Isolation and immunological characterization of the mammalian 32-34-kDa stress protein

Experimental

Isolation

Cell Research 178 (1988) 3 l-40

and Immunological Characterization Mammalian 32-/34-kDa Stress Protein

MADELYN

M. CALTABIANO,’ GEORGE and RUSSELL G. GREIG

of the

POSTE,

Department of Cell Biology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

19101,

Challenge of mammalian cells with heavy metals or sulfhydryl-reactive agents including sodium arsenite induces the de nouo synthesis of a 32-/34-kDa stress protein (~32) (M. M. Caltabiano, T. P. Koestler, G. Poste, and R. G. Greig (1986) J. Biol. Chem. 261, 13,381). Here we report that antibody prepared against p32/p34 purified from human A375 melanoma cells immunoprecipitated an antigen of similar molecular mass from a panel of human, rat, and murine cells following challenge with sodium arsenite. No reactivity was observed in lysates from control, uninsulted cultures. The precise molecular mass of the arseniteinduced antigen was species-specific: 32 kDa (human and rat) and 34 kDa (murine). Indirect immunofluorescence analysis using affinity-purified monospecific IgG demonstrated that p32/p34 was localized to the cytoplasm and displayed a perinuclear distribution.

0

1988 Academic

Press. Inc.

Challenge of procaryotic and eucaryotic cells with various chemical and physical insults induces a stress response characterized by the coordinate and selective synthesis of a group of highly conserved proteins commonly referred to as stress or heat-shock proteins [l-3]. The ubiquitous nature of this phenomenon suggests that the induced proteins play an important homeostatic function in protecting cells against environmental insults [4-6] but their precise role is not defined. In mammalian systems, the major stress proteins include the heat-shock proteins with molecular masses of 110, 90, 73, 72, and 22-28 kDa and the 95 and 78kDa glucose-regulated proteins [l-3, 71. Many of these polypeptides have been studied extensively and their genes isolated, cloned, and sequenced [l-3]. We have demonstrated recently that human and murine cells synthesize a 32and a 34-kDa protein, respectively (designated p32/p34), when challenged with heavy metals (Zr?+, Cu’+, and Cd’+) or sulfhydryl-reactive agents (disulliram, auranofin, arsenite, and iodoacetamide) [S]. p32/p34 is not induced by heat or calcium ionophore. Stress proteins with a molecular mass similar to that of ~321~34 have been identified in chick embryo fibroblasts and a variety of rat cells but have not been characterized thoroughly [9-l 11. As a first step toward defining its function we have purified human p32/p34, prepared polyclonal antibodies to it, and determined its subcellular location by indirect immunofluorescence. Antibodies raised against human p32/p34 cross-reacted with an antigen of similar ’ To whom reprint requests should be addressed at Department of Cell Biology, French Laboratories, 1500 Spring Garden Street, Philadelphia, PA 19101. 3-888339

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Smith Kline &

CopyrIght 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827188 $0303

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Caltabiano,

Poste, and Greig

molecular mass (32 to 34 kDa) in several human, indicating the presence of conserved epitopes. MATERIALS

