Departments of 1 Hygiene and
2 Pathology II, Protein content and mRNA expression of
extracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using the
affinity-purified IgG against native mouse EC-SOD. EC-SOD could be
detected in all of the tissues examined (lung, kidney, testis, brown
fat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach,
spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as µg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat had
a high content of EC-SOD in terms of micrograms per milligram protein,
which corresponded to that of lung. Kidney showed the strongest
expression of EC-SOD mRNA. Relatively strong expression of the mRNA was
observed in lung, white fat, adrenal gland, brown fat, and testis.
Heart and brain showed only weak signals, and no such expression could
be detected in either digestive organs or skeletal muscle.
Immunohistochemically, EC-SOD was localized mainly to connective
tissues and vascular walls in the tissues examined. Deep staining in
the cytosol was observed in the cortical tubular cells of kidney. These
results suggest that EC-SOD is distributed systemically in
mice and that the physiological importance of this enzyme may be a
compensatory adaptation to oxidative stress, particularly in lung and
kidney.
enzyme-linked immunosorbent assay; immunohistochemistry; lung; white fat; kidney
SUPEROXIDE DISMUTASES (SOD; EC 1.15.1.1) are
metalloenzymes capable of scavenging the free oxygen radical superoxide
(13). Mammals have three forms of SOD, which are characterized by their metal ions and their different localizations. Copper, zinc-SOD (CuZn-SOD) is found in the cytosol and contains copper and zinc ions in
the molecule, whereas manganese-SOD (Mn-SOD) is located in the
mitochondria, with a manganese ion. Much is known about the biochemical
and physiological properties of these two isoenzymes (6, 14, 16, 26).
The third SOD isoenzyme, extracellular SOD (EC-SOD) was discovered by
Marklund (9) in 1982. EC-SOD is a copper- and zinc-containing secretory
enzyme that is located in extracellular fluid such as plasma (10) and
in the extracellular matrix of tissues (10, 11, 18, 24).
Only limited information could be obtained concerning the detailed
characterization of native EC-SOD because of the technical difficulties
in the purification and activity assay, although several investigators,
including our group, succeeded in purifying EC-SOD from human (9, 15, 19), rat (27), or mouse (17) tissues.
EC-SOD is thought to be the least abundant form of SOD in mammalian
tissues. Although most species appear to have a relatively high
concentration of EC-SOD in the lung, the ratio of EC-SOD activity to
the total SOD activity is only 5-10% even in the lung (10, 17).
Therefore, the SOD activities derived from intracellular CuZn-SOD and
Mn-SOD interfere greatly with the EC-SOD assay. Chromatography on gel
filtration (4) or concanavalin A-Sepharose (10) has thus been employed
to separate EC-SOD from other SODs, but these procedures are
complicated and difficult for a bulk assay. An ELISA using the specific
antibody against EC-SOD appears to enable us to directly measure levels
of EC-SOD as distinct from other SOD isoenzymes.
One of the unique properties of EC-SOD is an affinity for heparin
analogs (9, 24). After being released to the extracellular space,
EC-SOD is thought to be distributed to specific regions of
extracellular matrix or on the cell surface by its affinity for heparin
analogs. This allows EC-SOD to efficiently scavenge superoxide in
specific regions of the extracellular matrix (18, 20).
To our knowledge, this is the first report on the quantification of
mouse EC-SOD by an ELISA. We have examined the level of immunoreactive
EC-SOD in 16 mouse tissues. In addition to evaluation of the expression
of mRNA for the enzyme, immunohistochemical studies with the specific
antibody have been done.
Materials.
Mouse EC-SOD type C [which has the strongest affinity for heparin
analogs (9)] was purified from lung tissue as described previously (17). Male mice of strain C57BL/6 were purchased from Japan
SLC (Shizuoka, Japan). Restriction endonucleases and DNA
modifying enzymes were purchased from Takara Shuzo (Tokyo, Japan),
Promega (Madison, WI), or New England Biolabs (Beverly, MA). The other
reagents were of the highest grade available.
