Vol. 265, No. 16, Issue of June 25, pp. 10&S10692,199O Printed in Ll, S.A.

Invasive Adenylyl Cyclase of Bordetella pertussis PHYSICAL, CATALYTIC, AND TOXIC PROPERTIES*

(Received for publication, January 10, 1990)

Fabrizio Gentile& Leslie G. Knipling, Dan L. Sackett, and J. Wolff5 From the National Znstitute of Diabetes and Digestive and Kidney Diseases, National Znstitutes of Health, Bethesda, Maryland 20892 ’

A rapid two-step purification to homogeneity of the calmodulin-activated adenylyl cyclase from urea ex- tracts of Bordetella pertussis organisms (strain 114) is described. Catalytic and invasive activities are pu- rified 30- and 177-fold, respectively, and virtually no degraded forms are found. Specific activities are 0.4 mmol/min/mg and 0.5 pmol/mg of enzyme proteinlmg of cell proteinlmin for catalytic and invasive activities, respectively. The 15 amino-terminal amino acids agree with those deduced from the DNA sequence, as does the molecular mass of 175 kDa (guanidine) or 177 kDa (urea) obtained by equilibrium sedimentation. The larger apparent molecular mass seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be as- cribed to anomalous migration. Half-maximal cyclase activation occurs at 3-4 x lo-” M calmodulin in the presence of Ca’+ and at 2 x lo-’ M calmodulin in its absence. Ca’+ activation is maximal at 60-100 &M free CaCIZ (at low calmodulin concentrations), and free Ca2+ concentrations above ~125 pM are inhibitory at any calmodulin concentration. Extracellular Ca2+ is essen- tial for intoxication. In Chinese hamster ovary cells, exogenous calmodulin does not inhibit penetration of the cyclase.

Genetic evidence has shown that the calmodulin-activated (l), extracellular (2) adenylyl cyclase of Bordetella pertussis is an important virulence factor (3). The enzyme can pene- trate or invade a number of cells and there produce toxic excesses of CAMP (4-6). The cDNA sequence for the enzyme has been established (7) and codes for a protein of 1706 amino acids (177 kDa). Expression in Escherichia coli failed to pro- duce an invasive enzyme that was toxic to host cells (8) whereas homologous expression produced invasive activity. On the other hand, protein purification attempts have led to the identification of small (<60 kDa), large (~200 kDa), and very large (>600 kDa) forms of catalytic activity, but these were not fully resolved. Goldhammer and co-workers (9) reported briefly a pure enzyme of high specific activity and good calmodulin sensitivity of 47 kDa molecular mass; this small form was extensively characterized by Ladant et al. (10) and identified also by others (11-14). In most of these studies invasiveness was either not tested or not found.

Invasive forms of the enzyme have been reported to be 190-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Centro di Endocrinologia e Oncologia Speri- mentale de1 Consiglio Nazionale delle Ricerche, II Policlinico, Via Pansini 5, Naples 80131, Italy.

§ To whom correspondence should be sent.

215 kDa (8, 13, 15) whereas other forms of ~200 kDa were found which were catalytically active but not invasive (8) or were thought to be inactive precursors that required process- ing to small forms (16, 17). Molecular masses of the activities that were both catalytically active and invasive were all sub- stantially larger on SDS-polyacrylamide gel electrophoresis than deduced from the cDNA sequence. Because of the mul- tiplicity of forms with variable function, the molecular mass discrepancies, the cumbersome nature of some of the purifi- cation steps employed, and the drastic reduction in calmodu- lin sensitivity (8), we report here a rapid preparation of invasive cyclase with a good yield, high calmodulin sensitivity, and a molecular mass that is consonant with the deduced sequence.

EXPERIMENTAL PROCEDURES

B. pertussis CuLtwe and Preparation o/ Urea Extracts-Cultures (24 h) were prepared from lyophilized strain 114 B. pertussis, plated onto blood agar dishes, and transferred to blood agar slants after 3 days. After 2 days, growth was transferred to 500-ml Erlenmeyer flasks containing 250 ml of Stainer-Scholte medium and incubated for 24 h at 35 OC, 110 rpm, to OD eU of 0.66-0.85. Aliquots of 125 ml of this culture were transferred to each of several Fernbach flasks containing 1.3 liters of Stainer-Scholte medium and incubated for 24 h. Cells were harvested by centrifugation at 4 “C (all subsequent steps were at 4 ‘C), and the combined wet paste was immediately extracted with 4 volumes of freshlv Drenared 4 M urea in 40 mM Tris-HCl, DH 7.5, and 1 mM MgCl* ii in bmni-Mixer with 4 x 30-s bursts. ‘Y!he mixture was centrifuged at 14,000 rpm, and the pellet was reextracted with 32 ml of 4 M urea. The combined supernatant solutions were pooled and frozen in liquid nitrogen as “urea extract.”

