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

Copper,zinc superoxide dismutase is primarily a cytosolic protein inhuman 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 IDepartment of Cell Biology, Medical School,University of Utrecht, Utrecht, The Netherlands

Communicated by Irwin Fridovich, July 27, 1992

ABSTRACT The intracellular lalization of human cop-per,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:su-peroxide oxidoreductase, EC 1.15.1.1) was evaluated by usingEM immunocytochemistry and both Isolated human cell linesand human tissues. Eight monoclonal antibodies raised againsteither native or recombinant human Cu,Zn-SOD and twopolyclonal antibodies raised against either native or recombi-nant human Cu,Zn-SOD were used. Fixation with 2% para-formaldehyde/0.2% glutaraldehyde was found necessary topreserve normal distribution of the protein. Monoclonal anti-bodies were less effective than polydonal antibodies in recog-nizing the antigen after adequate fixation oftissue. Cu,Zn-SODwas found widely distributed in the cell cytosol and in the cellnucleus, consistent with it being a soluble cytosolic protein.Mitochondria and secretory compartments did not label forthis protein. In human cells, peroxisomes showed a labelingdensity slightly less than that of cytoplasm.

The superoxide dismutases (SODs; superoxide:superoxideoxidoreductase, EC 1.15.1.1) are a family of enzymes com-monly characterized by the metals that they contain and bytheir function to dismute 2-, thus providing essential pro-tection of biological tissues against uncontrolled reactionswith oxygen-based radicals. The copper,zinc form of SOD(Cu,Zn-SOD) is a dimer having a molecular mass of 32,000Da. It has been identified as a soluble enzyme widelydistributed in the cytoplasm of all mammalian cells (1).Previous EM immunocytochemical localization of this pro-tein has been done on rat liver hepatocytes. The enzyme wasfound to be excluded from many membrane-bound compart-ments, such as nuclear envelope, endoplasmic reticulum,Golgi elements, secretory vesicles, and mitochondria (2).Quantitative immunocytochemistry done on rat Cu,Zn-SODidentified it to be widely distributed throughout the cyto-plasm and nucleus; the cytoplasmic matrix had a concentra-tion of 1.36 mg/ml, a concentration --50%o higher than that ofthe nuclear matrix (3). Lysosomes contained the highestconcentration (5.81 mg/ml). Rat hepatocyte peroxisomeswere found to contain the Cu,Zn-SOD in relatively lowconcentrations (0.27 mg/ml) (3).

Keller et al. (4) recently evaluated the intracellular local-ization of Cu,Zn-SOD in human fibroblasts and hepatomacells with four monoclonal antibodies (mAbs) raised againstrecombinant human (rh) Cu,Zn-SOD. Immunolocalizationwas done by using immunofluorescence (4, 5). The enzymewas reported to be localized only in punctate regions of thecells that were identified as peroxisomes because of colocal-ization of catalase to the same sites with a dual-immunoflu-orescence method. These authors speculated that the use ofmAbs raised against a recombinant protein insured the reli-ability of their studies localizing SOD only to peroxisomes.

They argued that the previous immunochemistry done byChang et aL (3) and Slot et al. (2), which reported a diffuseintracellular localization for Cu,Zn-SOD in rat tissues, mayhave been erroneous and caused by contaminating proteins inthe antigen used to prepare the polyclonal antibodies. Tworecent studies (6, 7) have provided biochemical evidence thatCu,Zn-SOD is found in peroxisomes, although not exclu-sively or predominantly so. The subcellular localization ofCu,Zn-SOD is of critical importance in evaluating manyaspects of intracellular metabolism and in understanding howcells are protected against intracellularly produced oxygen-based radicals. To evaluate the questions raised by Keller etal. (4), we obtained the fourmAbs used in their report, as wellas an additional four mAbs prepared by other laboratoriesagainst either native or rh Cu,Zn-SOD. In addition, polyclo-nal antibodies raised against both native and rh Cu,Zn-SODswere obtained for comparison with the mAbs. These antiserawere then used to localize this enzyme in a variety ofhumancells and tissues.