Animals and tissue preparations for ELISA and immunoblotting.
Male C57BL/6 mice were kept on a 12:12-h light-dark cycle at a room
temperature of 25°C. Mice had free access to standard diet and tap
water. The animals were cared for in accordance with the guiding
principles for the care and use of animals of the Helsinki Declaration.
For an ELISA, brain, lung, heart, stomach, ileum, colon, liver, kidney,
gastrocnemius muscle, interscapular brown fat, periepididymal white
fat, testis, adrenal gland, spleen, and pancreas were removed after
anesthesia. Blood was collected by a heart puncture before the tissue
collection. All of the tissues collected were homogenized in 9 vol of
Tris-buffered saline (20 mM Tris · HCl and 150 mM
NaCl, pH 7.4) with a Polytron homogenizer and then centrifuged at 5,000 g for 20 min. Protein concentration in
the supernatants was determined by a bicinchoninic acid protein assay
kit (Pierce, Rockford, IL), using BSA as a standard.
Preparation of polyclonal antibody.
The rabbit antiserum against the purified mouse EC-SOD was obtained as
described previously (17). An IgG fraction was precipitated with 33%
saturation of ammonium sulfate and then purified by a protein A
Cellulofine column (Seikagaku Kogyo, Tokyo, Japan). The protein A IgG
fraction was further purified by affinity chromatography with an
antigen-conjugated affinity column as follows.
Affinity purification of specific IgG.
The purified mouse EC-SOD (120 µg) was conjugated to a HiTrap
N-hydroxysuccinimide-activated Sepharose column
(Pharmacia, Uppsala, Sweden) according to the manufacturer's
instruction. The protein A IgG fraction was applied to a column
equilibrated with a binding buffer (50 mM sodium phosphate and 150 mM
NaCl, pH 7.4) at a flow speed of 0.1 ml/min and incubated at room
temperature for 20 min. After extensive washing with the binding
buffer, the bound fraction was eluted with an elution buffer (0.1 M
glycine hydrochloride, pH 2.5) at a flow speed of 0.5 ml/min. The
eluent was immediately neutralized by addition of 0.05 vol of 1 M Tris base solution. A preabsorbed IgG fraction was obtained as the flow-through fraction of the affinity chromatography by passes through
the column and then used for the control staining in an immunohistochemical study. Part of the affinity-purified antibody was
biotinylated using a biotin labeling kit (Boehringer Mannheim, Indianapolis, IN) as a secondary antibody in ELISA.
ELISA for mouse EC-SOD.
A double-antibody sandwich ELISA was developed to quantitate mouse
EC-SOD. All of the assay procedures were performed at room temperature.
A volume of 50 µl/well was used throughout unless otherwise
indicated. Plates were washed three times with a washing buffer (50 mM
sodium phosphate and 150 mM NaCl, pH 7.4, containing 0.05% Tween 20 vol/vol) between steps. The affinity-purified anti-mouse EC-SOD IgG was
coated onto 96-well MaxSorp immunoplates (Nunc, Roskilde, Denmark) at a
concentration of 2 µg/ml in a coating buffer (0.1 M bicarbonate
buffer, pH 9.6) for 2 h, followed by washing with a washing buffer
without Tween 20. Unoccupied binding sites on the immunoplates were
blocked by 2-h incubation with 1% gelatin and 0.5% BSA in the coating
buffer (300 µl/well). After a washing, plates were stored at
Immunoblotting.
The immunoblot detection of the samples separated on an SDS-PAGE gel
was performed as described elsewhere (17). Briefly, 30 µg of protein
from tissue homogenates and low-molecular-mass markers (Pharmacia,
Uppsala, Sweden) were separated on a 12.5% gel using a discontinuous
buffer system (8). Samples separated with SDS-PAGE were transferred to
ProBlott polyvinylidene difluoride membrane (Applied Biosystems, Foster
City, CA) with a semidry electroblotting apparatus, using an
Northern blot analysis.