AND

rat, and murine cell types

METHODS

Cell culrure. The following human cell lines were used: A375 (melanoma) [12], U-937 (histiocytic lymphoma) [13], CCD-21Sk (normal skin tibroblast) [14], HL-60 (promyelocytic leukemia) [15], SKCO-1 (colon adenocarcinoma) [16], and HCMC (normal colonic mucosa) [17]. Rat cell lines included NRK-52E (normal kidney epithelium) [18], NRK-49F (normal kidney flbroblast) [18], and IEC-6 (intestinal epithelium) [19]. The murine cell lines were B16-FlO (melanoma) [20], NCTC clone 929 (connective tissue) [21], CMT-93 (rectal carcinoma) [22], and Balb/3T3 clone A31 (embryonic tibroblast) [23]. Cell lines, obtained from the American Type Culture Collection (ATCC, Rockville, MD), were grown as monolayer or suspension cultures according to ATCC recommended protocols except for B16-FlO murine melanoma cells which were maintained as described previously [20]. Stress conditions and metabolic labeling. Cells were seeded into 35 mm diameter plastic dishes or T75 flasks in growth medium and incubated at 37°C overnight. Confluent cultures were rinsed three times with phosphate-buffered saline (PBS) (GIBCO) and challenged with sodium arsenite (25-50 FM) for 6 to 8 h in serum-free Dulbecco’s modified Eagle’s medium (GIBCO). In certain experiments, cells were also radiolabeled with 100 @i/ml [35S]methionine (L-[35S]methionine, >800 Ci/mmol, Amersham Corp.) for the last 4 h of exposure to the stress agent. In uifro translation. Total cellular RNA was isolated using a modified guanidinium isothiocyanate/2mercaptoethanol procedure [24]. Cells were homogenized with 4 M guanidinium thiocyanate, 0.1 M 2mercaptoethanol, and 25 mM citrate, pH 7.0, and the extract was sedimented through a 5.7 M cesium chloride cushion. Following an ethanol precipitation and passage over an oligo(dT)-cellulose column (Collaborative Research) the protein-free mRNA (1 ug) was translated in a nuclease-free rabbit reticulocyte preparation (BRL) with [35S]methionine according to the supplier’s protocol. Preparation ofantiserum. Human p32/p34 antigen was purified from arsenite-stressed A375 melanoma cells by successive cycles of preparative SDS-PAGE separations [8] and electroelutions using an ISCO Model 1750 Sample Concentrator. Isolated protein was mixed in Freund’s complete adjuvant (GIBCO) and injected subcutaneously into New Zealand White rabbits. Subsequent subscapular injections using Freund’s incomplete adjuvant (GIBCO) were performed every 2-3 weeks for several months. Immunoglobulin (IgG) was isolated by protein A-Sepharose 4B column chromatography (Pharmacia) and was purified further by adsorption against a cellular extract of unstressed A375 melanoma cells immobilized on CNBr-activated Sepharose 4B (Pharmacia). Unbound immunoglobulin from this column (referred to as IgG fraction I) was then affinity purified using a modified procedure of Horvat et al. [25]. Briefly, extracts from stressed A375 melanoma cells were separated by preparative one-dimensional SDS-PAGE and transferred to nitrocellulose paper. ~321~34 was localized by Western analysis (described below) of a thin nitrocellulose strip. The entire area containing p32/p34 was then excised and blocked for 1 h at 25°C with 0.5 % gelatin in PBS. Following several rinses with PBS supplemented with 0.02% gelatin and 0.25% Tiiton X-100, the nitrocellulose strip containing electrophoretically separated p32/p34 was incubated with IgG fraction I, diluted in 0.02% gelatin/0.25% Triton X-lOO/PBS for 16 h at 4”C, and washed with the same buffer. Bound IgG was eluted with 30 mM glycine-HCl buffer, pH 2.3, for 3 min at 4°C. The eluate, referred to as IgG fraction II, was adjusted to pH 7.5 with 1 M Tiis, lyophilized, resuspended in PBS, and dialyzed against the same buffer for 24 h at 4°C. Fluorography, immunoprecipitation, and Wesrern analysis. Radiolabeled cell extracts or immunoprecipitates were prepared and analyzed by one- or two-dimensional SDS-PAGE and fluorography as described previously [8]. For Western analysis, unlabeled cell lysates were analyzed on SDS-PAGE gels and transferred electrophoretically onto nitrocellulose membranes as described 1261. Transfers were rinsed twice in double distilled water and air dried. Following rehydration, nitrocellulose was blocked with PBS containing 0.5 % gelatin for 1 h at 25”C, rinsed twice with 0.02% gelatin and 0.25 % Triton X-100 in PBS (Western buffer), and incubated for 1 h at 25°C with crude antiserum (1 : 200 to 1 : 1000 dilution), IgG fraction I (10 ug protein/ml), or affinity-purified IgG fraction II (1 ug protein/ml) diluted in Western buffer. Membranes were rinsed, incubated with 0.1 @i/ml “‘I-protein A (>30 mCi/mg) in Western buffer for 1 h at 25”C, and rinsed again. Dried membranes were exposed to Kodak X-Omat AR film using DuPont Cronex Quanta III intensifying screens. Immunocytochemistry. Indirect immunofluorescence microscopy was performed according to Sun and Green [27] with the following modifications. Cells were seeded into eight-chamber glass Lab-Tek slides (Lab-Tek Products, Naperville, IL) and allowed to adhere overnight. The cultures were

