Bordetella pertussis Adenylate Cyclase - The Journal of Biological ...

THEJOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 263,No. 26,Issue of September 15,pp. 13310-13316,1988 Printed in U.S.A.

Bordetella pertussis Adenylate Cyclase IDENTIFICATION OF MULTIPLEFORMS

OF THE ENZYME BY ANTIBODIES* (Received for publication, August 31, 1987,and in revised form, January 14,1988)

Arie RogelS, ZviFarfelQ,Sara GoldschmidtS, Joseph Shiloachq, and Emanuel Hanski$)I From the $Department of Hormone Research, The Weizmann Institute of Science, Rehouot 76100, Israel, the §Clinical Pharmacology Unit and Department of Medicine E, Sheba Medical Center, Tel Auiv University, Tel Hashomer 51621, Israel, and the (INationul Institute of Arthritis and Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892 ~

Two forms of Bordetella pertussisadenylate cyclase mutants deficient in adenylate cyclase which are avirulent, of 200 and 47 kDa have been purified from dialyzed indicating that theenzyme contributes to thepathogenesis of urea extract of the bacteria to specific activities of 466 whooping cough.Recently we have shown unambiguously that and 1685 pmol*min”*mg”, respectively. Both forms the increase of cAMP observed in cells exposed to B. pertussis are activated 50-200-fold by calmodulin. The half- adenylate cyclase results from theentry of thisbacterial maximum concentration required for the activation ofenzyme into the host cell and the conversion of endogenous the 200-kDa catalyst is5.4. M, whereastheone ATP to cAMP (5). The bacterial invasive enzyme penetrates requiredfor activation ofthe 47-kDa catalyst is into a particulate fraction of the host cell, presumably the 1.8*10-’0 M. plasma membrane, where it produces cAMP and undergoes a Polyclonalantibodiesraisedagainstthe 47-kDa rapid process of inactivation (5, 6). Little is known about the catalyst specifically recognize both forms of the enzyme in purified state as well as in bacterial extracts structure and themolecular mechanism of penetration of the on immunoblots. The antibody inhibits at similartiter enzyme. Most of the invasive cyclase activity, i.e. the toxic as well form of the enzyme, is found in the bacterial cell and not in adenylate cyclase activity of the purified forms as the activity present in dialyzed urea extract of the the culture medium (7). Gel filtration of a bacterial extract bacteria. It also prevents the penetration of theinva- showed that the toxic form constitutes only a minor portion sive B. pertussis adenylate cyclase into human lym- of total cyclase activity found in the extract and has an phocytes.Theinhibitioninduced by theantisera is apparent size of 190 kDa (8).Recent purification attempts of specific toB. pertussis enzyme, since both calmodulin- B . pertussis adenylate cyclase conducted in several laboratodependent brain and sperm adenylatecyclase are not ries indicated molecular heterogeneity of the enzyme. Shattuck et al. (9) and Ladantet al. (10) have purified the extraaffected by the antibody. cellular enzyme to specific activities of 600 and 1,600 pmol. min” .mg”, respectively. Three forms of 43, 45, and 50 kDa were isolated; the former two forms were shown to be strucBordetella pertussis, the causative organism of whooping turally related (10). Kessin and Franke (11) demonstrated cough, produces an adenylate cyclase which has several unique that the extracellular enzyme apparently forms large aggreproperties. The enzyme is located predominantly in the peri- gates. A 50-kDa form can be derived from a 700-kDa aggregate plasmic space of the bacteria and a small portion of it is of the enzyme. Friedman (12) has isolated two species of the released to theculture medium during bacterialgrowth (1).It enzyme of 200 and 60 kDa from urea extract of the organism. isactivatedup to 1,000-fold by the Ca2+-bindingprotein Unfortunately, the specific activities of these species were calmodulin (CaM),’ which does not exist in the bacteria (2). about 10,000-fold lowerthan those reported for the extracelThus it was postulated that B. pertussis adenylate cyclase lular enzyme (9, lo), and both species were unresponsive to may affect eukaryotic cells and act as a toxin. Confer and CaM. Eaton (3) have shown that an extractof B. pertussis containIn the present paper we report the purification of a 200ing the enzyme increases cAMP levels in human neutrophils kDa catalyst of B. pertussis adenylate cyclase in a CaMand macrophages, andas a consequence their phagocytic sensitive form. The enzymatic and immunological properties functions areconsiderably impaired. Weiss et al. (4) produced of the 200-kDa catalyst are compared with other forms of B. pertussis adenylate cyclase, particularly to a highly enriched * This research was supported by grants from the Fund for Basic form of a 47-kDa species of the enzyme.

