A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized.
THEJOURNALOFBIOLOGICAL Q 1991 by
CHEMISTRY
Vol. 266. No. 4, Issue of February 5, pp. 2390-2396, 1991 Printed in U.S. A.
The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterizationof Pseudomonas aeruginosa Exotoxin A Binding Glycoprotein from Mouse LM Cells* (Received for publication, June 12, 1990)
Michael R. Thompson*§,Judith Forristals, Peter Kauffmanne, Terrance Maddens, Kenneth Kozaks, Randal E. Morrisv, and Catharine B. SaelingergII From the $Division of Digestive Diseases, §Department of Molecular Genetics,Biochemistry, and Microbiology and the TDepartmentof Anatomy and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
A Pseudomonas aeruginosaexotoxin A (PE) binding glycoprotein was affinitypurified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular massof greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10%of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.
of toxin to PE receptors of toxin sensitive cells. The second step is more complicated and comprises internalization of PE by receptor-mediated endocytosis, the conversion of toxin to an enzyme active form, and ultimately translocation of toxin across a vesicular or endosome membrane into the cytosol. The third eventis the inactivation of cytoplasmic elongation factor 2. The cellular sites at whichtoxin activationand translocation occur have not been clearly defined. For several years, we have examined the interactionof P E with mouse LM fibroblasts,a cell line which is highly sensitive t o P E (15, 16). On the ultrastructural level, we have shown that biotinyl-PE initially binds to sites randomly distributed on the LM cell surface (17). This binding is saturable, with native toxin competing with biotinyl-PEfor surface binding sites (18).Following warming of cells to 37 “C, surface-bound toxin moves rapidly to coated areas and is internalized into Pseudomonas aeruginosa is a significant opportunistic path- noncoated vesicles (endosomes). This pathway of internaliogen. The pathogenesisof Pseudomonas infection is complex zation is identical to the receptor-mediated endocytic pathway and involves the in uiuo production of several virulence fac- described for other ligands such as low density lipoprotein tors, including an extracellular protein, Pseudomonas exo- (19), transferrin (ZO), and growth hormones (21). From the toxin A (1). PE’ belongs to the family of bacterial toxins endosome, a fraction o f internalized PE is transported to the classified as ADP-ribosyltransferases(2). Specifically, the Golgi region of the cell and ultimately is delivered to lysotoxin acting on mammaliancells catalyzes the transferof the somes where it is degraded (14, 17). We also have measured ADP-ribose moiety of NAD to cytoplasmic elongation factor the binding of ‘251-labeledP E t oparaformaldehyde fixed LM 2, rendering elongation factor 2 inactive in protein synthesis; cells (22). Using this system, we have demonstrated specific the end result iscell death (3). binding of toxin. Saturation isachieved at approximately 5.4 PE is secreted by P. aeruginosa as a single polypeptide, nM toxin, which permits an estimate of approximately 100,000 which is toxic to cells, but is enzymatically inactive (4). In its receptors/LM cell. Toxin escaping this degradative pathway secreted form, the toxin is a 613-amino acid protein (5). X- is thought to causecell death by acid-mediated translocation ray crystallographic studies have shown that the toxin pos- from endosome to cytosol. sesses three structural domains: domain I (amino terminus), We havebegun to isolate the receptor for toxin from mouse contains the eukaryoticcell recognition site; domain I11 (car- LM cells. We chose t o work with these cells forseveral boxyl terminus), contains theenzyme active site; and domain reasons. First P E is routed into these cells via structures I1 (middle domain) is believed t o function in transport of P E associated with receptor-mediated endocytosis. Second, LM across mammalian cell membranes (6-11) and in secretionof cells possess high affinity P E binding sites. Third, we have recombinant P E into the periplasm of Escherichia coli (12, established that treatment of LM cellswith trypsin tran13). Three distinct steps have been implicated in cell intoxi- siently ablates toxin sensitivity.’ Finally, we recently showed process is the binding that a P E binding moiety can be solubilized from LM cell cation by PE (14). The first step in the extract^.^ In this paper we describe the purification and char* This work was supported by the National Institutes of Health acterization of a PE binding glycoprotein. Grants AI 17529 and G M 24028. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524. Tel.: 513-5580072; Fax: 513-558-8474. ’ The abbreviations used are: PE, pseudomonas exotoxin A; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; staph V8, staphylococcus aureus V8 protease; PNGase F, peptide N-glycosidase F; ELISA, enzyme-linked immunosorbent assay; Endo H, endo-P-N-acetylglucosaminidaseH.
EXPERIMENTAL PROCEDURES
Materials and Reagents-Purified P. aeruginosa exotoxin A and sheep Pseudomonas antitoxin A were obtained from Swiss Serum and Vaccine Institute, Berne, Switzerland. The toxin migrated as a single staining band on SDS-PAGE. PEwas resuspended according to the manufacturer’s specifications and stored as aliquots at -80 “C. Biotinylated PE was prepared as described previously (18). Rabbit anti-
* c. E. Saelinger, unpublished observation.