Total RNAs were isolated from each mouse tissue using TRIzol reagent
(Life Technologies, Gaithersburg, MD). RNA (11 µg) was fractionated
by electrophoresis on a denaturing formaldehyde-1.2% agarose gel with
a 0.24- to 9.5-kb RNA ladder (Life Technologies) as a size marker and
transferred to a positively charged nylon membrane (Hybond N+; Amersham
Life Science, Arlington Heights, IL) by capillary action in 20×
SSC overnight (1× SSC, 150 mM NaCl, and 15 mM trisodium
citrate) followed by ultraviolet irradiation fixation. A
541-bp DNA fragment was amplified by PCR from mouse genomic DNA using
the primers EC213 (dGAGAAGATAGGCGACACGCA) and EC793c
(dCTCCCGCCGCCGCTTCTTGC), oligonucleotides corresponding to mouse SOD3
gene nt 213-232 and nt 774-793, respectively (2). The PCR
product was inserted into the Sma I
site of pBluescript II KS+ vector (Stratagene, La Jolla, CA) and then
propagated by the standard procedure (23). The DNA fragment was excised
with EcoR I and
BamH I, purified through agarose gel
electrophoresis, and then used as a probe. The blotted membrane was
prehybridized in the prehybridization solution (50% formamide,
3× SSC, 5× Denhardt's solution, 0.1% SDS, and 100 µg/ml
denatured shared salmon sperm DNA) at 42°C for 8 h, followed by
hybridization at 42°C overnight in the same buffer containing 5%
dextran sulfate (wt/vol; Pharmacia) with 1 × 106
counts · min Immunostaining of mouse tissues.
Air-dried frozen tissue sections were fixed for 5 min at room
temperature in 4% paraformaldehyde-PBS (pH 7.3) and blocked in PBS
containing 5% skim milk (wt/vol) for 10 min. Sections were incubated
with 1 µg/ml affinity-purified anti-mouse EC-SOD IgG at 4°C
overnight. Bound antibodies were detected with HRP-labeled donkey
anti-rabbit IgG polyclonal antibody diluted 1:100 (Chemicon, Temecula,
CA). HRP activity was developed for 5 min in a 50 mM Tris · HCl buffer (pH 7.6) containing 0.1% DAB and
0.01%
H2O2. The sections were briefly washed with PBS between steps. Controls were
performed by parallel incubation with the preabsorbed IgG (see above)
or without the primary antibody. Counterstaining was carried out using
hematoxylin.
Purification of polyclonal antibody by immunoaffinity
chromatography.
The IgG fraction prepared by ammonium sulfate precipitation followed by
protein A Cellulofine chromatography was further purified on a native
mouse EC-SOD-conjugated Sepharose column. The typical elution profile
of the immunoaffinity chromatography is illustrated in Fig.
1A.
Approximately 4-5% of IgG was eluted in the bound fraction. A
small amount of IgG could be found in the bound fraction (Fig.
1B) by two passes through the
affinity column. Through this affinity purification of the antibody,
nonspecific signals confirmed by Western blotting were decreased
dramatically (data not shown).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
20°C until use. The samples were diluted appropriately with
a dilution buffer (50 mM sodium phosphate and 150 mM NaCl, pH 7.4, containing 0.5% BSA; this buffer was used for all subsequent
dilutions), and the assay standards (serially diluted purified mouse
EC-SOD) were added to the wells and incubated for 2 h. After another
washing, the immunoplates were incubated with 0.6 µg/ml biotinylated
secondary antibody for 2 h, followed by incubation with horseradish
peroxidase (HRP)-conjugated streptavidin (Dakopatts, Glostrup, Denmark)
for 15 min. The ELISA was developed with
o-phenylenediamine (Sigma, St. Louis,
MO) and H2O2
according to the instructions from Sigma. The enzyme reaction was
stopped by addition of 50 µl of 0.5 M
H2SO4,
and the optical absorbance was measured with a Multiscan Multisoft
plate reader (Labosystems, Helsinki, Finland) at 492 nm.