Research administered by the Israel Academy of Science and Humanities, from the Minerva Foundation, and from the Yeda Research and Development Co. (to E. H.). 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. (1 To whom correspondence should be addressed. The abbreviations used are: CaM, calmodulin; PBS, phosphatebuffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)-tetraaceticacid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; HP, high molecular weight peak of adenylate cyclase activity; LP, light molecular weight peak of adenylate cyclase activity; FIC, fractions inducing cAMP accumulation.

EXPERIMENTAL PROCEDURES

Culture of Organism and Preparation of Urea Extract The B. pertussis strain 165 was grown in 70 liters of fermentor (New Brunswick) in Stainer-Scholte liquid medium at 37 ‘C with good aeration. The fermentor was inoculated with a starter culture (10% volume) that grew for 24 h in Fernbach flasks. After 40-42 h the culture in the fermentor reached a stationary phase with approximately 4 g of cells, wet weight, per liter. The culture was cooled down and cells were collected using a AS 16P sharpless continuous centrifuge. Dialyzedurea extract of B. pertussis was prepared as previously described by us (5,8).Essentially, the bacterial cell pellet was admixed with 5.3 M urea and extensively dialyzed against buffer A (10 mM

13310

B.pertussis Adenylate Cyclase Hepes, 100 mM NaCl, 1mM CaCl,, 1mM 2-mercaptoethanol, pH 7.4). Then the pellet was separated from the cell-free supernatant by for 80 min. centrifugation at 143,000 X gmax

13311

Under these conditions the proteins were eluted from the paper strips and the catalytic activities were partially recovered. The yieldof activity after SDS-PAGEand electroblotting ranged from 10 to 20%.

Adenylate Cyclose Assay

Immunization, Immunoprecipitation, and Immunoblotting

Adenylate cyclase activity was determined in a total volume of 50 pl at 35 "C for 15 min. The assay mixture for B. pertussis adenylate cyclase contained, if not otherwise mentioned: 50 mM Hepes, pH 7.6, 1mM [w~'P]ATP (specific activity 20-100 cpm/pmol), 10 mMMgC12, 60 p~ CaCl,, and 10 p~ CaM. Brain adenylate cyclase was assayed as described (13), the assay mixture being supplemented with 5 mM MnC12, 100 p~ forskolin, and 10 p~ CaM. Sperm adenylate cyclase was assayed as described (14). [32P]cAMPformed was isolated according to Salomon et al. (15). All results are means of duplicate or triplicate determinations which differed by less than 10%.

The Coomassie Blue-stained gels containing the 47-kDa catalyst were excised by razor blade, and then minced and dispersed in PBS and mixed 1:1 (v/v) with complete Freund's adjuvant. Guinea pigs were immunized four times subcutaneously a t 2-week intervals with 1-4 pg of protein. 1 month after the last injection the animals were boosted with 2 pg of LP, which was purified as described and mixed with incomplete Freund's adjuvant in 1:l (v/v) ratio. Purified LP at 6 ng/ml was incubated for 18 h at 4 "C with nonimmune and immune serum in a final volume of 60 pl containing 100 mM NaC1, 10 mM Tris, pH 7.4, 2 mM EDTA, and 1%(v/v) of heatinactivated guinea pig non-immune serum. For determination of cyclase activity prior to immunoprecipitation, 20-pl aliquots were withdrawn and diluted with 10 pl of the above solution. Two samples of 10 pl each were then removed and assayed. To the rest of the incubation tubes 20 pl of undiluted goat anti-guinea pig antibodies were added and thetubes were further incubated for 4 hat 4 "C. After centrifugation in an Eppendorf centrifuge for 10 min two samples of 10 p1 were removed from the supernatant andassayed for adenylate cyclase activity. For immunohlotting, purified H P and LP were subjected to SDSPAGE and thenelectrophoretically transferred to nitrocellulose. The nitrocellulose was blocked for 30 min with PBS containing 2% (w/v) BSA, and then incubated overnight a t 4 "C with 20 ml of a 1:200 dilution of serum in PBS containing 2% (w/v) BSA. The strips were then washed four times for 10 min each with the following solutions: ( a ) PBS, (b) PBS containing 0.2 (w/v) Tween 20, (c) as in (b), and (d) PBS. The nitrocellulose was then incubated in PBS containing protein 2% (w/v) BSA and supplemented with lo5 cpm of 1251-labeled A/ml, for 2 h at 21 "C. Unbound protein A was removed by repeated washings with PBS containing 0.2% Tween 20. Strips of nitrocellulose were dried and autoradiographed a t -70 "C for 1-12 h.