M. R. Thompson, D. Volk, and C. B. Saelinger, submitted for publication.
2390
Characterization of Pseudomonas Exotoxin Binding Protein toxin was prepared against a purified glutaraldehyde-treated lot of PE (17). Diphtheria toxin (95% unnicked), cholera toxin B subunit, and goat anti-exotoxin A were obtained from List Biological Laboratories, Inc. Affinity purified rabbit anti-sheep IgG (h+l), horseradish peroxidase-laheled or alkaline phosphatase-labeled, was obtained from Kirkegaard and Perry Laboratories, Inc. Affinity purified swine anti-goat IgG labeled with horseradish peroxidase, PNGase F, Endo H, neuraminidases isolated from Vibrio cholerae and Arthrobacter ureafacians, calf intestine alkaline phosphatase, and the protease inhibitor leupeptin were obtained from Boehringer Mannheim. Phenylmethanesulfonyl fluoride, N-tosyl-L-phenylalanine chloromethyl ketone, dithiothreitol, immobilizedbovine pancreatic trypsin, and Staphylococcus aureus endoproteinase V8 were obtained from Sigma. Streptavidin-horseradish peroxidase was obtained from Zymed. Extractigel was obtained from Pierce Chemical Co. Molecular mass markers and materials for SDS-polyacrylamide gel electrophoresis were obtained from Bio-Rad. ~-[~H]Leucine was obtained from Amersham Corp. (45 Ci/mmol). Cells-Mouse LM Fibroblasts (ATCC CCL 1.2, L-M) were obtained from the American Type Culture Collection. Cells were maintained in McCoy's 5A medium (GIBCO) containing 10% heat-inactivated fetal calf serum, penicillin, and streptomycin and were grown 3 days at 37 "C in 10% CO, prior to harvest (17). National Institutes of Health/OVCAR-3 cells are isolated from malignant ascites of patients with ovarian carcinoma and were obtained from Dr. D. FitzGerald (National Cancer Institute, Bethesda, MD); they were grown in RPMI medium containing 10% fetal bovine serum, penicillin, and streptomycin. Preparation of Solubilized Binding Protein-PE binding protein was solubilized from LM cells as described previously.3 Briefly, eight 150-cm2flasks containing a total of 4 X lo7 LM cells were washed with 10 mM sodium phosphate-buffered saline (PBS),pH 7.2, at room temperature. Cells were collected by scraping in buffer A (0.125 M Tris maleate buffer, pH 6.0, containing 160 mM NaCl, 0.002 mM MgCI, 2.0mM CaC12, 1.0 mM EDTA, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, 0.1 mM phenylmethanesulfonyl fluoride, and 0.1 mM leupeptin). All additional steps were performed at 4 "C. Cells were centrifuged, resuspended in 6 ml of buffer A, and thendisrupted by repeated passage through an 18-gaugeneedle. The suspension was made 1.0% (v/v) Triton X-100. Detergent extraction continued with stirring for 45 min. The material was then centrifuged at 100,000 X g for 1h and thepellet discarded. The resulting supernatant, hereafter called detergent extract, was assayed for protein and frozen at -80 "C or applied to a PE-Sepharose affinity column. In some experiments, the detergent Nonidet P-40 was used in place of Triton X-100. Preparation of Affinity Column-One mg of purified P E was coupled to 2-ml bead volume of CnBr-activated Sepharose 4B (Pharmacia LKB Biotechnology Inc.) according to the manufacturer's instructions. The efficiency of PE coupling was virtually loo%, determined by ELISA and by silver-staining SDS-PAGE. This affinity column was washed routinely prior to use with buffer B (PBS, 0.1% Triton X-100, pH 8.5). After several uses, an additional wash was employed using 10 ml of 5 M NaSCN in buffer B followed by 50 ml of buffer B. The PE-Sepharose was stored in buffer B with 0.05% sodium azide a t 4 "C when not in use. Purification of PE Binding Protein-To affinity purify P E binding proteins, 5ml of the detergent extract (3.3 X lo7cell equivalents) was directly applied to a 2-ml bead volume PE-Sepharose column and recirculated through the column overnight at 4 "C. The column was washed with 100 ml of buffer B, followed by 15 ml of 1 M NaCl in buffer B to remove unrelated proteins. The binding protein was then eluted in a single peak with 12.5 ml of 1.0 M NaSCN dissolved in Buffer B. This eluate was immediately applied to Pharmacia PD-10 gel filtration columns equilibrated in buffer B. The binding proteincontaining fractions were then concentrated to a final volume of approximately 600 pl using centrifugal concentrators (Centricon 30, M, 30,000 cut-off microconcentrators, Amicon, Inc.). This affinity purified binding protein was used for further analysis or storedfrozen in aliquots at -80 "C. For some experiments, excess Triton X-100 was removed from concentrated purified binding protein with Extractigel. Proteinconcentrations were determined using a commercially available Bradford protein detection reagent (Bio-Rad Protein Assay Reagent). Affinity purified binding protein concentration was determined by fluorescamine protein assay to circumvent difficulty in protein quantitation inthe presence of high concentrations of Triton X-100 in these samples. BSA was used as the protein standard for these assays.
