From the
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.
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)
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
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
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).
E18 rat hippocampal cells were cultured in
serum-containing media using previously described
methods(40, 41) . Bilateral hippocampi were dissected in
Ca
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
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
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).
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
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 (
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?
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.
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, (
)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) .
=
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.
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.
, 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).
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.
/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).
, 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.
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.
= 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.
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.
-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) .
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.
Table: Relationship of cell-associated SOD:Tet451 to
total SOD activity in N18RE105 cells
Table: Association of native human SOD-1 or SOD:Tet451
with cultured hippocampal neurons
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.