Determination of Penetration Activity Penetration activity was determined by measurement of the intracellular cAMP produced in human lymphocytes exposed to the invasive enzyme, as described previously (5, 8). In all experiments the amount of cAMP produced was linearly proportional to the amount of the invasive enzyme added. All results are means of duplicate determinations, which differed by less than 10%. Purification of B. pertussis Adenylate Cyclose Gel Filtration-The supernatant of dialyzed urea extract (40 ml, 8-20 mg of protein .ml-') of specific activity of 0.2-0.4 pmol. min" . mg" was applied onto an Ultrogel AcA 34 column (5 X 82 cm) equilibrated and run in buffer A. 60 fractions of 25 ml each were collected; the flow rate was 1.0 ml/min. The two major peaks of cyclase activity of apparent high molecular weight (HP) and low molecular weight (LP) were pooled (see Fig. 1). CaM-Agarose Chromutography-Triton X-100,20% (w/v) in buffer A, urea, ATP, and MgClz were added to the pools of H P and LP to final concentrations oE 5% (w/v), 2 M, 1mM, and 1mM, respectively. The pools were applied to 5-8 ml of CaM-agarose column equilibrated in buffer A containing 5% (w/v) Triton X-100,2 M urea, 1mM ATP, and 1 mM MgC12; columns were run a t a flow rate of 1.5 ml/min. After application, the columns were washed successively with the following solutions (25ml each): ( a ) buffer A containing 0.5 M NaCl, 1 mM ATP, and 1mM MgC12; (b) buffer A withoutNaCl but containing 5% (w/v) Triton X-100, 1 mM ATP, 1 mMMgC1,; (c) 10 mM sodium acetate, 1%(w/v) Triton X-100, 100 mM NaCl, 1 mM 2mercaptoethanol, 1mM ATP, and1mM M&l2 at pH 4.0. The activity was eluted with 15 ml of a solution containing 10 mM glycine, 20 mM NaCl, 2 M urea, 1 mM ATP, 1 mM MgCl,, 1%(w/v) Triton x-100, and 1 mM 2-mercaptoethanol at pH 1.9. The pH of the eluted pool was immediately adjusted to pH 6.8 by the addition of 1 ml of 1 M Hepes-HC1 buffer of pH 7.5. Hydroxylapatite Chromatography-The 15-ml pools of activity eluted from the CaM-agarose column were diluted with 2 volumes of 20 mM Hepes-HC1, 0.5% (w/v) Triton X-100, pH 6.8, and applied onto hydroxylapatite columns (1.5-2 ml) whichwere equilibrated with the diluting buffer and run a t a flow rate of 0.2 ml/min. The columns were washed with 10 ml of the diluting buffer followed by elution with a solution containing 350 mM potassium phosphate, 0.1% (w/v) Triton X-100, 2 M urea, 1 mM MgC12, 1 mM ATP, and 1 mM 2-mercaptoethanol at pH7.8. 15 fractions of 1 ml were collected. The peaks of activity(about 3-4 ml) were loaded onto a 15-ml Sephadex G-25 column, equilibrated, and runin asolution containing 50 mM NHdHC03, 0.01 (w/v) Triton X-100, to remove potassium phosphate, urea, and thebulk of the Triton X-100.

Adenylate Cyclose from Eukaryotic Cells CaM-sensitive and -insensitive forms of purified bovine brain adenylate cyclase were kindly provided by Dr. A. G. Gilman, Health and Science Center at Dallas. The two forms were purified through fo-skolin-agarose chromatography as described by Smigel (13), followed by chromatography on CaM-agarose. Partially purified CaMsensitive bovine sperm adenylate cyclase (17) was provided by Dr. Y. Salomon, Weizmann Institute of Science. Other Procedures Proteins were determined by the methods of Bradford (18) or by Schaffner and Weizmann (19). Human lymphocytes were isolated from heparinized whole blood as described (20). Materials ATP, CAMP,bovine brain calmodulin, calmodulin-agarose,protein A, the protein standards for SDS-PAGE, isobutylmethylxanthine, and EGTA were obtained from Sigma. Ficoll-Hypaque was obtained from Pharmacia LKB Biotechnology Inc., Ultrogel AcA 34 from LKB, and hydroxylapatite from Bio-Rad. Freund's complete and incomplete adjuvants were purchased from Difco, goat anti-guinea pig IgG was from BioMakor, and guinea pigs were from local breed. [c~-~'P]ATP (80 Ci/mmol), [3H]cAMP (27 Ci/mmol), and NalZ5I(1000 Ci/mmol) were obtained from the Radiochemical Centre (Amersham, United Kingdom).

Identification of the Purified Catalysts after SDS-PAGE

RESULTS

Purified HP and LP were lyophilized, solubilized in sample buffer, and subjected to 9% SDS-PAGE according to Laemmli (16). The proteins were electroblotted onto nitrocellulose for 2 h at a constant current of 190 mA in buffer containing 120 mM glycine and 15.6 mM Tris-base at pH8.3. Major bands were visualized on the nitrocellulose paper by staining with 5% Panceau-Sdye dissolved in 10 mM sodium acetate, pH 4.0. The staining was carried out for 5-10 min and the paper was washed several times in double-distilled water to reduce background. This method of proteinstaining did not affect the catalytic activity of the enzyme. 3-mm strips of the nitrocellulose paper were cut and incubated for 24 h at 4 "C in 1 ml of buffer A containing 1%(w/v) Triton X-100, 1 mM MgCl,, and 1 mM ATP.