32-l34-kDa stress protein

B I

Frucrlon 2 3

C 4

T

33

Fraction 2 2ss

5

,p32/p34

i -p32

1~34

Fig. 1. Preparation of p32/p34 antigen. p32/p34 was isolated from arsenite-stressed, [3sS]methionine-labeled human A375 melanoma cells by preparative SDS-PAGE and electroelution as described under Materials and Methods. (A) Aliquots of five electroeluted fractions analyzed on a 10% acryhunide gel and visualized by fluorography. (B) Aliquots of fraction 2 analyzed on a 12% acrylamide gel and visualized by either fluorography (2) or silver stain (2SS). Total cellular extracts from radiolabeled control (C) and arsenite-stressed (7) A375 cells are included for comparison.

stressed, rinsed briefly with PBS, and fixed with 2% paraformaldehyde and 0.01% glutaraldehyde for 30 min at 25°C or with absolute methanol for 10 min at -20°C. Paraformaldehyde-fixed cells were permeabilized with digitonin (10 &ml) in 30 m&f Hepes buffer, pH 7.0, containing 100 mFt4 KCl, 20 mM NaCl, and 1 mM EGTA for 30 min at 25°C. Samples were rinsed in PBS, blocked with 5 % normal goat serum/l % BSmBS, and washed three times in PBS containing 1% BSA. Each chamber was incubated with affinity-purified IgG fraction II for 60 min at 25°C. Samples were rinsed, incubated with a 1 : 20 dilution of rhodamine-conjugated goat anti-rabbit IgG (Accurate Chemical and Scientific Corp., Westbury, NY) for 60 min at 25”C, rinsed again, mounted in glycerol (50% in PBS), and examined with a Leitz Vario-Orthomat fluorescence microscope.

RESULTS Metabolic labeling and in vitro translation. As shown previously [8], challenge of human A375 melanoma cells with 48 u&I sodium arsenite enhanced the synthesis of a 32-/34-kDa stress protein as assessed by one-dimensional SDS-PAGE analysis of [3SS]methionine-labeled cell extracts (Fig. 1A). mRNA isolated from arsenite-stressed, but not control, A375 melanoma cells also encoded a protein with molecular mass of 32 kDa as determined by in vitro translation using a protease- and nuclease-free rabbit reticulocyte system (data not shown). Antigen preparation. Radiolabeled extracts from stressed A375 cultures were

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Caltabiano,

Poste, and Greig

C

TI

TI

C

BLK

C

TI

T2

C

TI

SE

C

TI

SE

%s:

C. Crude

Antiserum

IgG Fraction

I

D. IgG

Fraction

II

Fig. 2. Purihcation of anti-p32/p34 antibody. Human A375 melanoma cell extracts containing equivalent amounts of protein (100 ug) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with either (A) crude antiserum, (B and C) IgG fraction I, or (D) affinity-purified monospecific IgG (IgG fraction II) as described under Materials and Methods. [‘%i]methionine-labeled A375 lysates are included for comparison. C, control; TI, test, 48 w sodium arsenite; BLK, blank: SE, human skin extract (20 ug).

