Proc. Nat!. Acad. Sci. USA Vol. 89, pp. 10405-10409, November 1992 Cell Biology

Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells (antioxidant enzyme/peroxlsm/catalase/hnmunocytocemistry)

JAMES D. CRAPO*tt, TIM OURYf, CATHERINE RABOUILLE§, JAN W. SLOT§, AND LING-YI CHANG* Departments of *Medicine and *Pathology, Duke University Medical Center, Durham, NC 27710; and University of Utrecht, Utrecht, The Netherlands

IDepartment of Cell Biology, Medical School,

Communicated by Irwin Fridovich, July 27, 1992

ABSTRACT The intracellular lalization of human copper,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both Isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polydonal antibodies in recognizing the antigen after adequate fixation oftissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

They argued that the previous immunochemistry done by Chang et aL (3) and Slot et al. (2), which reported a diffuse intracellular localization for Cu,Zn-SOD in rat tissues, may have been erroneous and caused by contaminating proteins in the antigen used to prepare the polyclonal antibodies. Two recent studies (6, 7) have provided biochemical evidence that Cu,Zn-SOD is found in peroxisomes, although not exclusively or predominantly so. The subcellular localization of Cu,Zn-SOD is of critical importance in evaluating many aspects of intracellular metabolism and in understanding how cells are protected against intracellularly produced oxygenbased radicals. To evaluate the questions raised by Keller et al. (4), we obtained the four mAbs used in their report, as well as an additional four mAbs prepared by other laboratories against either native or rh Cu,Zn-SOD. In addition, polyclonal antibodies raised against both native and rh Cu,Zn-SODs were obtained for comparison with the mAbs. These antisera were then used to localize this enzyme in a variety of human cells and tissues.

The superoxide dismutases (SODs; superoxide:superoxide oxidoreductase, EC 1.15.1.1) are a family of enzymes commonly characterized by the metals that they contain and by their function to dismute 2-, thus providing essential protection of biological tissues against uncontrolled reactions with oxygen-based radicals. The copper,zinc form of SOD (Cu,Zn-SOD) is a dimer having a molecular mass of 32,000 Da. It has been identified as a soluble enzyme widely distributed in the cytoplasm of all mammalian cells (1). Previous EM immunocytochemical localization of this protein has been done on rat liver hepatocytes. The enzyme was found to be excluded from many membrane-bound compartments, such as nuclear envelope, endoplasmic reticulum, Golgi elements, secretory vesicles, and mitochondria (2). Quantitative immunocytochemistry done on rat Cu,Zn-SOD identified it to be widely distributed throughout the cytoplasm and nucleus; the cytoplasmic matrix had a concentration of 1.36 mg/ml, a concentration --50%o higher than that of the nuclear matrix (3). Lysosomes contained the highest concentration (5.81 mg/ml). Rat hepatocyte peroxisomes were found to contain the Cu,Zn-SOD in relatively low concentrations (0.27 mg/ml) (3). Keller et al. (4) recently evaluated the intracellular localization of Cu,Zn-SOD in human fibroblasts and hepatoma cells with four monoclonal antibodies (mAbs) raised against recombinant human (rh) Cu,Zn-SOD. Immunolocalization was done by using immunofluorescence (4, 5). The enzyme was reported to be localized only in punctate regions of the cells that were identified as peroxisomes because of colocalization of catalase to the same sites with a dual-immunofluorescence method. These authors speculated that the use of mAbs raised against a recombinant protein insured the reliability of their studies localizing SOD only to peroxisomes.