Purification of 200- and 47-kDaSpecies of B. pertussis Adenylate Cyclase-As previously reported, gel filtration of dialyzed urea extract of B. pertussis organism resulted in the separation of apparent high (HP) and low (LP) molecular weight peaks of adenylate cyclase activity (8). The peak of fractions inducing cAMP accumulation in human lymphocytes (FIC), i.e. the penetration activity, was associated with a minor peak of adenylate cyclase activity which migrated between H P and LP (Fig. 1). By the two-step procedure which is detailed under "Experimental Procedures" and summarized

Adenylate B. pertussis

13312

‘H‘

Cyclase

kDa (Fig. 2 A , top). Electroblotting of H P and LP followed by incubation of individual strips of nitrocellulose in a solution containing 1%(w/v) Triton X-100 resulted in elution and recovery of 10-20% of the adenylate cyclase activity which was initially applied to SDS-PAGE. This procedure allowed a positive identification of catalysts. As shown in Fig. 2A (bottom), adenylate cyclase activity was associated with the 200- and 47-kDa polypeptides. The tiny peaks of activity detected along the blot of the HPmaterial probably represent residual amounts of breakdown products. Different preparations of purified H P contained small amounts of 190-, 120-, and 99-kDa polypeptides bearing adenylate cyclase activity. Both H P and LP, in addition to the200- and 47-kDa catalysts, contained a major protein band of29 kDa which was not 20 40 50 associated with adenylate cyclase activity (Fig. 2, A and 23). Froclion Nurnbel In addition, the silver-stained gels shown in Fig. 2B reveal FIG. 1. Gel filtration chromatography of B. pertussis di- the presence of 33- and 24-kDa polypeptides in purified LP alyzed urea extract. Chromatography of dialyzed urea extract on and the presence of a 36-kDa polypeptide in purified HP. AcA34 column was performed as describedunder“Experimental to These polypeptides were devoid of adenylate cyclase activity, Procedures.” Fractionswere assayed for: adenylate cyclase, ability and their relation to the enzyme remains to be determined. induce cAMPaccumulation in lymphocytes (penetrationactivity) and protein. HP and LP denotethe apparenthigh and low molecular Some preparations of LP also contained active polypeptides weight peaks of cyclase activity; FIC stands for the peak of fractions of molecular weight of45 and 43kDa. Similar species of inducing cAMP accumulation in cells. 40 ml of dialyzed extract with adenylate cyclase were purified by Ladant et al. (10) from specific activity of 0.3 pmol .min” .mg” were applied to the column, and the column was run as described under “Experimental Condi- culture medium of B. pertussis. Although the K , for ATP of tions.” 10 pl samples of 1:lOO dilution from each fraction were used the isolated catalysts was similar (1.5 and 2.0 mM for H P and for adenylate cyclase activity determination whereas 80 pl samples LP, respectively’), the specific activity of the 200-kDa catalyst from each fraction were utilized for determination of penetration. was always 2-5-fold lower than that of the 47-kDa catalyst. Attempts to purify the catalyst of FIC led to variable results; TABLEI several active polypeptides of molecular weight in the range of 200-47 kDa were identified. Their relative amounts Purification of B. pertussis adenylate cyclase from LP and HP Dialyzed urea extract was run on an Ultrogel AcA 34 column and changed among different preparations, not allowing concluthe pools of LP and HP were identified as described in the legend to sive identification of FIC catalyst. Fig. 1. LP and HP pools were subjected to chromatography on CaMThe Differential Sensitivity of the 200- and the 47-kDa agarose and hydroxylapatite as described under “Experimental Pro- Catalyst to Actiuation by CaM-The dose-response curves of cedures.” Recoveriesare cumulative throughthe preparation. activation of the 200- and the47-kDa catalysts by calmodulin Specific Total are shown in Fig. 3. The CaM concentration required for halfactivitv Recovery Protein maximum activation of the 200-kDa catalyst was 30-fold higher than theone required for the activation of the 47-kDa catalyst (5.4. lo-’ M uersus 1.8.10-’0 M2). Both catalystscould be activated by CaM in the absence of Ca2+and the presence LP pool 165.0 97.500 100 1.7 of EGTA. The half-maximum concentration of CaM required 47 863 0.090 77.7 CaM-agarose 29 1685 45.5 0.027 for the activation of the 47-kDa catalyst in the presence of Hydroxylapatite EGTA was 1.1.lo-’ M, whereas the one required for activation 100 5.1 60.0 307.0 HP pool of the 200-kDa catalyst was higher by at least 3 orders of 23 198 71.4 0.36 CaM-agarose magnitude; its exact value could not be determined since no 14 466 0.09 42.0 Hydroxylapatite saturation was reached. The Vmax value of the 47-kDa catalyst in the presence of EGTA was 1.5-fold higher than the V,, in Table I, we have purified the catalysts from the H P and value obtained in the presence of Ca2+.This enhancement of the LP. Adenylate cyclase strongly binds to theCaM-agarose activity by EGTA probably occurs through a direct effect on column, and once the complex is formed it can be efficiently the enzyme, since the activity in the absence of CaM was eluted with solutions of pH 1.9 or 8.8 M urea. Inclusion of 2- increased to the same extent (not shown). Similar observa5% (w/v) Triton X-100 and 1-2 M urea in the loading buffer tions of EGTA-mediated activation were made for the crude increased on the one hand the amount of HP and FIC cata- B. pertussis adenylate cyclase (21, 22). lysts that bound to the CaM-agarose columns, and on the Immunological Studies-Guinea pigs were immunized with other hand itsignificantly decreased the adsorption of unre- purified LP as described under “Experimental Procedures.” lated proteins. Following loading, three consecutive washes Sera were tested bothfor their ability to precipitate adenylate with high-salt 5% (w/v) Triton X-100 and sodium acetate of cyclase and theirability to inhibit cyclase activity. One of the pH 4 were performed. After these steps, 60-95% of cyclase sera examined, efficiently inhibited, and precipitated adenylactivity remained bound to thecolumn. The activity could be ate cyclase (Fig. 4). About 50% inhibition of LP activity (0.04 efficiently eluted at pH 1.9 or with a solution of 8.8 M urea. ng in the assay) was obtained at a titer of 1:40,000. NonimATP and MgC12 were included during chromatography on mune serum neither affected the activity nor precipitated the CaM-agarose and hydroxylapatite to protect against possible enzyme. Since an excess of CaM was always present in the decrease of catalytic activity. ATP, MgC12, andthe bulk assays, it is most likely that the antibody is not directed portion of Triton X-100 wereremovedby passage of the against the CaM-binding site. Table I1 demonstrates that the purified material on a Sephadex G-25 column (see “Experimental Procedures”). Analysis of the purified H P and LP Determined by linear regression analysis of Lineweaver-Burk catalyst by SDS-PAGE revealed major bands at 200 and 47 plots. I