analyzed on preparative SDS-PAGE and ~321~34 was localized by fluorographic examination of a thin acrylamide strip. The p32/p34-containing region of the gel was horizontally sectioned into live strips, 2 mm wide. Each strip was electroeluted and the resulting supernatant was separated on SDS-PAGE. The five fractions yielded proteins that differed slightly in their molecular masses, which ranged from 28 to 34 kDa (Fig. 1 A). Although fraction 2 appeared as a single band with molecular mass of 32 kDa, low amounts of contaminants were detected upon silver staining (Fig. IB). This fraction was used without further purification for immunization. Antibody purification. Six weeks postimmunization, serum was collected and analyzed for antibody titers by Western analysis and immunoprecipitation. Western analysis of A375 cellular extracts with crude antiserum from one animal detected a single 31-kDa band in control lysates and two bands of 31 and 32 kDa in extracts of arsenite-stressed cultures (Fig. 2A). Nonspecific reactivity with a 68-kDa band was also observed. Preimmune serum failed to react with the 31and 32-kDa proteins but showed weak recognition of the 68-kDa material (data not shown). To minimize reactivity with control cell lysates (31-kDa band), the crude antiserum was fractionated by protein A-Sepharose chromatography followed by adsorption of the purified IgG against extracts from unstressed A375 cells. IgG not bound during the adsorption step (referred to as IgG fraction I) reacted with a 32-kDa band in extracts of arsenite-stressed A375 melanoma cultures but failed to bind the 31-kDa component in lysates of both control and stressed cultures (Fig.

32-134-kDa stress protein IEF -

control

489M

~BJ.N

S&urn

35

Sodium Arsenita

Arsenite

C

Fig. S. Immunoprecipitation and two-dimensional SDS-PAGE analysis of p32ip34. [35S]methioninelabeled A375 lysates were prepared as described under Materials and Methods. Samples containing equivalent amounts of TCA-insoluble radioactivity (500,000 cpm) were either analyzed by twodimensional SDS-PAGE and fluorography directly (A and B) or first incubated with anti-p32/p34 IgG fraction 11 (c) and immune complexes precipitated by Sraphylococcus protein A. (A and B) total cell lysates; (c) immunoprecipitate.

2B). However, this pm-Cation step failed to eliminate reactivity against the 68kDa band. Artifacts similar to the 68-kDa material have been attributed to contaminating epidermal keratins [28]. To investigate this possibility the reactivity of IgG fraction I against extracts of human skin was examined. Western analysis revealed several immunoreactive proteins with molecular masses ranging from 50 to 68 kDa (Fig. 20. A similar pattern was observed with anti-keratin antiserum (data not shown). The source of this contamination was subsequently traced to various reagents, in particular the glycine used in preparing acrylamide gels and electrophoresis buffers (data not shown). Final purification of anti-p32/p34 immunoglobulin was achieved by adsorption of the antibody to human p32/p34 immobilized on nitrocellulose. This preparation, containing affinity-purified monospecitic IgG (referred to as IgG fraction II),

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Fig. 4. Immunoprecipitation of human, rat, and murine p32/p34 stress proteins. 13’Slmethioninelabeled human, rat, and murine cell extracts were prepared as described under Materials and Methods. Samples containing equivalent amounts of TCA-insoluble radioactivity (100,000 cpm) were incubated with anti-p32/p34 IgG fraction I. Immune complexes were precipitated by Staphylococcus protein A and analyzed by one-dimensional SDS-PAGE and fluorography. C, control; T, test, 48 pJ4 sodium arsenite.

reacted with only a single 32-kDa protein in extracts of stressed A375 cultures (Fig. 20). There was no reactivity with unstressed cells, epidermal keratins, or other stress proteins. Similar results were observed in cultures challenged with other sulfhydryl-reactive agents (data not shown). The specificity of the antiserum was further evaluated by immunoprecipitation of arsenite-stressed A375 extracts with anti-p32/p34 IgG fraction II and subsequent analysis by two-dimensional SDS-PAGE. p32/p34 in total cell extracts failed to enter the isoelectrofocusing gel when analyzed by equilibrium methods (Fig. 3B). Similar results were obtained using nonequilibrium conditions (data not shown). Incubation of stressed A375 cell lysates with affinity-purified antip32/p34 IgG precipitated a 32-/34-kDa protein with an identical electrophoretic mobility (Fig. 3C). Species-specljk irnmunoreactivity. To investigate ~321~34 expression in different cell types, extracts of various arsenite-challenged human, rat, and murine cell lines were immunoprecipitated with anti-p32/p34 IgG fraction I. A 32-kDa protein was consistently detected in human and rat cell lysates whereas a 34-kDa protein was found in murine cultures (Fig. 4). Little or no reactivity was observed in unstressed cultures. The antibody also precipitated less prominent bands of 31 and 28 kDa in stressed human and rat cells and 33- and 27-kDa components in arsenite-challenged murine cultures (Fig. 4). These proteins may be either degradation products of p32/p34 or polypeptides closely associated with the stressinduced proteins. Preimmune serum failed to precipitate proteins from either unstressed or stressed cell lysates (data not shown). Zmmunocytochemistry. Human CCD-21Sk skin tibroblast and A375 melanoma