Purification of the Znvasive Adenylyl Cyclase of B. pertu.ssis-5 ml of urea extract was diluted 4-fold to bring the urea concentration to 1 M. The final Tris-HCl concentration was adjusted to 20 mM and titrated to pH 7.9 with HCl. The sample was loaded onto a column packed with 12.5 ml of phenyl-Sepharose CL-4B (Pharmacia LKB &otechnology Inc.) (2.5 X 2.6.cm) Equilibrated with 1 M urea, 20 mM Tris-HCl. DH 7.9: several more bed volumes of this buffer were applied. ii ascen&ng urea gradient was then started using 10 hed volumes of loading buffer and 10 bed volumes of 4 M urea, 20 mM Tris-HCI, pH 7.9; the elution was continued with the latter buffer. The flow rate was 130 ml/h. 2.5.ml fractions were collected and monitored for protein concentration, adenylyl cyclase activity, and intracellular CAMP production in CHO cells as detailed below. All urea solutions were made fresh.

The active fractions from phenyl-Sepharose were stored at -70 ‘C until use and then pooled ana dialyzed-against 150 mM NaCl, 15 mM Tris-HCl, pH 7.8, 1 M glycerol (buffer A) (50 volumes X 1 h X 3). The dialyzed pool was loaded onto a column (1.5 X 1.4 cm) packed with 2.5 ml of DEAE-Senharose Fast Flow (Pharmacia) equilibrated with buffer A. Several bed volumes of buffer ‘A were applied: and then a gradient from buffer A to 300 mM NaCl in 15 mM Tris-HCl, pH

’ The abbreviations used are: SDS, sodium dodecyl sulfate; CHO, Chinese hamster ovary; EGTA, [ethyienebis(oxyethylenenitrilo)]tet- raacetic aci& EMEM. Eagle’s minimum essential medium with Earle’s salts;’ HMEM: Eai1e.s minimum essential medium with Hanks’ salts.

10686

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

Bordetella Adenylyl Cyclase

7.8, 1 M glycerol (buffer B) was applied (12 bed volumes); the elution was continued with buffer B. The flow rate was 60 ml/h. 0.5-ml

10687

glycan kit from Boehringer Mannheim according to the instructions supplied.

fractions were collected and analyzed for protein activity, adenylyl cyclase activity, and intracellular CAMP production in CHO cells as detailed below. All chromatographic procedures were performed at 4OC.

Adenylyl Cyclase Assuy-Adenylyl cyclase activity was measured at 30 ‘C for 10 or 12 min in 60 mM Tris-HCl, oH 7.9,l mM ATP and 2 mM MgC&, or 5 mM ATP and 10 mM I&$& and 120 mM Tris- HCl, 10 iM or 100 fiM added CaCIZ, 1 pM bovine brain calmodulin, and 0.33 &i of la-3*PlATP in a volume of 60 or 100 ~1 as indicated in the legends. Tubes *were prewarmed for 2 min, and the reactions were started either by the addition of prewarmed substrate mix or by enzyme. Subsequent processing was as described (18), and samples were counted and calculated on a Packard 19OOCA liquid scintillation counter. Free calcium concentrations were computed by the “Cafree” program of Fabiato and Fabiato (19).

Assay of Zntracelluhr CAMP Production-CHO cells (kindly pro- vided by Dr. April Robbins) were grown in EMEM with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and strepto- mycin, and nonessential amino acids in a 95% air, 5% CO2 humidified atmosphere at 37 “C. Cells were seeded in 12-well plates and used X3- 24 h later before confluence was reached. At the time of the experi- ment, cells were washed once with prewarmed EMEM at 37 OC containing 0.3 mM isobutylmethylxanthine and then were preincu- bated with 1 ml of the same medium at 37 ‘C for 30 min in mixed atmosphere; after another change of medium, incubations were car- ried out in 1 ml at 37 “C. Incubations at different temperatures were performed with HMEM in normal atmosphere.

For routine assay of column fractions, 30-~1 aliquots were incubated for 40 min at 37 ‘C. Urea was removed from phenyl-Sepharose fractions by dialysis against buffer A (75 min at 4 ‘C in a microdialysis apparatus, Bethesda Research Laboratories Life Technologies). Any change in the volume of samples was corrected before assay. The concentrations of glycerol did not affect the invasive properties of impure or pure preparations of the adenylyl cyclase. DEAE-Sepharose fractions were assayed as such immediately after elution from the column. A linear dose-response relationship was observed throughout the concentration range of the urea extract and purified preparations, whereas saturation was observed with large aliquots or longer times.

At the end of incubations, cells were washed twice with ice-cold EMEM and quickly frozen on dry ice. Intracellular CAMP was ex- tracted with 1 ml of absolute ethanol overnight at -20 ‘C. Extracts were collected, and cells were rinsed with 0.5 ml of 50 mM sodium acetate, 2 mM EDTA, pH 4.7. The extract and rinse were pooled after standing for 1 h at -20 ‘C, they were centrifuged at 1800 X g for 15 min; the supernatants were dried in a vacuum centrifuge and assayed for CAMP content by radioimmunoassay (20).