-amino-n-caproic acid-Tris buffer
system according to instructions in a newsletter from Bio-Rad (Hercules, CA). The membrane was blocked with 3% BSA (fraction V;
Sigma) in a TN buffer (20 mM Tris · HCl and 500 mM
NaCl, pH 7.5) and then incubated with 2 µg/ml affinity-purified
rabbit anti-mouse EC-SOD IgG, followed by incubation with
HRP-conjugated goat anti-rabbit IgG polyclonal antibody (Dakopatts).
The antibody against EC-SOD was detected with a
3,3'-diaminobenzidine tetrahydrochloride (DAB)-H2O2
coloring system. Immunoblots were quantitatively analyzed with a GS700
imaging densitometer and Multi Analyst software (Bio-Rad).
1 · ml
1
of the 32P probe labeled with
[32P]dCTP (DuPont NEN,
Boston, MA) using the antisense sequence-specific primer EC793c and
Klenow fragment. The membrane was washed twice in 1× SSC-0.1%
SDS at room temperature and once in 0.1× SSC-0.1% SDS at
60°C. The membrane was exposed to X-ray film (Hyperfilm-MP; Amersham Life Science) with dual intensifying screens at
80°C for 3 days. Hybridization with a mouse
-actin cDNA
was performed to check the quality and quantity of RNA present in each
lane of the blots. Autoradiograms were quantitatively analyzed with a
BAS2000 phosphorimager (Fujix, Tokyo, Japan).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (12K):
[in a new window]
Fig. 1.
Affinity purification of rabbit anti-mouse [extracellular
superoxide dismutase (EC-SOD)] IgG on EC-SOD-conjugated Sepharose
column. Protein A-purified IgG fractions
(A), preabsorbed IgG
(B), and water blank
(C) were applied to a native mouse
EC-SOD-conjugated Sepharose column. A:
a big peak appeared as flow-through fractions after ~25 min of
retention time. After extensive washing with a binding buffer, ~5%
of protein A-purified IgG was eluted by an elution buffer with a sharp
peak (fractions collected are indicated by horizontal bar).
B: a very small part of IgG appeared
in binding fractions of preabsorbed IgG.
C: elution buffer shows an absorbance
at 280 nm (dashed horizontal bar).
Development of ELISA for mouse EC-SOD. Figure 2 shows a typical standard curve for EC-SOD obtained with ELISA. The working range appeared to be from 1 to 100 ng/ml, thereby indicating that this ELISA system enables us to measure EC-SOD levels in tissues and serum. All of the samples, including serum, needed an appropriate dilution (20-500 times) for the assay.
|
Tissue distributions of immunoreactive EC-SOD. Table 1 summarizes immunoreactive EC-SOD content in various mouse tissues. EC-SOD could be detected in all of the tissues examined, and the differences among individuals were very small. Lung showed the highest value both in terms of micrograms per gram of wet tissue and in terms of nanograms per milligram of protein. Kidney, testis, brown fat, and adrenal gland showed relatively higher contents, whereas brain, heart, stomach, ileum, skeletal muscle, spleen, and thymus belonged to the group with lower contents, particularly in terms of micrograms per gram of wet tissue. Except for contents in white fat, EC-SOD contents expressed in both of the two different units showed the same trend in all of the tissues examined. White fat showed a rather lower content in terms of micrograms per gram of wet tissue, probably because of the large mass of lipid within the tissue; however, it had a high content of EC-SOD in terms of nanograms per milligram of protein, which corresponded to that in lung.