32-l34-kDa

stress protein

31

CCD-21%

Control

Fig. 5. Localization of p32/p34 by indirect immunofluorescence. Human CCD-2lSk fibroblasts (A and B) and human A375 melanoma cells (C and D) were incubated in either serum-free DME (B and D, control) or serum-free DME plus sodium arsenite (25 m (A and C, test) for 8 h, fixed with methanol, and stained with anti-p32/p34 affinity-purified monospecific IgG (IgG fraction II) as described under Materials and Methods. Phase-contrast photomicrographs are shown in the right column for comparison. A, bar = 22 urn; C, bar = 18 urn. Arrow in (C) indicates ring-like pattern of fluorescence in A375 cells.

cells were used to examine the subcellular location of the 32-/34-kDa stress protein because of their flattened and well-spread morphology. Analysis of methanol-fixed, arsenite-stressed cultures, using affinity-purified anti-p32/p34 (IgG fraction II), revealed a granular cytoplasmic fluorescence (Figs. 4A and C). Intense perinuclear staining was also observed in a majority of the cells examined

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although nuclear fluorescence was absent. The ring-like pattern surrounding the nucleus was particularly prominent in the less flattened A375 melanoma cells (arrow, Fig. 5 C). In contrast to the distribution of p32/p34 staining, treatment of arsenite-stressed cultures with a polyclonal antiserum specific for the 72-kDa stress protein [29] resulted in diffuse cytoplasmic and intense nuclear fluorescence (data not shown). DISCUSSION In a previous study we described the specific induction of a 32-/34-kDa protein in both normal and neoplastic human (~32) and murine (~34) cells stressed with heavy metals (Zn*+, CL?, and Cd’+) or sulfhydryl-reactive agents (disulfiram, auranofin, arsenite, and iodoacetamide) [8]. However, synthesis of p32/p34 was not enhanced by conditions used routinely to induce other stress molecules such as the major heat-shock and glucose-regulated proteins (e.g., hyperthermia, amino acid analogs, glucose deprivation, and calcium ionophores), suggesting that this polypeptide belongs to a novel subset of stress-induced gene products. This hypothesis was further supported by the observation that preincubation of arsenite-stressed cells with either actinomycin D or cycloheximide blocked synthesis of p32/p34 suggesting that expression of this protein was controlled at both transcriptional and translated levels [8]. We have further demonstrated by in vitro translation that a novel gene encoding the p32/p34 transcript is activated in mammalian cells upon exposure to heavy metals and sulfhydryl-reactive agents. We have proposed previously that p32 (human) and p34 (murine) are closely related molecules encoded by species-specific variants of similar genes. This hypothesis was supported by shared biochemical properties between p32 and p34 and the observation that antibodies against human p32 cross-react with a 34-kDa protein in murine melanoma cells challenged with arsenite. As shown in the present study, a polypeptide of similar molecular mass (32-34 kDa) is detectable in a histologically diverse range of stressed human and rodent cell types, but not in control cultures. In fact, expression of this molecule is a uniform response to cellular challenge with arsenite and no exception has been identified to date. Cultures stressed with other sulfhydryl-reactive agents (auranofin and disultiram) also express an immunoreactive antigen of identical molecular mass. The precise molecular mass of the 32- to 34-kDa protein is species-specific. In the present survey of 13 cell lines, the 32-kDa antigen was expressed by 6 human and 3 rat cell types and the 34-kDa component by 4 murine cultures. p32/p34 is probably related to stress proteins with similar molecular masses expressed in chick embryo tibroblasts (35 kDa) and various rat cell lines (2.5 to 30 kDa) induced by challenge with arsenite [9-l I]. These results are consistent with the proposal 181 that a specific aspect of the stress response in mammalian and avian cells exposed to heavy metals and sulfhydryl-reactive agents involves the activation of a conserved gene encoding a 30- to 35-kDa protein. As a prerequisite for studying the subcellular localization of p32/p34, confirmation of the specificity of the polyclonal antibody produced against this protein was required. Preliminary analysis indicated the crude antiserum recognized two