2391
E~ctrophoresis-SDS-PAGE was performed using the method of Laemmli (23). Samples were prepared for electrophoresis by mixing with sample solution with or without reducing agent. Depending on the requirements, samples were either incubated at 55 or 100 "C for the times indicated or incubated a t room temperature before application to the gel.After SDS-PAGE, proteins were stained using Coomassie Brilliant Blue R-250, followed when applicable by silver staining using the Bio-Rad silver staining kit, or transferred by electroblotting to nitrocellulose sheets for further analysis (24). PE Binding Protein Blot Assay-PE binding proteins were detected after SDS-gel electrophoresis separation and electroblotting to nitrocellulose sheets. All incubations were performed at room temperature except where indicated. The nitrocellulose sheet was incubated in nonfat dry milk dissolved in 0.3% BSA-PBS buffer, pH 7.2, for 1 h at 37 "C to block residual protein binding sites on the nitrocellulose (25). The sheet was washed with PBS and then incubated in 0.3% BSA-PBS containing PE at0.5 pg/ml for 1 h at 37 "C. PE routinely was detected by reaction with sheep anti-PE followedby affinity purified alkaline phosphatase-labeled rabbit anti-sheepIgG. Proteins which bind P E were detected with the substrate 5-bromo-4-chloro-3indolylphosphate (Kirkegaard and Perry Laboratories). ELZSA for PE Binding Protein-We have developed an ELISA to detect P E binding to immobilized solubilized binding p r ~ t e i n This .~ direct assay was employed to detect and titratebinding protein during purification, to study the binding characteristics of PE toimmobilized binding protein in a nondenaturing environment, and to study the effects of a variety of pretreatment conditions on the binding protein. Briefly, flat bottom microELISA plates (Immulon 11) were sensitized with appropriate dilutions of detergent extract or purified preparations. 100 pl of binding protein diluted in PBS buffer, pH 7.1, containing 0.01-0.02% Triton X-100 (sensitizing buffer) was applied to wells and dried by incubation overnight at 37 "C in a circulating air incubator. Wells were rinsed five times with buffer C (154 mM NaC1, 0.001% thimerosal, 0.1% Triton X-100,0.05% Tween-20, in 10 mM KH,PO,-Na,HPO, buffer, pH 7.2) and blocked for 1 h at 37 "C with 7% nonfat dry milk (w/v) in PBS. Plates were washed five times with this buffer between each incubation step. To obtain the optimum binding conditions for PE to the immobilizedpurified binding protein, P E binding was performed with 4 pg/ml P E for 3.5 h in the presence of either 50 mM sodium acetate or sodium phosphate buffers encompassing a pH range from 4.0 to 9.0, in the presence of 0.154 M NaC1. For all other experiments, sensitized plates were incubated with PE diluted in buffer D (3% BSA-PBS buffer, pH 7.2) for 3.5 h at 37 "C. After washing, plates were incubated with sheep anti-PE for 1 h a t 37"C. After washing, horseradish peroxidase-labeled rabbit antisheep IgG diluted 1:lOOO in buffer D was added for an additional hour. After washing, substrate (ortho-phenylamine diamine and H202) was added and color development allowed to proceed for 30 min at room temperature. The reaction was halted and absorbance measured at 492 nm. Typically, determinations were made in quadruplicate. A competition format of the ELISA was employed to show specificity of PE binding. Wells were sensitized as above with 1 pgof purified binding protein. In these experiments, 0.1 pg of biotinylated PE was incubated in wells with or without various concentrations of unlabeled PE, in 3% BSA-PBS for 3 h. Plates were washed to remove free PE, and the biotinylated P E remaining was detected by subsequent incubations with streptavidin-horseradish peroxidase diluted in the same buffer, followed by ortho-phenylamine diamine. Modification of Purified Binding Protein-The influence of different modifying agents or preincubation conditions on PE binding to detergent extract or purified binding protein was as follows. Affinity purified binding protein was diluted to a stock concentration of 100 pg/ml in buffer B (using the fluorescamine protein assay). For modification experiments, the binding protein was diluted 10-fold into the incubation mix yielding a concentration during treatment of 10 pg/ml. Proteolytic digestion with staph V8 protease was performed either in 50 mM ammonium bicarbonate buffer, pH 7.8, or in 50 mM sodium phosphate buffer, pH 7.8. All incubations were performed in a water bath for the appropriatetimes and temperatures. After treatment, purified binding protein was again diluted 10-fold into the appropriate buffer. For ELISA determinations, treated binding protein was diluted in sensitizing buffer, prior to assay. Serial dilutions of the treated binding protein were then tested, starting with 0.1 pg in 100 p1 dried onto the ELISA plate wells as described above. When treated samples were assayed by SDS-gel electrophoresis, they were diluted into sample buffer either with or without reducing agent prior to application to gels.
2392
Characterization of Pseudomonas Exotoxin Binding Protein
Glycosylation of Binding Protein-Commercially available glycan detection and differentiation kits were employed to identify carbohydrate and sialic acid constituents of purified binding protein (Boehringer Mannheim). Purified binding protein was electrophoresed as described above and then transferred to nitrocellulose sheets. Glycoproteins on the nitrocellulose were detected in two ways. Using the glycan detection kit, hydroxyl groups were oxidized and then labeled with digoxigenin through hydrazide reaction. The digoxigenin was then detected by enzyme immunoassay, according to the manufacturer’s instructions. By using the glycan differentiation kit, glycan constituents were identified by a similar enzyme immunoassay, employing group-specific lectins. In some experiments, binding protein was incubated with Endo H or PNGase F prior to electrophoresis, electroblotting, and glycan detection to distinguish N-linked and 0linked glycan structures.PNGaseF was incubated with binding protein in 50 mM EDTA, 0.05% sodium azide, 20 mM potassium phosphate buffer, pH 7.2. Endo Hwas incubated with binding protein in PBS, pH 7.2. When binding protein preparations obtained from Nonidet P-40 extractswere digested, the buffers in addition contained 0.1%Nonidet P-40. Gel Filtration of Detergent Extracted Receptor-Gel filtration was performed using a Pharmacia Superose 12 fast protein liquid chromatography column a t room temperature. Receptor purified from I 1 I I I I Triton X-100 extracts was chromatographed in PBS buffer, pH 7.1, 15 010 5 25 20 containing 0.01% Triton X-100. Individual fractions were either Fraction Number assayed directly for receptor by ELISA, by directly staining SDSPAGE separation of the fractions, or by SDS-PAGE blot assay for FIG. 1. Purification of PE binding protein by PE-Sepharose P E binding protein (see above). The column was calibrated with the affinity chromatography. 42.5 mg of detergent-solubilized protein following protein standards to correlate retention times with molec- was applied to theaffinity column, as described under “Experimental ular mass: Apoferritin (440,000 Da), P-amylase (200,000 Da), alcohol Procedures.” After overnight recycling at 4 “C, the column was dehydrogenase (150,000 Da), bovine serum albumin (66,000 Da), washed with PBS-Triton X-100 (fractions 1-20, 5.0 ml/fraction) and carbonic anhydrase (29,000 Da), andcytochrome c (12,400 Da). then eluted with 1 M NaSCN (6 X 2.5 ml; fractions 21-26). Fractions Assay of Toxin Biological Actioity-Inhibition of LM cell protein were desalted, diluted to 1:200, and then assayed by ELISA for PE synthesis was used as an assay for PE biological activity. Protein binding. Background (wells not sensitized, A = 0.150) was subtracted synthesis was measured by determining the amount of ~-[~H]leucine from each reading. incorporation into trichloroacetic acid-insoluble material during a 60min pulse at 37 “C (15). RESULTS