MATERIALS AND METHODSmAbs. Four mAbs raised against rh Cu,Zn-SOD were

obtained from Robert A. Hallewell (Chiron) and were des-ignated CZSOD F2, CZSOD A3, CZSOD A6, and CZSODA7, as reported by Keller et al. (4). A mAb prepared againstrh Cu,Zn-SOD was obtained from Tomas Porstmann (Hum-boldt University of Berlin; designated Porstmann M5) (8).Two mAbs raised against human erythrocyte Cu,Zn-SODwere obtained from Kumeo Ono (Gunma University, Mae-bashi, Gunma, Japan) (9) (designated Ono 13 and Ono F,3,4).A single mAb raised against native human Cu,Zn-SOD wasobtained from Tetsuo Adachi (Gifu Pharmaceutical Univer-sity, Gifu, Japan) (10).

Polyclonal Antibodies. A polyclonal antibody against rhCu,Zn-SOD was obtained from Tomas Porstmann. A poly-clonal antibody raised against native human Cu,Zn-SODpurified from erythrocytes was obtained from Larry Oberly(University of Iowa, Iowa City) (11). Our polyclonal antise-rum against rat Cu,Zn-SOD has been described (2, 3). Apolyclonal antiserum against native baboon liver Cu,Zn-SODwas prepared by immunizing rabbits using described methods(2, 3). Anti-catalase antibodies were obtained by immunizingrabbits with purified bovine catalase.

Cells and Tissues. HepG2 cells were obtained from theAmerican Type Culture Collection. These cells were eithergrown on slides and fixed in 3% paraformaldehyde in phos-phate-buffered saline (pH 7.2) for 15 min or grown in 25-cm2flasks 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

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

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for cryo-ultrathin sectioning. Cells were also cultured onchamber slides and prepared for light microscopic immuno-fluorescence.Human lung was obtained as part of a surgical biopsy

specimen. The patient was a 65-yr-old man with a large cellcarcinoma in the left upper lobe and a 40-pack per yr smokinghistory but no other form of clinical lung disease. The entireleft upper lobe was removed, and lung tissue away from thecarcinoma was obtained. This tissue was preserved by usingintra-airway instillation of 2% paraformaldehyde/0.2% glu-taraldehyde for 1 hr, after which the tissue was diced andprepared for cryo-ultrathin microtomy. Baboon lung wasfixed by inflation with 2% paraformaldehyde/0.2% glutaral-dehyde. Human placenta, human leukocytes, baboon stom-ach, and baboon liver were also obtained and fixed byimmersion in 2% paraformaldehyde/0.2% glutaraldehyde.After 1 hr of fixation, each of the tissues was immediatelyprocessed for cryo-ultramicrotomy.EM Immunocytochemistry. Ultrathin cryosections of tis-

sues and cells were prepared and immunolabeled as de-scribed (2). For multiple labeling, second and/or third anti-bodies followed by treatment with protein A-gold of distinctsize were used before staining with uranyl acetate (12).

Light Microscopic Immunofluorescence. Slides of fixedHepG2 cells were prepared for light microscopic immuno-fluorescence with methods similar to those described byKeller et al. (4). Cells were permeabilized with 1% TritonX-100 for 5 min and washed extensively with phosphate-buffered saline. Cells were then incubated sequentially withmAbs against rh Cu,Zn-SOD, fluorescein-conjugated goatanti-mouse IgG, rabbit polyclonal antisera against catalase,and rhodamine-conjugated goat anti-rabbit IgG (Jackson Im-munoResearch). The two secondary antisera used do notreact with animal tissues other than the target IgG source. In

control preparations, the primary antisera were each omittedto verify specificity of the reaction of each probe.SDS/Gel Electrophoresis, Imnunoblots, and Dot-Blots.

HepG2 cell lysates were fractionated on SDS/polyacrylam-ide gel and transferred to nitrocellulose paper. Strips of theblot, each containing a lane of molecular-weight markers anda lane of cell lysate, were incubated with each of the eightmAbs and the two polyclonal antibodies against humanCu,Zn-SOD. These blots were then incubated with 125I-labeled protein A, and autoradiographs were prepared. Poly-clonal antibodies against rat Cu,Zn-SOD and baboon Cu,Zn-SOD were also evaluated for specificity and purity usingimmunoblots of their respective purified Cu,Zn-SOD andliver homogenates. Dot-blots of human lung supernatantagainst both monoclonal and polyclonal antisera were pre-pared to evaluate the ability of the antisera to bind afterdifferent types of tissue fixation. The dot-blots were incu-bated with 17-5I-labeled protein A, and autoradiographs wereprepared.