I

I

B. pertwsis Adenylate Cyclase

13313 I

LP

FIG.2. SDS-PAGE and silver-

ZOSK 116K

staining of HP and LP and identification of the catalysts.A, HP and LP were purified as described in the text. Lyophilized samples of purified HP (4 pg) andLP (10 pg) were dissolved in sample buffer, boiled for4 min, and run on 9% acrylamide gels by the method of Laemmli (16). The Coomassie Blue staining patterns are shown at the top. Arrows indicate the migrationof calibrating proteins: carbonic anhydrase, 29 kDa; egg albumin, 45 kDa; bovine serum albumin, 66 kDa; phosphorylase b, 97.4 kDa; &galactosidase, 116 kDa; and myosin, 205kDa.Purified HP and LP (4 pg) weresubjected to SDS-PAGE, as described above and electroblotted, and the nitrocellulosewas stained with Panceau-S. Strips of 3 mm each were cut and the proteins were eluted by incubation in a buffer containing 1% (w/v) of Triton X-100 as describedunder ”Experimental Procedures.”Two samples of 10 pl werewithdrawnfrom each tube and assayed for adenylate cyclase activity. For silver-staining ( B ) , HP and LP werepurified as described in the text. HP (2 pg) and LP (4 pg) were subjected to 10% SDS-PAGE according tothe method of Laemmli (16), and then silver stained. Arrows indicate the positions of molecular weight standardswith masses in kDa.

