetanus toxoid to elicit circulating antibody. Indeed, TTC has been extensively evaluated as an alternative immunizing agent to tetanus toxoid(67, 68, 69) . Such circulating antibodies might interfere with the interaction of systemic SOD:Tet451 and its neuronal cell-surface receptor.

In the present report, we have clearly shown that SOD:Tet451 dramatically increases the delivery of human SOD-1 to neurons compared to the native enzyme. Future studies of this hybrid protein will allow us to answer two important questions. 1) What is the protective value of supplemental cellular SOD in the form of SOD:Tet451 in neurological diseases? 2) Are hybrid proteins containing the neuronal binding domain of tetanus toxin an effective means of functional enzyme delivery to the central nervous system?

  
Table: Relationship of cell-associated SOD:Tet451 to total SOD activity in N18RE105 cells

Cells were incubated with 25 µg/ml SOD:Tet451 in binding buffer for 1 h at 37 °C. Thereafter, half the cells were treated briefly with pronase. Cell-associated SOD:Tet451 was measured by EIA while total SOD enzyme activity was determined by extrapolation from a human SOD-1 standard curve (see ``Experimental Procedures''). Results are means ± S.E. of 4 replicates for each treatment.


  
Table: Association of native human SOD-1 or SOD:Tet451 with cultured hippocampal neurons

Hippocampal cultures (8 days in vitro) were incubated with either human SOD-1 or SOD:Tet451 in DMEM for 1 h at 37 °C. Cell lysates were analyzed for cell-associated ligand by enzyme immunoassay for hSOD-1.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Veterans Administration Merit Review Award.

Supported by National Institutes of Health Grant NS31248-01.

**
Supported by the Muscular Dystrophy Association, the Amyotrophic Lateral Sclerosis Association, the Pierre L. de Bourgknecht ALS Research Foundation, the Myrtle May MacLellan ALS Research Foundation, the C. B. Day Investment Company, Inc., the Abramson A. L. S. Research Fund, and National Institutes of Health Grant 1P01NS31248.

§§
To whom correspondence and reprint requests should be addressed. Tel.: 410-605-7000 (Ext. 6612); Fax: 410-605-7937.

The abbreviations used are: SOD-1, CuZn superoxide dismutase; TTC, tetanus toxin C-fragment; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; EIA, enzyme immunoassay; ALS, amyotrophic lateral sclerosis; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. J. Halpern for providing the cDNA for tetanus toxin fragment C, Dr. R. Schnaar for N18-RE-105 cells, Dr. T. Rogers for his helpful advice on hybrid protein internalization studies, and Wendy Chin for checking the partial sequence of two plasmid inserts.


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