Isolation of PE Binding Protein-PE binding protein was solubilized from mouse LM fibroblasts and purified by single step affinity chromatography on PE-Sepharose affinity columns. PE binding as measured by ELISA was used to follow the purification of potential receptor and to calculate approximate recovery during purification. PE binding activity was solubilized from LM cells with a variety of nondenaturing detergents including Triton X-100, Nonidet P-40, Chaps, and n-octyl glucoside. In several experiments, PE binding protein was purified from detergent extracts of a 33,000 x g (60 min) membrane pellet. The PEbinding characteristics of the protein purified from these crude membranes were identical to those of the protein purified from detergent extractsof whole cell homogenate (not shown). From approximately 40 mg ofprotein solubilized from eight 150-cm2flasks of LM cells,the procedure consistently yielded 50-100 pgof purified binding protein. P E binding activity measured by ELISA was found to elute in a single peak using 1.0 M NaSCN (Fig. 1).The recovery of PE binding activity was typically 30% of the total applied in the detergent extracts. Higher yields were obtained (up to 50%)when binding protein was eluted with 3 or 5 M NaSCN rather than 1 M NaSCN (not shown). However, elution under these conditions caused a fraction of the purified protein to spontaneously aggregate. Although very little protein and almost no PE binding activity was detected in a 1.0 M NaCl wash, this step was subsequently incorporated just prior to SCN elution to remove trace quantities of nonspecifically bound proteins. The affinity columns characteristically improved with use, initially demonstrating tracelevels of PE in the final product. Repeated NaSCN regeneration of affinity columns did not alter the binding capacity of the PE-Sepharose affinity column. Protease inhibitors were required during chromatogra-
phy to minimize degradation of immobilized PE by the LM cell detergent extract. SDS-PAGE Analysis of the Purified PE Binding ProteinMultiple proteins were extracted by Triton X-100 (Fig. 2.4). Purified binding protein migrated as one species. The affinity purified material showed a single protein band when stained by CoomassieBrilliant Blue (not shown). Contamination with other proteinswas quite low as evidenced from silver staining of the purified preparation (Fig. 2 A ) . The PEbinding protein was affinity purified equally well from Triton X-100 and Nonidet P-40 detergent extractsof LM cells (Fig. 2B). Preincubation of the affinity purified preparation with reducing agent slightly increased the apparent size of the molecule. This treatment did not generate lower molecular weight species detectable by silver staining, suggesting that the toxin binding protein is a single peptide, rather than an aggregate or multimeric structure. Incubation with reducing agent at 100 “C for 10 min resulted in significant reduction in staining intensity of the protein with either Coomassie or silver. Again, no breakdown products of molecular weights greater than 10,000 were observed after this pretreatment. The strongest staining was observed with toxin binding protein that was neither incubated with reducing agent, nor heated. The toxin binding protein extracted and purified in Nonidet P-40 had an identical electrophoretic mobility as the protein extracted with Triton X-100. Fig. 2C shows that the PEbinding site of the purified preparation was retained after SDS-PAGE. The strongest toxin binding occurred when purified protein was neitherheated nor reduced prior to electrophoresis. This corresponds with the staining behavior of the receptor. PE binding was greatly reduced but not abolished by heating the PE binding protein in SDS or reduction with 2-mercaptoethanol prior to electrophoresis. Fig. 2 0 shows that an identical toxin binding protein is identified with antitoxin prepared in several species. In addition, none of the second antibodies
2393
Characterization of Pseudomonas Exotoxin Binding Protein B
A
4
ORIGIN
--
1.5 1
200 kDa
2
3
4
5
c
6
-c
116 97.4
D
66.2
7 8 910
FIG.2. SDS-polyacrylamide gel electrophoresisof PE binding protein purified from mouse LM cells. A , Triton X-100 detergent extract and affinity purified binding protein identified by silver staining.B , affinity purified binding protein identified by silver staining. Samples inlanes 1-3, Nonidet P-40-extractedprotein; lanes 4-6, Triton X-100 extracted protein. Lanes 1 and 4, samples not heated or treated with reducing agent; lanes 2 and 5,samples incubated with 2-mercaptoethanol priorto electrophoresis;hnes 3 and 6, samples heated for 10 min at 100 “Cin the presence of 2-mercaptoethanol. C, SDS-PAGE PE bindingblotassay;samples in lanes correspond to those in B. PE binding was performed as described 2.0 1.0 0 -1.0 -2.0 -3.0 under “Experimental Procedures.”D, PE blot assay, Nonidet P-40Log pg ProteinlWell extracted protein, reduced lanes 7 and 9,binding protein identified with rabbit antitoxin, affinity purified horseradish peroxidase-labeled FIG.3. PE binding to immobilized detergent extracted and goat anti-rabbit IgG; lanes 8 and 10, binding protein identifiedwith affinity purified binding protein. MicroELISA plate wells were goat antitoxin horseradish peroxidase-labeled swine anti-goat IgG. sensitized with dilutions of Triton X-100 detergent extract (A) or The PE incubation step was omitted from lanes 9 and 10 to serve as affinity purified binding protein (0).After washing to remove uncontrol. Samples were electrophoresed in 6% separating polyacryl- bound protein and detergent, the PE binding of the immobilized amide gel. The molecularmassmarkersmyosin (200,000 Da), p- material wasmeasured as describedunder“ExperimentalProcegalactosidase (116,000Da), phosphorylase b (97,400 Da), and bovine dures.” The background absorbance (unsensitized wells) was 0.13. serum albumin (66,200 Da)are indicated to the right of A. employed reacted nonspecifically with the toxin binding protein (Fig. 2 0 , lanes 9 and 10).Thus thesignal depends entirely upon incubation with PE. The PE binding protein migrated with a n R F identical to fibronectin (-440,000 Da)whenelectrophoresedin 4-8% 0 acrylamide gels. The PE bindingprotein molecular mass estimate from protein