2

6

Fraction Number

LSK

29K

10 14 18 22 26 30 Fraction Number

-205

HP L P

antibody raised against LP inhibited all species of the enzyme including those associated with penetration activity. The interaction of antibody with B. pertussis adenylate cyclase is specific, since no inhibition of the CaM-dependent adenylate cyclase from either brain or sperm was observed (Table 11). To demonstrate that theantiserum interactsdirectly with B. pertussis adenylate cyclase, immunoblotting was performed. Both purified LP and HP were subjected to SDS-PAGE and immunoblotted, and the polypeptides interacting with the antibodies were identified by autoradiography using ‘2sI-labeled protein A, as described under “Experimental Procedures.” Fig. 5A demonstrates that the immune serum interacted both with the 200- and the 47-kDa polypeptides (lanes 1 and 3),whereas no interaction was observedfor nonimmune serum (lanes2 and 4 ) . The purified H P preparation used for immunoblotting contained also a small amount of 120-kDa polypeptide with cyclase activity. This polypeptide is also recognized bythe antibodies (lane 1 ). An immunoblot of crude dialyzed extract is shown in Fig. 5B. Three major bands of 200, 190, and 47 kDa were visualized. In addition, several minor bands inthe molecular weight range of 200-47 kDa were detected by antibodies. Presently, we cannot exclude the possibility that some of them are not related to the enzyme. To assess the ability of the anti-cyclase antibodies to inhibit penetration, FIC was incubated with immune and nonimmune serum for 2 h at 4 “C, and then samples were withdrawn for determination of cyclase activity and the ability to generate CAMP in lymphocytes. As shown in Fig. 6, the anti-cyclase antibody inhibited penetration slightly less effectively than the adenylate cyclase activity. Nonimmune serum did not affect penetration at all. It is important to stress that the

different titer of the antiserum required for the inhibition of adenylate cyclase and for penetration cannotbe explained by the additional incubation time (30 min a t 37 “C), which was required for the penetration assay, since controls demonstrated that inhibition of cyclase was preserved under those conditions. DISCUSSION

This study describes the purification of a 200-kDa catalyst of B. pertussis adenylate cyclase fromcrude extract in a CaMsensitive form, and with a specific activity of 466pmol-min” mg”. In addition, a 47-kDa catalyst is purified from the extract to a specific activity of 1685 pmol-min” mg”. Both the 200- andthe 47-kDa catalyst strongly bind to CaMagarose column and once the complex is formed in the presence of Ca2+,it essentially cannot be eluted, unless harsh conditions such as pH 1.9 or 8.8 M urea (10) are applied. This fact allowed us to perform thorough washings of the column with detergent, salt, and 2 M urea prior to the elution. This washing minimized the adsorption of unrelated proteins and thus improved the CaM-agarose chromatography which is the most powerful step in the purification of the enzyme.Although the K, values for ATP of the 200- and the 47-kDa catalysts aresimilar, their specific activity and thesensitivity to CaM activation are different. The 2-5-fold increase in the specific activity of the 47-kDa catalyst compared with the specific activity of the 200-kDa catalyst is consistent with a degradation of the latter. All known examples of degradation of CaM-sensitive enzymes result in an enhancement of activity (23-27). However, proteolysis usually leads to a formation of CaM-independent activities, in contrast to the preserved CaM dependence of the 47-kDa catalyst. In fact, the sensitiv-

-

B. pertussis Adenylate Cyclase

13314

TABLEI1 The antibody raised against LP inhibits all f o r m of B. pertussis

1

1

1

0

9

8

-Log calmodulin

1

6

5

adenylate cyclase HP, LP, FIC, and dialyzed urea extract of B.pertussis were diluted with PBS containing 1%(v/v) nonimmune serum to final activity of 6.5 nmol.min-' 'm1-I. The specific activities of the above species were: 300, 1100, 1.2, and 0.2 #mol. min" .mg", respectively. Sample of 15 p1 was incubated with 15 p1 of immune serum which wasdiluted with PBS containing 1% (v/v) of nonimmune serum. After 18 h incubation at 4 "C, two samples of 10 pl each were assayed for adenylate cyclase activity as described under "Experimental Procedures.'' Samples of 80 pl of brain and sperm adenylate cyclase were incubated with 20 pl of immune serum as described above. Three samples of25 p l each were withdrawn and assayed for adenylate cyclase activity as described under "Experimental Procedures." The dilutions indicated in the table representthe final dilution of immune serum in the assay.

4

[MI

Adenylate cyclase/dilution of immune serum

1:1,000

1:10,000

1:100,000

% inhibition of cyclase aetiuity

42.5

-Log colmodulm [ M]

FIG. 3. Differential sensitivity of HP and LP catalyst to activation by CaM. Purified H P ( A ) and LP ( B ) a t a final concentration of 0.4 and 0.1 ng/ml, respectively, were assayed for adenylate cyclase activity at theindicated concentrations of CaM, either in the presence of 60 p~ CaC12or in theabsence of CaC12 and thepresence of 2 mM EGTA. The basal activities of H P were 4.5 and 5.8 pmol. min".mg" in the presence ofCa2' and the presence of EGTA, respectively. The basal activities of LP under the above conditions were 12.5 and 19.3 pmol.min" .mg-', respectively. These values were subtracted from the data presented.

-

) .