mAbs. Four mAbs raised against rh Cu,Zn-SOD were obtained from Robert A. Hallewell (Chiron) and were designated CZSOD F2, CZSOD A3, CZSOD A6, and CZSOD A7, as reported by Keller et al. (4). A mAb prepared against rh Cu,Zn-SOD was obtained from Tomas Porstmann (Humboldt University of Berlin; designated Porstmann M5) (8). Two mAbs raised against human erythrocyte Cu,Zn-SOD were obtained from Kumeo Ono (Gunma University, Maebashi, Gunma, Japan) (9) (designated Ono 13 and Ono F,3,4). A single mAb raised against native human Cu,Zn-SOD was obtained from Tetsuo Adachi (Gifu Pharmaceutical University, Gifu, Japan) (10). Polyclonal Antibodies. A polyclonal antibody against rh Cu,Zn-SOD was obtained from Tomas Porstmann. A polyclonal antibody raised against native human Cu,Zn-SOD purified from erythrocytes was obtained from Larry Oberly (University of Iowa, Iowa City) (11). Our polyclonal antiserum against rat Cu,Zn-SOD has been described (2, 3). A polyclonal antiserum against native baboon liver Cu,Zn-SOD was prepared by immunizing rabbits using described methods (2, 3). Anti-catalase antibodies were obtained by immunizing rabbits with purified bovine catalase. Cells and Tissues. HepG2 cells were obtained from the American Type Culture Collection. These cells were either grown on slides and fixed in 3% paraformaldehyde in phosphate-buffered saline (pH 7.2) for 15 min or grown in 25-cm2 flasks and fixed with either 3% paraformaldehyde or 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr, detached, and centrifuged to form a pellet. The pellets were processed

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Abbreviations: SOD, superoxide dismutase; Cu,Zn-SOD, copper, zinc SOD; rh, recombinant human; mAb, monoclonal antibody. tTo whom reprint requests should be addressed. 10405

MATERIALS AND METHODS

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control preparations, the primary antisera were each omitted to verify specificity of the reaction of each probe. SDS/Gel Electrophoresis, Imnunoblots, and Dot-Blots. HepG2 cell lysates were fractionated on SDS/polyacrylamide gel and transferred to nitrocellulose paper. Strips of the blot, each containing a lane of molecular-weight markers and a lane of cell lysate, were incubated with each of the eight mAbs and the two polyclonal antibodies against human Cu,Zn-SOD. These blots were then incubated with 125Ilabeled protein A, and autoradiographs were prepared. Polyclonal antibodies against rat Cu,Zn-SOD and baboon Cu,ZnSOD were also evaluated for specificity and purity using immunoblots of their respective purified Cu,Zn-SOD and liver homogenates. Dot-blots of human lung supernatant against both monoclonal and polyclonal antisera were prepared to evaluate the ability of the antisera to bind after different types of tissue fixation. The dot-blots were incubated with 17-5I-labeled protein A, and autoradiographs were prepared.

for cryo-ultrathin sectioning. Cells were also cultured on chamber slides and prepared for light microscopic immunofluorescence. Human lung was obtained as part of a surgical biopsy specimen. The patient was a 65-yr-old man with a large cell carcinoma in the left upper lobe and a 40-pack per yr smoking history but no other form of clinical lung disease. The entire left upper lobe was removed, and lung tissue away from the carcinoma was obtained. This tissue was preserved by using intra-airway instillation of 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr, after which the tissue was diced and prepared for cryo-ultrathin microtomy. Baboon lung was fixed by inflation with 2% paraformaldehyde/0.2% glutaraldehyde. Human placenta, human leukocytes, baboon stomach, and baboon liver were also obtained and fixed by immersion in 2% paraformaldehyde/0.2% glutaraldehyde. After 1 hr of fixation, each of the tissues was immediately processed for cryo-ultramicrotomy. EM Immunocytochemistry. Ultrathin cryosections of tissues and cells were prepared and immunolabeled as described (2). For multiple labeling, second and/or third antibodies followed by treatment with protein A-gold of distinct size were used before staining with uranyl acetate (12). Light Microscopic Immunofluorescence. Slides of fixed HepG2 cells were prepared for light microscopic immunofluorescence with methods similar to those described by Keller et al. (4). Cells were permeabilized with 1% Triton X-100 for 5 min and washed extensively with phosphatebuffered saline. Cells were then incubated sequentially with mAbs against rh Cu,Zn-SOD, fluorescein-conjugated goat anti-mouse IgG, rabbit polyclonal antisera against catalase, and rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch). The two secondary antisera used do not react with animal tissues other than the target IgG source. In vi

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RESULTS SDS/gel electrophoresis of cell lysates of HepG2 cells followed by immunoblotting and reaction with antisera demonstrated that all eight mAbs reacted with a 16-kDa protein consistent with the monomer form of CuZn-SOD. Single bands were found with all mAbs used. The two polyclonal antibodies against Cu,Zn-SOD were also immunoblotted against the HepG2 cell lysate after SDS/gel electrophoresis. Both polyclonal antibodies displayed a single band on the immunoblot after SDS/gel electrophoresis at a molecular mass of 16 kDa (data not shown). The anti-rat Cu,Zn-SOD antiserum and anti-baboon Cu,Zn-SOD antiserum were immunoblotted against both their respective purified proteins and rat or baboon liver homogenates. These antibodies