32-/34-kDa

stress protein

39

bands of 31 and 32 kDa in extracts of arsenite-stressed human melanoma cells and a single band of 31 kDa in lysates from untreated control cells. Nonspecific reactivity against contaminating epidermal keratins was observed in both preimmune and immune serum. Monospecific polyclonal antibody was obtained by sequential purification on protein A-Sepharose chromatography, adsorption of the isolated IgG against control cell lysates, and final affinity separation against ~321~34 immobilized on nitrocellulose. The resulting affinity-purified antibody specifically recognized p32/p34 as determined by Western analysis of total cellular proteins derived from control and arsenite-challenged cultures (Fig. 2). The specificity of this antibody was further evaluated by immunoprecipitation of [35Slmethionine-labeled cell extracts and subsequent analysis by two-dimensional SDS-PAGE. Incubation of arsenite-stressed A375 human melanoma cell lysates with affinity-purified anti-p32/p34 IgG precipitated a 32-kDa protein with an electrophoretic mobility identical to that of the major 32-kDa arsenite-induced protein observed in total cell extracts. Immunocytochemical analysis of arsenitestressed human fibroblasts and melanoma cells with the purified monospecific antibody indicates that p32/p34 is a cytoplasmic protein with a predominantly perinuclear distribution. This pattern is similar to that of the heat-shock protein hsp 90, a cytoplasmic polypeptide [30], and the glucose-regulated protein grp 78 which localizes to the endoplasmic reticulum [31]. Since ~321~34 required mild detergent for solubilization [8], it may also be peripherally associated with the endoplasmic reticulum membrane. In contrast to another major stress protein of mammalian cells, hsp 72, p32/p34 was not detected in the nucleus [29]. Although the present study does not address the function of p32/p34, the ubiquitous induction of this protein in diverse cell types by heavy metals and sulfhydryl-reactive reagents and its cytoplasmic/endoplasmic reticulum location suggest it may play a role in detoxification. Consistent with this speculation are the observations that several detoxification enzymes have similar molecular masses and are distributed in cytoplasmic and microsomal compartments (e.g., methyltransferase, 28 kDa; acetyltransferase, 33 kDa; and sulfurtransferase, 33 kDa) [32]. In addition, many xenobiotics are capable of enhancing, in a dose- and time-dependent manner, their own metabolism by eliciting de noun synthesis of the enzymes responsible for their detoxification [33]. Whether induction of p32/p34 by sodium arsenite is a further example of xenobiotic metabolism will require isolation of native p32/p34 in sufficient quantities for analysis of enzymatic activity. Human skin extract and rabbit anti-hsp 72 serum were kindly provided by Dr. Robert Clark, Thomas Jefferson University, Philadelphia, Pennsylvania, and Dr. William Welch, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, respectively. We thank Jo Anne Mackey for excellent secretarial assistance, Scot Gerding and Ray Gues for photographic services, and Dr. Thomas Koestler for helpful discussions.

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31. Munro, S., and Pelham, H. R. B. (1986) Cell 46, 291. 32. Jakoby, W. B. (Ed.) (1980) Enzymatic Basis of Detoxification. 33. Klinger, W. (1982) Pharmac. Ther. 16, 377. Received November 11, 1987 Revised version received March 15, 1988

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