-

> .e

"0

c-4 NonImmune

serum Immune serum PO Resldual a c t d y after nmmunoprecipitotian W

-Log Dllutlon

FIG. 4. Inhibition of adenylate cyclase activity by antibodies. Purified L P was incubated with sera at the indicated dilutions for 18 h at 4 "C. Aliquots were removed and assayed for adenylate cyclase. Immunoprecipitation was performed and residual activity was determined inthe supernatants as described under "Experimental Procedures." The sera dilutions represent final dilutions in the assay which contained 0.04 ng of LP.

ity of the 47-kDa catalyst to CaM is higher than that of the 200-kDa catalyst by 30-fold in the presence of Ca2+,and by at least 2000-fold in itsabsence. The reasons for this increased sensitivity are asyet unknown. Direct binding studies of CaM

HP 82.0 94.8 LP 88.0 99.0 FIC 96.3 86.6 B. pertussis extract 94.8 82.4 0 0 Sperm" Brain, CaM-sensitive" 0 0 Brain, CaM-insensitive" 0 0 "No inhibition of cyclase activity was observed also 1:25 and 1:lOO.

46.0 45.1 35.4 0 0 0 at titers of

to the two catalysts under a variety of conditions should elucidate this phenomenon. Nevertheless, the different sensitivity of the two catalysts to CaM may explain the extended dose-response curves, over 3-4 orders of magnitude, observed by investigators using crude or partially purified preparations of the enzyme (2,9, 22). Several lines of evidence suggest that theisolated catalysts are related and the small form of the enzyme is probably derived from the larger form. B. pertussis adenylate cyclase was recently cloned and sequenced by Glaser et al. (28). The bacterial gene codes for a polypeptide of approximately 180 kDa. In addition, antibody raised against the 47-kDa form of the enzyme recognizes both the purified 47-kDa catalyst and the 200-kDa catalyst on immunoblots. Furthermore, immunoblots of crude extract of the bacteria reveal the presence of the 200- and the47-kDa polypeptides. Moreover, the antibody inhibits the activity of all forms of the enzyme at similar titers. Since the inhibition induced by the antibody is specific to B. pertussis adenylate cyclase, this result indicates that all species of the enzyme possess immunologically related regions. The multiple forms of the enzyme observed by us and by others (29) probably result from a degradative process which occurs in the bacteria during secretion of the enzyme into the culture medium. Indeed, recently Ladant et al. (10) have purified adenylate cyclase from culture medium of B. pertussis which is similar to the catalyst isolated from LP in molecular weight and specific activity. The penetration activity migrates on gel filtration in association with a minor peak of adenylate cyclase activity, whose position is between the HP and the LP. Purification of the catalyst of the minor peak by the methods described here lead to enormous variation in the size of the catalyst. Several active polypeptides in the molecular weight range from 200 to 47 kDa are identified and their relative amounts change among different preparations. Nevertheless, the antibody raised against the 47-kDa catalyst inhibitsthe catalytic activity of FIC at simiIar titer and prevents its penetration into lymphocytes, Thissupports the notion thatthe catalytic


13315

I -180 OILUrIoN FIG.6. Inhibition of penetration of the invasive enzyme into

-

36.5

-26.6 1 2 FIG. 5. Identification of HP and LP on immunoblots ( A )and immunoblotting of dialyzed urea extract ( B ) .Purified LP and H P (2 pgllane) were subjected to 9% SDS-PAGE. The resolved proteins were then electrophoretically transferred from the gels to nitrocellulose. The nitrocellulose blots were incubated with immune serum (A, lanes 1 , 3 ) , or nonimmune serum (A, lanes 2 , 4 ) . Visualization of the proteins interacting with antibodies was performed using 12SI-labeledprotein A followed byautoradiography as described under "Experimental Procedures." Arrows indicate the migration of the visualized proteins a t their respective molecular weights. In E, samples of crude dialyzed extract (50pgllane) were subjected to 10% SDS-PAGE. The proteins were electrophoretically transferred to nitrocellulose and visualized as described for A . The nitrocellulose blots were incubated with immune serum ( B , lane 1 ) or nonimmune serum ( B , lane 2 ) . Arrows indicate the positions of molecular weight standards with masses in kDa.

moiety of the invasive enzyme does not constitute a distinct form of B. pertussis adenylate cyclase. What causes FIC to be invasive? It is possible that a separate factor which migrates on a gel filtration column between HP and LP is required for penetration. Alternatively, trimming of the 200-kDa catalyst to a certain size and/or secondary modifications is required to produce the invasive form. Further studies are needed to solve this question. Both B. pertussis and Bacillus anthracis produce calmodulin-dependent adenylate cyclase enzymes which are capable of penetration into eukaryotic cells (30). CaM, however, does not exist in bacteria; thus itmay bespeculated that the CaMdependent bacterial adenylate cyclases originated from eukaryotes and were incorporated by the bacteria during evolution. Antibody directed against B. pertussis adenylate cyclase recognized neither CaM-dependent and-independent brain cyclases nor sperm cyclase. Therefore, these findings seem not to support the above hypothesis. Acknowledgments-We would like to thank Dr. A. G. Gilman for providing us with purified brain adenylatecyclase and Dr. Y. Salomon and N. Garty for providing us with partially purified sperm adenylate cyclase. We also thank Drs. J. E. Schultz and R. Kessin for helpful comments and Rona Levin for excellent secretarial assistance.