|
Western blotting. The content of EC-SOD was also confirmed by Western blotting (Fig. 3A). The results of the immunostaining coincided roughly with those of ELISA. Our previous study revealed that mouse EC-SOD has a heterotetramer structure with subunits of two different sizes (33 and 35 kDa), both of which have equal reactivity to the antibody (17). The current study also confirmed this, the 35-kDa band being relatively stronger than the 33-kDa band. As shown in Fig. 3, B and C, densitometric quantification of EC-SOD in four tissues also indicated that the highest EC-SOD content was in lung. White fat and kidney showed 80 and 60%, respectively, of the lung value. These results also coincided well with the results of the ELISA (Table 1).
|
Tissue-specific expression of EC-SOD mRNA. To investigate the expression of mouse EC-SOD mRNA, 11 µg of total RNA from 16 different tissues were analyzed by Northern blot hybridization. As shown in Fig. 4A, a discrete band could be seen in some tissues at 1.8 kb. Kidney showed the strongest expression. Relatively strong expressions were also observed in lung, white fat, adrenal gland, brown fat, and testis. Heart and brain showed only weak signals, and no such expression could be detected in either digestive organs or skeletal muscle.
|
Immunohistochemistry. Immunohistochemical examination was done in some selected tissues, and the results are summarized in Table 2. In the mouse lung, deep staining could be observed in extracellular portions such as connective tissue around blood vessels and bronchi, alveolar septa, and vascular walls (Fig. 5A). In kidney, all tubular cells in cortical areas were clearly positive in the cytoplasm as well as in the extracellular portions (Fig. 5C). Glomeruli were negative, except that nuclei faintly stained in some cells. Medullary areas contained sparsely stained tubules. In some tissues, peripheral nerve fibers and cell nuclei were deeply stained.
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DISCUSSION |
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Although several investigators have reported an ELISA for human EC-SOD using a monoclonal antibody (1) or a combination of polyclonal and monoclonal antibodies (5), the lack of a reliable quantitative assay for convenient laboratory animals has limited study of the role of EC-SOD in mammals other than humans. This is the first report about ELISA for mouse EC-SOD. Immunochemical assay methods such as ELISA offer some advantages over enzymatic methods, especially for EC-SOD, because the determinations are specific to the protein moiety so that they are not interfered with by other SOD isoenzymes (5). On the other hand, in addition to its complexity, EC-SOD activity assay still appears to have some reliability problems due to the presence or absence of cofactors, activators, and inhibitors (14).
The distribution of EC-SOD in mouse tissues agreed well with those reported by Marklund (estimated by the enzyme activity) (10). Because we omitted perfusion in the tissue collection protocols, contamination from the remaining blood should be considered. The serum level of EC-SOD was 4.05 ± 0.21 µg/ml, so, if blood occupies 5% of the tissue weight at the maximum, the contribution of the blood contamination is estimated to be <12% even in the tissue with the lowest content, brain. Thus, even if there was blood contamination to a greater or lesser degree in the current study, the influences on the actual values of EC-SOD in each tissue are not considered to be potent.
Mammals seem to have acquired the latest SOD, EC-SOD, about four hundred million years ago to prevent increasing oxidative stress as the oxygen content of the air gradually increased to the present level (20.9%). Lung is a unique organ that is always exposed to much oxygen, implying a sustained occurrence of reactive oxygen species (ROS) (7). It appears, therefore, to stand to reason that lung has the highest content of EC-SOD.
Moreover, lung is an organ rich in vessels and connective tissues. This also seems to be a reason for the high level of EC-SOD in lung, because EC-SOD is ample in the connective tissues and vascular smooth muscles with its affinity for heparin analogs (20). However, the bulk of SOD activity in tissues, including lung, is derived from CuZn-SOD and Mn-SOD. EC-SOD accounts for only 5-10% of the total SOD activity even in lung (10, 17). So, one may deduce that EC-SOD counts for nothing as a protector against ROS. On the other hand, Oury et al. (19, 21) hypothesized that scavenging of extracellular superoxide by EC-SOD is likely to play an important role in mediating the nitric oxide responses of vascular smooth muscles.