RESULTSSDS/gel electrophoresis of cell lysates of HepG2 cells fol-lowed by immunoblotting and reaction with antisera demon-strated that all eight mAbs reacted with a 16-kDa proteinconsistent with the monomer form of CuZn-SOD. Singlebands were found with all mAbs used. The two polyclonalantibodies against Cu,Zn-SOD were also immunoblottedagainst the HepG2 cell lysate after SDS/gel electrophoresis.Both polyclonal antibodies displayed a single band on theimmunoblot after SDS/gel electrophoresis at a molecularmass of 16 kDa (data not shown). The anti-rat Cu,Zn-SODantiserum and anti-baboon Cu,Zn-SOD antiserum were im-munoblotted against both their respective purified proteinsand rat or baboon liver homogenates. These antibodies

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FIG. 1. Localization ofCu,Zn-SOD in HepG2 cells.(a) HepG2 cells fixed with 3% paraformaldehyde for 15min labeled with CZSOD A7 mAb against rh Cu,Zn-SOD. Heavy labeling (10-nm gold) was found in thenucleus (nu), and none was found over mitochondria(m). Notice the lack of ultrastructural details due to theweak-fixation conditions. (b) HepG2 cells fixed with

m 2% paraformaldehyde/0.2% glutaraldehyde for 1 hrand labeled with CZSOD A7 mAb. The ultrastructurewas greatly improved, but immunolabeling was absent.rer, Rough endoplasmic reticulum. (c) HepG2 cell fixedwith 2% paraformaldehyde/0.2% glutaraldehyde for 1hr and then triple-labeled with Porstmann's rabbitpolyclonal antibody against rh Cu,Zn-SOD (15-nmgold), cathepsin D (10-nm gold), and catalase (5-nmgold). Lysosomes (ly) were labeled for cathepsin D,and peroxisomes (p) were labeled for catalase. SomeCu,Zn-SOD labeling does occur in these structures, but

P its density there does not exceed the cytoplasmicCu,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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Table 1. Distribution of Cu,Zn-SOD labeling on subceliular compartments of HepG2 cells

Labeling density

Antibody Nucleus Cytosol Peroxisome MitochondrionTissue fixed with 3% paraformaldehyde

Monoclonal* +++ + +++tPolyclonalt +++ + +++t

Tissue fixed with 2% paraformaldehyde/0.2% glutaraldehydeMonoclonal*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 andwere monospecific for Cu,Zn-SOD on immunoblots of liverhomogenates.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 1hr. In cryosections HepG2 cells fixed with 3% paraformal-dehyde were found to have an electron-lucent cytosol withsubstantial loss of cytoplasmic matrix and loss of ultrastruc-tural details of the subcellular organelles (Fig. la). Mem-brane-bound organelles could not be easily distinguishedfrom each other. In contrast, cells fixed with 2% paraformal-dehyde/0.2% glutaraldehyde displayed a' normal, dense cy-tosolic matrix and well-preserved subcellular organelles (Fig.lb).EM Immunochemistry. The ability of various mAbs and

polyclonal antibodies to label human Cu,Zn-SOD was testedon cryo-ultrathin sections of both HepG2 cells and humanlung. All antibodies showed positive labeling for Cu,Zn-SODon cells fixed with 3% paraformaldehyde (Fig. la), althoughnone of the mAbs gave positive labeling with either HepG2cells or human lung when 2% paraformaldehyde/0.2% glu-taraldehyde was used as fixative (Fig. lb). Each polyclonalantibody gave high-quality labeling on HepG2 cells andhuman lungs under the more stringent fixation technique.

Table 1 gives the relative distribution of Cu,Zn-SOD la-beling 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 thecytoplasm. Cu,Zn-SOD was not detectable in other cellularstructures with any ofthe antibodies used. Using HepG2 cellsfixed with 2% paraformaldehyde/0.2% glutaraldehyde, weobserved no reaction after incubation with any of the mAbsagainst Cu,Zn-SOD (Fig. lb). To confirm the apparent in-ability of these monoclonal antisera to bind to tissues fixedwith glutaraldehyde, we performed dot-blots of the antiseraversus human lung tissue fixed by each technique (Fig. 2). Allmonoclonal antisera showed markedly decreased bindingafter the tissue extracts were fixed with glutaraldehyde,whereas the polyclonal antisera maintained a high level ofbinding.With polyclonal antibodies, we encountered a diffuse la-