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nucleus (nu), and none was found over mitochondria (m). Notice the lack of ultrastructural details due to the weak-fixation conditions. (b) HepG2 cells fixed with 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr and labeled with CZSOD A7 mAb. The ultrastructure was greatly improved, but immunolabeling was absent. rer, Rough endoplasmic reticulum. (c) HepG2 cell fixed with 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr and then triple-labeled with Porstmann's rabbit polyclonal antibody against rh Cu,Zn-SOD (15-nm gold), cathepsin D (10-nm gold), and catalase (5-nm gold). Lysosomes (ly) were labeled for cathepsin D, and peroxisomes (p) were labeled for catalase. Some Cu,Zn-SOD labeling does occur in these structures, but its density there does not exceed the cytoplasmic Cu,Zn-SOD labeling. None of the gold markers were present in the extracellular spaces (ex) or mitochondria (m). (Bars = 200 nm.)

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Proc. Natl. Acad. Sci. USA 89 (1992)

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Table 1. Distribution of Cu,Zn-SOD labeling on subceliular compartments of HepG2 cells Labeling density Mitochondrion Peroxisome Cytosol Antibody Nucleus Tissue fixed with 3% paraformaldehyde +++ + Monoclonal* +++t + +++ +++t Polyclonalt Tissue fixed with 2% paraformaldehyde/0.2% glutaraldehyde Monoclonal* ++ Polyclonalt ++ *Similar labeling densities were found for all eight mAbs. tLabeling over peroxisomes under this fixation condition was highly variable, ranging from 0 to + ++. tSimilar labeling densities were found for the two polyclonal antibodies.

reacted with the appropriate form of native Cu,Zn-SOD and were monospecific for Cu,Zn-SOD on immunoblots of liver homogenates. EM of HepG2 Cells. HepG2 cells were fixed both by 3% paraformaldehyde for 15 min [as reported by Keller et al. (4, 5)], and by 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr. In cryosections HepG2 cells fixed with 3% paraformaldehyde were found to have an electron-lucent cytosol with substantial loss of cytoplasmic matrix and loss of ultrastructural details of the subcellular organelles (Fig. la). Membrane-bound organelles could not be easily distinguished from each other. In contrast, cells fixed with 2% paraformaldehyde/0.2% glutaraldehyde displayed a' normal, dense cytosolic matrix and well-preserved subcellular organelles (Fig.

cytes, and rat liver hepatocytes (Fig. 3a) all also showed cytoplasmic and nuclear labeling for Cu,Zn-SOD similar to that found for human lung cells. One difference in rat tissues was an apparent accumulation ofCu,Zn-SOD in lysosomes as we reported earlier (2, 3). Light Microscopy with HepG2 Ce*l. With light microscopic immunofluorescence, the pattern of Cu,Zn-SOD and catalase labeling was similar to that seen with EM. HepG2 cells demonstrated diffuse nuclear labeling for Cu,Zn-SOD and a faint but diffuse distribution of labeling for Cu,Zn-SOD in the cytoplasm (data not shown). Cu,Zn-SOD was not found to colocalize with catalase, which was used as a marker for peroxisomes.

lb). EM Immunochemistry. The ability of various mAbs and polyclonal antibodies to label human Cu,Zn-SOD was tested on cryo-ultrathin sections of both HepG2 cells and human lung. All antibodies showed positive labeling for Cu,Zn-SOD on cells fixed with 3% paraformaldehyde (Fig. la), although none of the mAbs gave positive labeling with either HepG2 cells or human lung when 2% paraformaldehyde/0.2% glutaraldehyde was used as fixative (Fig. lb). Each polyclonal antibody gave high-quality labeling on HepG2 cells and human lungs under the more stringent fixation technique. Table 1 gives the relative distribution of Cu,Zn-SOD labeling for the two different fixatives. After fixation with 3% paraformaldehyde, labeling was most intense in the nucleus, present in some peroxisomes, and consistently light in the cytoplasm. Cu,Zn-SOD was not detectable in other cellular structures with any of the antibodies used. Using HepG2 cells fixed with 2% paraformaldehyde/0.2% glutaraldehyde, we observed no reaction after incubation with any of the mAbs against Cu,Zn-SOD (Fig. lb). To confirm the apparent inability of these monoclonal antisera to bind to tissues fixed with glutaraldehyde, we performed dot-blots of the antisera versus human lung tissue fixed by each technique (Fig. 2). All monoclonal antisera showed markedly decreased binding after the tissue extracts were fixed with glutaraldehyde, whereas the polyclonal antisera maintained a high level of