lymphocytes by anti-cyclase antibodies. Samples of 10 pl of FIC (dialyzed against PBS and concentrated 10-fold) were preincubated a t 4 "C for 2 h with the indicated dilutions of immune serum in a final volume of 20 pl. Immune serumwas diluted with PBS containing 1% (v/v) of heat-inactivated nonimmune serum. Aliquots of 5 p1 were withdrawn and diluted 100-fold in PBS containing 1% (v/v) heatinactivated nonimmune serum, and 3 samples of 10 pl were assayed for cyclase activity. To the rest, human lymphocytes (lo6cells in 0.5 ml of PBS) were added and themixture was incubated further for 30 min a t 37 "C. cAMP was determined as explained under "Experimental Procedures." 100% represents adenylate cyclase activity of 0.75 pmol.min" .mg-l and penetration activity of 52 nmol. 10" cells. mg", as determinedin the presence of nonimmune serum. The intracellular level of cAMP in control cells that were exposed to the same amount of boiled FIC was 0.1 nmol. lo" cells. mg-'. REFERENCES 1. Hewlett, E. L., Urban, M.A., Manclark, C. R., and Wolff, J. (1976)Proc. Natl. Acad. Sei. U.S. A. 73,1926-1930 2. Wolff, J., Cook, G. H., Goldhammer, A. R., and Berkowitz, S. A. (1980)Proc. Natl. Acad. Sei. U. S. A. 77, 3841-3844 3. Confer, D. L., and Eaton, J. W. (1982)Science 217,948-950 4. Weiss, A. A., Hewlett, E. L., Myers, G. A., and Falkow, S. (1984) J. Infect. Dis. 160,219-222 5. Friedman, E., Farfel, Z., and Hanski, E. (1987)Biochem. J. 243, 145-151 6. Farfel, Z., Friedman, E., and Hanski, E. (1987)Biochem. J. 243, 153-158 7. Weiss, A. A., and Hewlett, E. L.(1986)Annu. Rev. Microbiol. 40, 661-686 8. Hanski, E., and Farfel, Z. (1985)J. Biol. Chem. 260,5526-5532 Oldenberg, D. J., and Storm, D. R. (1985) 9. Shattuck, R.L., Biochemistry 24,6356-6363 10. Ladant, D., Brezin, C., Alonso, J.-M., Crenon, I., and Guiso, N. (1986)J. Biol. Chem. 261, 16264-16269 11. Kessin, R. H., and Franke, J. (1986)J. Bacterid. 166,290-296 12. Friedman, R. L. (1987)Infect. Immun. 55, 129-134 13. Smigel, M. D. (1986)J. Biol. Chen. 261, 1976-1982 14. Hanski, E., and Garty, N. (1983)FEES Lett. 162,447-452 15. Salomon, Y.,Londos, C., and Rodbell, M. (1974)Anal. Biochem. 68,541-548 16. Laemmli, U. K. (1970)Nature 227,680-685 17. Garty, N. B., and Salomon, Y.(1986)6th ZnternationulConference on Cyclic Nucleotides, Calcium, and Protein Phosphorylation, Bethesda, MD, Sept. 1986,Abstr. 9 18. Bradford, M. M. (1976)A d Biochem. 72, 248-254 19. Schaffner, W., and Weizmann, C. (1973)Anal. Biochem. 56,502514 20. Bojum, A. (1968)Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 7789 21. Greenlee, D. V., Andreasen, T. J., andStorm, D. R. (1982) Biochemistry 21,2759-2764 22. Kilhoffer, M.-C., Cook, G.H., and Wolff, J. (1983) Eur. J. Biochem. 133,ll-15

13316 23. 24. 25. 26. 27.


Gietzen, K., Sadorf, I., and Bader, H. (1982) &o&em. J. 207, 541-548 Niggli, U., Adunyah, E. S., and Carafoli, E. (1981) J. Biol. Chem. 256,8588-8592 Walsh, M. P., Dabrowski, R., Hinkins, S., and Hartshorne, D. J. (1982) Biochemistry 21,1919-1925 Meijer, L., and Currier, R. (1982) Biochim. Biophys. Acta 702, 143-146 Keller, C. H., Laporte, D. C., Toscano, W. A., Jr., Storm, D. R.,

and Westcott, K. R. (1980) Ann. N. Y. Acad. Sci. 356, 205219 28. Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbid. 2, 19-30 29. Masure, H. R., Shattuck, R. L., and Storm, D. R. (1987) Microbial. Reu. 5 1,60-65 30. Leppla, S. H. (1982) Proc. N&l. Acad. Sci. U. S. A. 79, 31623166