beling ofapproximately equivalent density for Cu,Zn-SOD inthe cytoplasm, in the euchromatin fields of the nucleus, andin peroxisomes (Table 1). Most lysosomal structures had arelatively low degree of labeling for Cu,Zn-SOD (Fig. ic).These observations in HepG2 cells were consistent withfindings in human lung tissue. Human lung cells evaluatedinclude alveolar macrophages (Fig. 3c) type I and type IIepithelial cells, interstitial fibroblasts (Fig. 3d) and macro-phages, capillary endothelial cells, and polymorphonuclearleukocytes. All these cell types demonstrated a similar in-tracellular distribution of Cu,Zn-SOD. Human placenta tro-phoblasts, baboon lung alveolar septal cells (Fig. 3b), baboonstomach chief cells and parietal cells, baboon liver hepato-

cytes, and rat liver hepatocytes (Fig. 3a) all also showedcytoplasmic and nuclear labeling for Cu,Zn-SOD similar tothat found for human lung cells. One difference in rat tissueswas an apparent accumulation ofCu,Zn-SOD in lysosomes aswe reported earlier (2, 3).

Light Microscopy with HepG2 Ce*l. With light microscopicimmunofluorescence, the pattern ofCu,Zn-SOD and catalaselabeling was similar to that seen with EM. HepG2 cellsdemonstrated diffuse nuclear labeling for Cu,Zn-SOD and afaint but diffuse distribution of labeling for Cu,Zn-SOD in thecytoplasm (data not shown). Cu,Zn-SOD was not found tocolocalize with catalase, which was used as a marker forperoxisomes.

DISCUSSIONThese studies show that Cu,Zn-SOD is widely distributed inthe nucleus and cytosol of human cells. HepG2 cells and avariety of cell types evaluated in human tissue all demon-strated diffuse cytoplasmic and nuclear labeling. A lowerdegree of labeling over the cell cytosol occurred when cellswere fixed for only 15 min with 3% paraformaldehyde. Thisweaker fixation, particularly if combined with a detergent,leads to loss ofproteins from the cell cytosol and thereby lossof immunolabeling over the cytosolic compartment. Thiseffect may be exaggerated when the cells are stored in bufferfor significant periods of time after fixation. Loss of immu-nolabeling over the cytoplasm can cause an apparent pre-dominance of labeling over subcellular organelles, such asperoxisomes, although we found strong labeling to persist

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FIG. 2. Autoradiographs of dot-blots on nitrocellulose papershowing sensitivity of the binding of Cu,Zn-SOD antiserum tofixatives. All wells were originally filled with 5 jig of protein from a20,000 x g supernatant from human lung tissue. The protein was thenexposed to normal saline for 60 min (row 1), normal saline for 45 minfollowed by 3% paraformaldehyde for 15 min (row 2), and 2%paraformaldehyde/0.2% glutaraldehyde for60 min (row 3). Dot-blotswere then incubated with the following antisera: CZSODF2 (columnA), Ono 13 (column B), Porstmann MS (column C), and Porstmannpolyclonal 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-ratCu,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 ofCu,Zn-SOD (arrowheads) in the cytosol, its virtual absence in mitochondria, and its presence (arrow) in peroxisomes at concentrations lowerthan 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) Humanlung alveolar macrophage labeled with rh polyclonal antisera to SOD (15-nm gold). (d) Human lung fibroblast labeled with rh polyclonal antiserato 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 varietyof tissues were studied, including human lung, baboon lung,and rat liver. Polyclonal antibodies raised against both nativeand recombinant proteins consistently demonstrated thatCu,Zn-SOD was diffusely located in the nucleus and cellcytosol. Peroxisomes do contain Cu,Zn-SOD. Because per-oxisomes represent a tiny fraction of the cell volume [1.2% inrat hepatocytes (3)], their total contribution to cellular Cu,Zn-SOD is small [estimated at <1% in rat hepatocytes (3)]. Theseimmunolocalization studies are consistent with previous bio-chemical studies in which enzyme distribution has beenmeasured in cell-fractionation experiments (1, 6, 7). Wandersand Denis (6), using analytical cell-fractionation experi-ments, clearly identified Cu,Zn-SOD to be associated withperoxisomes but that distribution represented only a tinyfraction of total cellular Cu,Zn-SOD.Our results disagree with those of Keller et al. (4), who