binding. With polyclonal antibodies, we encountered a diffuse labeling of approximately equivalent density for Cu,Zn-SOD in the cytoplasm, in the euchromatin fields of the nucleus, and in peroxisomes (Table 1). Most lysosomal structures had a relatively low degree of labeling for Cu,Zn-SOD (Fig. ic). These observations in HepG2 cells were consistent with findings in human lung tissue. Human lung cells evaluated include alveolar macrophages (Fig. 3c) type I and type II epithelial cells, interstitial fibroblasts (Fig. 3d) and macrophages, capillary endothelial cells, and polymorphonuclear leukocytes. All these cell types demonstrated a similar intracellular distribution of Cu,Zn-SOD. Human placenta trophoblasts, baboon lung alveolar septal cells (Fig. 3b), baboon stomach chief cells and parietal cells, baboon liver hepato-

DISCUSSION These studies show that Cu,Zn-SOD is widely distributed in the nucleus and cytosol of human cells. HepG2 cells and a variety of cell types evaluated in human tissue all demonstrated diffuse cytoplasmic and nuclear labeling. A lower degree of labeling over the cell cytosol occurred when cells were fixed for only 15 min with 3% paraformaldehyde. This weaker fixation, particularly if combined with a detergent, leads to loss of proteins from the cell cytosol and thereby loss of immunolabeling over the cytosolic compartment. This effect may be exaggerated when the cells are stored in buffer for significant periods of time after fixation. Loss of immunolabeling over the cytoplasm can cause an apparent predominance of labeling over subcellular organelles, such as peroxisomes, although we found strong labeling to persist fAt

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FIG. 2. Autoradiographs of dot-blots on nitrocellulose paper showing sensitivity of the binding of Cu,Zn-SOD antiserum to fixatives. All wells were originally filled with 5 jig of protein from a 20,000 x g supernatant from human lung tissue. The protein was then exposed to normal saline for 60 min (row 1), normal saline for 45 min followed by 3% paraformaldehyde for 15 min (row 2), and 2% paraformaldehyde/0.2% glutaraldehyde for60 min (row 3). Dot-blots were then incubated with the following antisera: CZSODF2 (column A), Ono 13 (column B), Porstmann MS (column C), and Porstmann polyclonal antibody to rh Cu,Zn-SOD (column D).

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FIG. 3. Localization of Cu,Zn-SOD on animal and human tissues fixed with 2% paraformaldehyde/0.2% glutaraldehyde for 1 hr. (a) Localization of Cu,Zn-SOD and catalase in rat liver hepatocyte. Cryo-ultrathin sections of rat liver were incubated with rabbit anti-rat Cu,Zn-SOD (1:400) and 15-nm protein A-gold followed by rabbit antibovine catalase (1:500) and 5-nm protein A-gold. Note abundance of Cu,Zn-SOD (arrowheads) in the cytosol, its virtual absence in mitochondria, and its presence (arrow) in peroxisomes at concentrations lower than that in cytoplasm. (b) Baboon lung alveolar type II cells labeled with rabbit anti-baboon Cu,Zn-SOD and 20-nm protein A-gold. (c) Human lung alveolar macrophage labeled with rh polyclonal antisera to SOD (15-nm gold). (d) Human lung fibroblast labeled with rh polyclonal antisera to SOD (15-nm gold). m, Mitochondria; p, peroxisome; lb, lamellar body; g, granule; ga, Golgi apparatus. (Bars = 200 nm.)

over the cell nucleus under these same conditions. A variety of tissues were studied, including human lung, baboon lung, and rat liver. Polyclonal antibodies raised against both native and recombinant proteins consistently demonstrated that Cu,Zn-SOD was diffusely located in the nucleus and cell cytosol. Peroxisomes do contain Cu,Zn-SOD. Because peroxisomes represent a tiny fraction of the cell volume [1.2% in rat hepatocytes (3)], their total contribution to cellular Cu,ZnSOD is small [estimated at