standards in SDS-PAGE is only approximate, since the PE binding protein is so much larger than available molecular mass markers. Extrapolation from protein standards comprising the molecular mass range of 1 I I I 29,000-200,000 Da yields a significantly lower molecular mass 0.01 10 0.1 1.0 estimate (approximately 300,000-350,000 Da). pg Native ToxinlWell The apparent size of theproteinwasnotartifactually FIG.4. Competition inhibition of biotinylated PE binding increased in the presence of high concentrationsof Triton X- to purified binding protein by PE. Immobilized binding protein 100,since removal of excess Triton X-100 prior to electro- was incubated with 100 ng of biotinylated PE and various concentraits electrophoreticmobility(not phoresisdidnotchange tions of native PE for 4.5 h and then developed as described under “Experimental Procedures”(0).In other experiments increasing conshown). Occasionally, detergent extractsshowed smaller PE binding centrations of choleratoxin B (0)ordiphtheriatoxin (A) were fragments detected by SDS-PAGE blot. Storage of purified substituted for native PE. The data presented are the averages for six experiments, each consistingof triplicate or quadruplicatedeterprotein on ice or at 4 “C for prolonged periods (2-4 weeks) minations, and are expressed as percent bindingof control (no added spontaneously generated at least one PE binding fragment competing PE). The absorbance obtained with biotinylated PE was with an apparent massof about 100,000 Da. typically 0.9 in sensitized wellsin the absence of competing PE, Characterization of Purified PE Binding Protein by compared with backgroundof 0.15 in unsensitizedwells. ELISA-The binding characteristicsof detergent extract and PE progressively inhibited the bindingof biotinylated PE. I n affinitypurifiedbindingproteins were virtuallyidentical when compared by ELISA (Fig. 3). 0.1 pg of purified PE contrast, two other toxins, diphtheria toxin and the binding binding protein yielded approximately 0.75 in the ELISA. domain of cholera toxin, did not displace biotinyl-PE. Solubilized affinity purified protein was preincubated under Under identical ELISA conditions, the same signal was obPE a variety of conditions to study their influence on subsequent tainedwith 25-30 pg of detergentextract.Therefore binding to immobilized purified binding protein was about PE binding measured by ELISA. The toxin binding protein at 250-fold greater than in the detergent extract. The similar wasstabletofreeze-thawing(threetimes)andstorage 3 h. The dose-response curves for these preparations (Fig. 3) suggest -80 “Cand was stableat room temperature for up to PE binding characteristics after that the PE binding site was not altered by the purification purified material retained its storage on ice a n d a t 4 “C overnight.Toxinbindingwas procedure. 5 min prior to immobiliThe affinity purified immobilized preparation specifically abolished by boiling the protein for bound PE as shown in Fig. 4. Increasing concentrations of zation.
E
z
2394
Characterization of Pseudomonas Exotoxin Binding Protein
We looked at the pH stability of purified toxin binding protein. Prior exposure to pH4.0 for 30 min at 37 “Cdecreased the ELISA signal to approximately 55% that of affinity purified protein incubated at pH 7.0. The PE binding of a preparation incubated in sensitizing buffer (PBS, pH 7.2) at 37 “Cwas reduced 10-20%after 10 min, but remained at that level for at least 90 min. We also examinedthe effect of different incubation conditions during PE binding to immobilized affinity purified protein. Fig. 5 shows that PE binding was optimal between pH 5.0 and 5.5 in 50 mM sodium acetate buffer. Binding at pH 7.0 was approximately 40% of binding at pH 5.5. This contrasts with our earlier findings that PE binding to detergent extract was maximalat pH 5.0 and below.3 Enzymatic Treatment of PE Binding Protein-The sensitivity of purified toxin binding protein to degradation by several proteases with defined specificities was compared in ELISA and by SDS-PAGE. Preincubation of the purified preparation with staph V8 protease reduced the binding of PE to immobilized treated material, compared with an untreated preparation (Fig. 6A). Interestingly, even under the most severe conditions tested, i.e. overnight incubation of purified protein with equivalent weight of protease, PE binding as measured by ELISA was not completely abolished. These results were obtained when the purified PE binding protein was incubated under conditions that favor cleavageat only Glu-C bonds (26). Proteolytic digestion was also monitored by SDS-PAGE. As shownin Fig. 6B,discrete lower molecular mass fragments retaining PE binding sites were detected. Virtually identical fragmentation patterns were observed when the PE binding protein was digested under conditions favoring either Glu-C or both Arg-C and Glu-C activities. Fragments with apparent mass of 280,000 and 200,000 retained significant PE binding capacity. Smaller discrete proteolytic fragments observed on stained gels showed little or no appreciable toxin binding. At the highest concentration of staph V8 protease tested, PE binding was greatly reduced and only observedon the molecular mass 200,000 Da fragment. This reduced PE binding is in contrast to theELISA results after thesame pretreatment, where significant toxin binding was retained by the receptor. The purified toxin binding protein also was sensitive to digestion by trypsin in a dose-dependent fashion (not shown). In contrast to theprofile observedafter digestion with staph V8 protease, SDS-PAGE showed a smear of PE binding activity suggesting a continuum of proteins ranging from less than 100,000 Da upto thesize of the native material. Although two discrete products were observedat low concentrations of
-
0.11 I
l
0
l
.l
l .4
l l l l .3 .2 .5 .6
l
l
l
l
.7
.B
.9
1.0
Staph. VB Protease (pg)
B 1 2 3 4 5 6 7 8 91011
FIG. 6. A, the effect on PE binding of Staphylococcal protease V8 digestion of purified binding protein. Toxin binding protein was preincubated with indicated concentrations of enzyme for 24 h at 25 “C.Treated protein was then diluted 1:lO in sensitization buffer and dried onto ELISA plates prior to PEbinding as described under “Experimental Procedures.” Background absorbance (unsensitized wells) was approximatelyA492= 0.28. W, untreated protein. B, cleavage of PE binding protein by Staphylococcal protease V8: SDS-PAGE PE blot assay. Purified toxin binding protein (1.0 pg) was incubated with various concentrations of protease a t 25 “Cfor 24 h. Samples were then made 2% with SDS, electrophoresed in 6% SDS-polyacrylamide gels, electroblotted, and then assayed for PE binding as described under “Experimental Procedures.” Lane l , purified protein, no treatment. Lanes 2 and 3, toxin binding protein incubated in ammonium bicarbonate and sodium phosphate buffers, respectively. Lanes 4-7,1.0 pg of toxin binding protein treated with 0.001, 0.01, 0.1, and 1.0 pgof Staph V8 protease, respectively, in ammonium acetate buffer. Lanes 8-11, toxin binding protein incubated with identical concentrations of protease in sodium phosphate buffer.