Unexpectedly, fat tissues, particularly white fat tissue, had a higher content of EC-SOD. One reason for the high content of EC-SOD in fat tissues may be that the tissues are rich in vessels and connective tissues. On the other hand, white fat tissues occupy a large mass in the body as subcutaneous and intra-abdominal fat pads. They are also scattered all over the areolar connective tissues. Moreover, fat tissues per se seem to be vulnerable to oxidative stress because they contain a high percentage of unsaturated fatty acids. These observations suggest that EC-SOD in fat tissues makes a considerable contribution to protection against ROS in the body.
Although brain is one of the main organs involved under oxidative stress, consumes 20% of the oxygen received by the body, and has the highest blood flow, it had the lowest EC-SOD content in terms of micrograms per gram of wet tissue among the 16 tissues examined. This low content of EC-SOD may be due mainly to the existence of the blood-brain barrier, as well as to low EC-SOD expression. Moreover, Oury et al. (22) reported that the overexpression of EC-SOD in brain increased central nervous system (CNS) oxygen toxicity in transgenic mice. Thus the low content of EC-SOD in the CNS appears to be physiologically reasonable.
The mRNA levels of EC-SOD did not always parallel the enzyme protein levels (determined by ELISA and Western blotting) in several tissues; for instance, kidney had the strongest expression of EC-SOD mRNA, but its EC-SOD content was not very high, and, despite the lack of expression of mRNA, digestive organs and skeletal muscle showed low but detectable contents of the enzyme. However, such discrepancies between gene expression and protein content remain to be elucidated.
The pattern of EC-SOD mRNA expression in mice obtained in the current study was different from the finding in humans by Folz and Crapo (3) that expression of mRNA was detectable in heart, placenta, pancreas, lung, and kidney. The reason for the difference in EC-SOD mRNA expression between humans and mice remains unclear, although it would probably not be denied that the difference is attributable to an evolutionary divergence of rodents and primates.
Effects of cytokines or growth factors on EC-SOD expression were
investigated by Marklund (12) using a human skin fibroblast cell line.
He has reported that EC-SOD is induced by interferon (IFN)- and
interleukin (IL)-1
and depressed by tumor necrosis factor-
and
transforming growth factor-
but not affected by IFN-
, IL-2, IL-3,
IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, or
growth hormone. On the other hand, no effects of oxidative stress were
observed in the study of Strålin and Marklund (25). It would
probably not be denied that EC-SOD expression in mouse kidneys may be
induced by cytokines and/or growth factors; however, clearly
further work needs to be done.
Imunohistochemical examinations showed deep staining of connective tissues and vascular walls in lung. Lung is the organ that has been most extensively examined as to the immunohistochemical localization of human EC-SOD. Our findings are consistent with those of previous works (18, 21).
There have been no reports, to our knowledge, of detailed studies of EC-SOD in kidney. The current study revealed that kidney expressed EC-SOD mRNA in bulk. Immunohistochemically, all tubular cells in cortical areas of the kidney were clearly positive in the cytoplasm. In situ analysis of the mRNA for EC-SOD will further clarify the origin of the mRNA in the tissue.
In conclusion, EC-SOD is distributed systemically in mice, and the physiological importance of this enzyme may be a compensatory adaptation to oxidative stress, particularly in lung and kidney, although more detailed information is still needed.
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ACKNOWLEDGEMENTS |
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We thank Dr. Noriaki Kinoshita for helpful technical advice on ELISA and Masahiko Segawa for skillful technical assistance.
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FOOTNOTES |
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This work was supported in part by a grant from the Kawano Memorial Foundation for Promotion of Pediatrics.
Address for reprint requests: T. Ookawara, Dept. of Hygiene, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.
Received 8 December 1997; accepted in final form 13 May 1998.
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