concluded that Cu,Zn-SOD is located only in peroxisomes inhuman cells. We evaluated this conclusion by using bothimmunofluorescent light microscopic immunochemistry andEM immunocytochemistry and by using both the same an-tibodies used by Keller et al. (4) and a variety of antibodiesraised against recombinant or native human SOD by differenttechniques and by different laboratories. The weak fixationmethods used by Keller et al. (4, 5) probably led to loss ofcytoplasmic proteins and thereby an artifactual enhancement

of the relative density of fluorescent labeling of SOD overperoxisomes. The absence ofnuclear labeling in these studiesis less easily explained because nuclear labeling was well-preserved when we repeated these experiments. Possibly thefixation and storage techniques used led to a virtual loss ofCu,Zn-SOD from both the nucleus and cytoplasm in the cellsstudied by Keller et al. (4).We found that polyclonal antibodies were consistently

more reliable than mAbs for immunolocalization studies.mAbs are less likely to maintain effective labeling underconditions of adequate fixation where antigenic loci on theprotein are being modified. Polyclonal antibodies show a fallin labeling density as proteins are modified during fixation;however, the capacity of polyclonal antibodies to recognizemultiple epitopes maintains higher quality labeling undermore stringent fixation conditions. Polyclonal antibodieshave been shown to give a labeling response that is linear andproportional to protein density for both purified SOD and ratliver fixed with glutaraldehyde under controlled conditions.This reproducible pattern of labeling permits quantitativeimmunocytochemistry to be reliably done (3). We concludethat immunolocalization studies require appropriate fixationof tissue and that polyclonal antibodies of proven specificityare effective probes for immunolocalization studies. TheCu,Zn-SOD is diffusely distributed throughout the cell cyto-sol and nucleus of all studied mammalian cell lines. It is

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present in peroxisomes, although not in a particularly highconcentration.

We thank Drs. Tetsuo Adachi, Robert A. Hallewell, Larry Oberly,Kumeo Ono, and Tomas Porstmann for generously providing mono-clonal or polyclonal anti-Cu,Zn-SOD antibodies for these studies.This work was supported, in part, by National Heart, Lung, andBlood Institute Grants P01 HL31992 and R01 HL42609.

1. Fridovich, I. (1986) in Advances in Enzymology, ed. Meister, A.(Wiley, New York), pp. 61-97.

2. Slot, J. W., Geuze, H. J., Freeman, B. A. & Crapo, J. D.(1986) Lab. Invest. 55, 363-371.

3. Chang, L., Slot, J. W., Geuze, H. J. & Crapo, J. D. (1988) J.Cell Biol. 107, 2169-2179.

4. Keller, G., Warner, T. G., Steimer, K. S. & Hallewell, R. A.(1991) Proc. Natl. Acad. Sci. USA 88, 7381-7385.

Proc. NatL Acad. Sci. USA 89 (1992) 10409

5. Keller, G., Gould, S., Deluca, M. & Subramani, S. (1987) Proc.Nat!. Acad. Sci. USA 84, 3264-3268.

6. Wanders, R. J. A. & Denis, S. (1992) Biochim. Biophys. Acta1115, 259-262.

7. Dhaunsi, G. S., Gulati, S., Singh, A. K., Orak, J. K.,Asayama, K. & Singh, I. (1992) J. Biol. Chem. 267, 6870-6873.

8. Porstmann, T., Wietschke, R., Schmechta, H., Grunow, R.,Porstmann, B., Bleiber, R., Pergande, M., Stachat, S. & vonBaehr, R. (1988) Clin. Chim. Acta 171, 1-10.

9. Ono, K., Kimura, S., Nakano, M. & Naruse, T. (1991) FEBSLett. 282, 115-118.

10. Adachi, T., Usami, Y., Kishi, T., Hirano, K. & Hayashi, K.(1988) J. Immunol. Methods 109, 93-101.

11. Oberley, T. D., Gonzalez, A., Lauchner, L. J., Oberley, L. W.& Jonathan, J. L. (1991) Cancer Res. 51, 1922-1929.

12. Slot, J. W., Geuze, H. J., Gigangack, S., Lienhard, G. E. &James, D. E. (1991) J. Cell Biol. 113, 123-135.

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