trypsin, it is clear that this enzyme efficiently attacks the molecule at multiple sites. The trypsin-treated preparation showed a protein-staining profile that was similar to the toxin binding profile. The susceptibility of affinity purified binding protein to other enzymes was examined. Electrophoretic behavior and PE binding characteristics were unchanged after extended incubation with alkaline phosphatase, suggesting that the protein was not highly phosphorylated (not shown). Additionally, purified binding protein was not altered by preincubation with chondroitinase or phospholipase C. Identification of Sugar Residues-The affinity purified toxin binding protein is glycosylated. Sugars in PE binding protein electroblotted from SDS-PAGE were detected both by direct oxidation and by specific lectin binding characterL I istics. PNGase F pretreatment of Nonidet P-40 extracted PE binding protein reduced its apparent molecular mass about 30,000-40,000 Da as measured by SDS-PAGE (Fig. 7 A ) and removed sugar residues detected by hydrazide-digoxigeninand lectin-digoxigenin labeling (not shown). The contribution of carbohydrate to themolecular massof the PE binding protein can only be roughly estimated from the SDS-PAGE profiles. Toxin binding was minimally reducedby PNGase treatment (Fig. 7B).Preincubation with Endo H prior to electrophoresis did not alter either the apparent size of their protein or its 4.0 5.0 6.0 7.0 8.0 9.0 PE binding characteristics. pH Specific glycosylationstructures of the LM cellPE binding FIG.5. The effect of pH on binding of PE to immobilized protein were identified by a panel of digoxigenin-labeled lecpurified binding moiety. P E binding was performed ineither sodium acetate buffer (A) or sodium phosphate buffer (A) at the tins. The material was analyzed withand without preincubaindicated pH. PE binding was performed in 3% BSA-PBS buffer (0). tion with PNGase F prior to electrophoresis (Table I). From strong reactivity with Galanthus niualis, Sambucus nigra,and Each point represents the average of quadruplicate determinations.
Characterizationof Pseudomonas Exotoxin
FIG. 7. Effect of glycosidase treatment on PE binding protein. A, purified PE binding protein silver-stained polyacrylamide gel. Lane 1, no reducing agent;lane 2,treated with 2-mercaptoethanol, to electrophoresis;lane 3, binding protein heated 10 min at 55 "C prior incubated with glycosidaseincubationbuffer, 1 h, 37 "C; lane 4, PNGase F, 1 h, 37 "C;lane 5,Endo H, 1 h, 37 "C.Lanes 6-8, identical to samples in lanes 3-5, but heated for 10 min at 55 "Cprior to electrophoresis. B , PE blot assay of samples treated as in A . Samples were electrophoresedin 6%separating gel.
TABLE I Lectin binding characteristics of purifiedPE binding protein Purified bindingprotein was preincubatedwith PNGase F or buffer and then analyzed by SDS-PAGE. Electrophoresed proteins were transferred to nitrocellulose and sugars were identified by specific lectin binding as described under "Experimental Procedures." GNA, Galanthus nivalis agglutinin;SNA, Sambucus nigra agglutinin; MAA, Maackia amurensis agglutinin; DSA, Datura stramonium agglutinin; PNA, peanut agglutinin. Treatment GNA Lectin
None PNGase F
+-
SNA
MAA
DSA
PNA
+-
+
+
-
-
If:
Binding Protein
2395
TABLE I1 Neutralization of PE toxicity 40, 100, or 200ngof PE were incubated with 12 pg of affinity purified binding protein in 3% BSA-PBS for 3 h at 37 "C.The mixture was then diluted 1:4 in Hanks' balanced salt solution to the toxin concentrations indicated, cooled, and incubated withLM cell monolayers for 45 min at 4 "C.Cells were washed twice and reincubated in McCoy's 5A medium for3 hat 37 "C.Protein synthesis was measured. Data are expressed as percent of ~-[~H]leucine incorporation in toxin treated cells compared with control cells. ~-[~H]Leucine incorporation into LM cells treated with binding proteinalone or with buffer is45.3 cpm/pg cell protein and 50.5 cpm/pg, respectively. PE PE
Protein synthesis and binding PE alone protein' % control
nglwell
37
10 25 50
100
86
74 38
20 PE incubated with binding protein prior to incubation with LM cells. a
OVCAR-3 cells in a manner identical to LM cells and analyzed by SDS-PAGE PE blot assay showed no PE binding activity. DISCUSSION
We reported previously the presence of high affinity binding sites for P. aerugimsa exotoxin A on the surface of mouse Maackia amurensis agglutinins, we conclude that the toxin LM fibroblasts (22). Here we report affinity purification esbinding protein contains easily detected N-linked high man- sential to homogeneity of a large glycoproteinthat specifically nose glycan chains, and sialic acid terminally linked both (2- binds PE. The proceduredescribed is rapid and efficient, 6) and (2-3) to galactose in complex chains. Reaction with recovering approximately 30% of the PE binding capacity Datura stramonium agglutinin suggested the existence of solubilized from whole cell homogenate. The estimated enGalp-(ld)GlcNAc in either N- or 0-linked glycans or GlcNAc richment of approximately 250-fold for this procedure might in 0-glycans. Pretreatment with PNGase F removed all re- be an underestimate, since different protein assays were emactivity to G. nivalis agglutinin, s. nigra agglutinin, and M. ployed for the two preparations. The PE binding properties amurensis agglutinin in thisassay, suggesting that little if any of the purified protein are virtually identical to those of the sialic acid residues are linked through 0-linked glycans. D. detergent extract. The purified glycoprotein saturably and stramonium agglutinin, which strongly reacted with untreated specifically bound PE. material, weakly identified the toxin binding core protein The effect of pH on PE binding characteristics of the remaining after PNGase F treatment. This result suggests detergent extract and purified binding glycoprotein differed. that thepurified toxin binding moiety contains N-linked Gal We showed previously that binding of PE to whole LM cell (1-4)-GlcNAc and a small amount of 0-linked GlcNAc resi- detergent extracts at and below pH 5.0 occurs with proteasedues. sensitive and protease-insensitive components. This suggests Gel Filtration of PE Binding Moiety-Affinity purified Tri- that PEmay bind to membrane lipids extracted by the deterton X-100 extracted binding protein was analyzed by Superose gent as well as to the PE binding protein.' We believe that 12 gel filtration chromatography to independently determine the optimum pH for PE binding to the purified protein is the size of the detergent receptor complex. Only one toxin between 5.0 and 5.5. binding peak was identified by SDS-PAGE and ELISA. Gel The sensitivity of LM cell binding protein to glycosidase filtration in 0.01% Triton X-100yielded an estimate of treatment, its lectin binding characteristics, and glycan-spe300,000-400,000 Da for the apparent mass of the soluble cific SDS-PAGE staining profile showthat itis a monomeric detergent-receptor complex. This agrees with the apparent glycoprotein that is extensively modified during biosynthesis. mass estimated from SDS-PAGE. Removal of excess Triton We did not precisely determine the carbohydrate content of X-100 prior to gel filtration did not alter its retention time. the purified binding protein, but it comprised about 40,000 Neutralization of PE Toxicity for LM Cells-The biological Da or 10% of the molecule's overall mass.It appears that the activity of PE is inhibition of cellular protein synthesis. principle constituents areN-linked high molecularmass manTherefore to determine if the toxin binding protein is essential nose chains. Reactivity with the lectin D. stramonium agglufor toxicity, PE was incubated with affinity purified binding tinin suggests that 0-linked N-acetylglucosamine or glycan protein for 3 h at 37 "C; the mixture was then added to cell chains also may be present. The role of these sugars in the monolayers, and protein synthesis was measured (Table 11). conformation or function of the receptor has not been asIncubation of PE with binding protein resulted in decreased sessed. The datamake it apparent, however, that PE binding ability of the toxin to inhibit LM cellprotein synthesis. to receptor is not dependent upon the presence of N-linked PE-resistant Cells-OVCAR-3 cells are at least three logs carbohydrate. more resistant to PE intoxication than are LMcells. In Triton X-100 does not dissaggregate protein complexes as addition, they do not bind PE at 4 "C when monitored by efficiently as other detergents. We considered the possibility electron microscopy.2 Detergent extracts prepared from that thelarge apparent size of the Triton X-100 extracted PE
2396
Characterization of Pseudomonas Exotoxin Binding Protein
binding moiety might be due to artifactual detergent-protein complexes. However, extraction and purification using Nonidet P-40 yielded a purified material with an identical apparent molecular mass. Additionally, multiple lines of evidence indicate that the purified PE binding moiety is a large monomeric glycoprotein. This evidence includes electrophoretic behavior after reduction of disulfides in thepresence of SDS, sensitivity to protease treatment, and agreement in size estimates after SDS-PAGEand gel filtration. The specificity of staph V8 protease is pH dependent (26). Since littledifference was seen on the SDS-PAGE blotprofile when PE binding protein was digested under conditions favoring Glu-C or both Arg-C and Glu-C activities, Arg-C sites may not be available in the detergent solubilized molecule. Since staph V8 protease generates discrete fragments that retain the integrity of the PEbinding site, this approach may prove useful to generate fragments suitable for further purification and peptide sequencing. Under the most stringent proteolytic conditions tested (Fig. 6), we generated a single fragment which retained minimal PE binding activity. In contrast, under these same proteolytic conditions, significant P E binding was retained as measured in the ELISA. The difference in PE binding characteristics of denatured (SDSPAGE) and non denatured (ELISA) material after identical protease treatment suggests the toxin binding moiety can be nicked, but remains intact sufficiently well for toxin to bind in a non denaturing environment. An important unanswered question is whether thePE binding glycoprotein we have purified is the putative receptor involved in the internalization of PE molecules responsible for mammalian cell death. There are several observations that suggest that this might be so. Preliminary studies show that treatment of intact cells with trypsinsignificantly reduces the amount of toxin binding protein detectable by SDS-PAGE and by the PE blot assay and at the same time ablates the sensitivity of these cells to toxin (not shown). In addition, preincubation of P E with purified binding protein reduces its toxicity for LM cells (Table 11). Thusthe toxinbinding protein appears to be, at least in part, a surface associated proteinaceous moiety (trypsin sensitive) and canprotect cells from the cytotoxic effects of PE. Furthermore, the cytotoxicity of PE correlates with the demonstration of toxin binding activity incells. Detergent extracts of Vero cells, which are of intermediate sensitivity to PE, have a toxin binding component which comigrates on SDS-PAGE with the affinity purified protein from LM cells (not shown). We have recently established the presence of a PE binding component inmouse liver extracts.* This is of potential significance since the liver is a target organ for PE produced during fatal P. aeruginosa infection of burned animals (27-29). In contrast, OVCAR-3 cells, which are resistantto PEand which do not bind appreciable toxin, have no demonstrable toxin binding moiety by PE blot assay. This would suggest that a PE receptor is absent in OVCAR-3 cells. Thus the presence of a detectable M. R. Thompson, J. Forristal, R. E. Morris, and C. B. Saelinger, manuscript in preparation.
toxin bindingmoiety parallels cell sensitivity to Pseudomonas exotoxin A. The PEbinding moiety is larger than most but notall other known receptors involved in receptor mediated endocytosis. The low density lipoprotein receptor, for example, is a 164,000-Damonomer (30), and the transferrinreceptor exists as a dimer of 90,000 Da (31). Recently Ashcom et al. (32) identified a human a,-macroglobulin receptor of 429,000 Da, and Herz et al. (33) described a large (500,000Da) membrane protein of undefined function, closely related to the low density lipoprotein receptor. Thus there is precedence for large molecular mass receptors of physiological ligands on different cell types. Further characterization of this PEbinding moiety will permit evaluation of its function in the intoxication of mammalian cells in uitro and during Pseudomonas infections. Acknowledgment-Thetechnicalassistance gratefully acknowledged.
of Debbie Volk is
REFERENCES 1. Holder, I. A. and Neely, A. N. (1989) J. Burn Care Rehabil. 9 , 285-289 2. Saelinger, C.'B. (1990) in Trafficking of Bacterial Toxins (Saelinger, C. B., ed)p 1 14 CRC Press Boca RatonFL 3. Iglews&; B. H:, and Kabat, D. (1975) broc. Natl. Acad. Sci. U. S. A. 72, 2284-2288 4. Leppla, S. H., Martin, 0. C., and Muehl, L. A. (1978) Biochem. Biophys. Res. Commun. 81,532-538 5. Gray, G. L., Smith, D.H., Baldridge J. S. Harkins R.N. Vasil M. L., Chen, E. X., and Heyneker, H. L. (1984)'Proc. Natl. Acd. Sci. b. S. A. 81,2645-2649 6. Allured, V. S., Collier, R. J., Carroll, S. F., and McKay, D. B. (1986) Proc. Natl. Acad. Set. U. S. A. 83,1320-1324 7. Hwang, J., Fitzgerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 4 8 , 1291 .?I?
8. Siigil, C. B., Chaudhary, V. K., FitzGerald, D. J., and Pastan, I. (1989) J. Biol. Chem. 264,14256-14261 9. Jinno Y Chaudhar V. KKondo T. Adhya S FitzGerald, D. J.,and Paitan: I. (1988) YBiol. haem. 2 6 3 , i3203-i3267 10. Chaudry, G. J., Wilson, R. B., Draper, R. K., and Clowes, R. C. (1989) J. Biol. Chem. 2 6 5 , 15151-15156 11. Guidi-Rontani, C., and Collier, R. J. (1987) Mol. Micro. 1,67-72 12. Chaudhary, V. K., Xu, Y., FitzGerald, D., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sei. U. S. A. 86,2939-2943 13. Hamood, A. N., Olson, J. C., Vincent, T. S., and Iglewski, B. H. (1989) J. Bacteriol. 1 7 1 , 1817-1824 14. Morris, R. E. (1990) in Trafficking of Bacterial Toxins (Saelinger, C. B., ed) pp. 49-70, CRC Press, Boca Raton, FL 15. Michael, M., and Saelin er, C B (1979) Curr. Microbiol. 2 , 103-108 16. Middlebrook, J. L., and%orland; R. B. (1977) Can. J. Microbiol. 2 3 , 183189 17. FitzGerald, D., Morris, R. E., and Saelinger, C. B. (1980) Cell 21,867-873 18. Morris, R. E., and Saelinger, C. B. (1984) J. Histochem. Cytochem. 32, 3Q "134-1 ,=
19. Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russel, D. W., and Schneider, W. J. (1985) Annu. Reu. Cell Biol. 1 , 1-39 20. Ciechanover, A., Schwartz, A. L., and Lodish, H. F. (1983) J. Cell Biochem. 2% 1n7-1m ", "" 21. ca Carpenter G., and Zende i J G . (1986) Exp. Cell Res. 1 6 4 , 1-10 &entre:PyF., 22. M Manhart, E D., Morns, &E.,.Bonventre, P. F., Leppla, S., and Saelinger, C. B. (1984) Infect. Immun. 45,596-603 23. Laemmli U. K. (1970) Nature 227,680-685 Towbin H.. H. Staehelin. Staehelin T., T.,and and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. 24. Towbin. S. A. 36,'4350-4354 25. Johnsor Johnson, D. A., Gautsch, J. W., Sportsman, J. R., and Elder, J. H. (1984) Gene Anal. Tech. 1,3-8 26. Houmard, J., and Drapeau, G. R. (1972) Proc. Natl. Acad. Sci. U. S. A. 6 9 , 3506-3509 27. Saelinger, C. B., Snell, K., and Holder, I. A. (1977) J . Infect. Dis. 1 3 6 ,
".
h.
28. Snell, K., Holder, I. A,, Leppla, S., and Saelinger, C.B. (1978) Infect. Immun. 19,839-845 29. Pavloskis. 0. R.. Ielewski. B. H.. and Pollack.. M. (1978) Immun. . . Infect. . 19.29-33 30. Schneider, W. J., Beisiegel, U., Goldstein, J. L., and Brown, M. S. (1982) J.Biol. Chem. 257,2664-2673 31. Schneider, C., Sutherland, R., Newman, R., and Greaves, M. (1982) J. Biol. Chem. 257,8516-8522 32. Ashcom, J. D., Tiller, S. E., Dickerson, K., Cravens, J. L., Argraves, W. S., and Strickland, D. K. (1990) J. Cell B~ol.1 1 0 , 1041-1048 33. Herz,J.,Hamamann, U., Ro e, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1989) EMBPJ. 7,4119-4127 I
-