E@Z~rmm Centr$ugutmn-Analytical ultracentrifugation was performed using a Beckman model E equipped with the standard Beckman scanner and monochromator and a computer-based data acquisition system, essentially as described in (21). Samples of the cyclase were pooled from the peak of the DEAE column, concentrated with an Amicon Centricon 30, and dialyzed against the appropriate solvent. Samples of about 50 ~1 were loaded into double-sector scanner cells and centrifuged in a four-place Ti AN-F rotor. Data collection was performed, and attainment of equilibrium was demonstrated by the difference scan methods described in (21). The partial specific volume was calculated as 0.726 according to (22) using the sequence reported in (7).

Other Methods-Standard polyacrylamide gel electrophoresis in the presence of SDS and &mercaptoethanol was performed using a modification of the method of Laemmli as described in (23). Column fractions were subjected to chloroform-methanol precipitation (24), resolubilized in loading buffer at 100 ‘C for 3 min, and then loaded onto the gels. Some gels were destained and restained with silver 05).

Digestions with glycopeptidase F (Boehringer Mannheim) were conducted according to Tarentino et al. (26). A sample of 3 pg of purified cyclase was precipitated with chloroform-methanol, redis- solved in 1% SDS, heated at 100 ‘C for 3 min, and incubated with 1 unit of glycopeptidase F for 16 h at 37 OC in 150 mM sodium phos- phate, pH 7.5, 0.1% SDS, 0.5% octyl glucoside, 1% fl-mercaptoetha- nol. After another precipitation in chloroform-methanol, -samples were analyzed by SDS-polvacrvlamide gel electronhoresis. For car- bohydrate detection by enzyme immunoassay, serial dilutions of purified cyclase were electrophoresed on 5% gels and blotted onto Immobilon paper (Millipore Corp.). Detection was performed by a

Column fractions were analyzed for protein content by a bicin- choninic acid micromethod (Pierce Chemical Co.) using bovine serum albumin as a standard. Phenyl-Sepharose fractions were dialyzed before assay against 150 mM NaCl, 15 mM Tris-HCl, pH 7.8, whereas DEAE-Sepharose fractions were assayed as such using a proper blank correction. Cell monolayers were dissolved in 1 N NaOH; lo-p1 aliquots were assayed at a final NaOH concentration of 0.1 N by a dye-binding method (Bio-Rad). Siliconized tubes and pipette tips were used for most procedures.

The analysis of the amino-terminal sequence was performed at the Protein Structure Laboratory of the University of California at Davis using an ABl470 gas phase sequenator.

M&eri&s-Reagems used -were obtained as follows: Stainer- Scholte medium and calcium-free EMEM. from the NIH media unit: all other cell culture media, from ABI, Inc.; fetal calf serum, from GIBCO; phenyl-Sepharose CL-4B and DEAE-Sepharose Fast Flow, from Pharmacia: ATP and isobutvlmethvlxanthine. from Sigma: la- “P]ATP (800 &/mmol) and [3B]cAMP (30.5 Ci;mmol), corn bu Pant-New England Nuclear; 1*51-monosuccinyladenosine-cyclic AMP tyrosyl methyl ester and anti-CAMP antibodies, from Georgetown University, Washington, D. C.; Pansorbin, from Calbiochem; rabbit anti-goat IgG, from ICN Immunobiologicals; glycopeptidase F, octyl glucoside, a*-macroglobulin, aprotinin, and leupeptin, from Boehrin- ger Mannheim. All other chemicals were of the best quality available.

RESULTS

Purification Pi-ocedures-Purification of the adenylyl cy- clase was started from 4 M urea extracts of the fresh paste of 22-24-h cultures of B. pertussis strain 114. 5 M urea yielded extra protein peaks but failed to enhance the yield. A single experiment using 42-h culture yielded markedly reduced ad- enylyl cyclase activity. Extracts could be stored in 4 M urea in liquid nitrogen for 3-4 months with only small losses of activity. Because the enzyme lost substantial activity when stored in liquid nitrogen after urea had been dialyzed out (3), one of our aims was to keep the enzyme in 4 M urea for as many of the early manipulations as feasible.

A convenient and reproducible first purification step was by hydrophobic chromatography with phenyl-Sepharose CL- 4B. It was difficult to elute the activity from octyl-Sepharose. The 4 M urea extract was adjusted to 1 M and 20 mM Tris- HCl, pH 7.9, applied to a phenyl-Sepharose column, and subsequently eluted as a broad peak of activity between 2.7 and 35 M urea in a l-4 M urea gradient as depicted in Fig. 1. The active fractions were pooled for subsequent purification. We have routinely observed greater than 100% recovery in the invasive activity during this step whereas catalytic activity showed no such increase; we have tentatively ascribed this to the removal of an inhibitor of the penetration process.

For further purification, DEAE-Sepharose chromatography was found to produce catalytic and invasive activities of the highest purity. Urea had to be removed since it reduced recoveries of both activities. The salt concentration of the dialysis and loading buffer was adjusted to 150 mM NaCl. In addition, substantial improvement in the yields, both after the dialysis and the DEAE chromatography, was brought about by the inclusion of 1 M glycerol in the buffers. Dialysis was brief and with frequent buffer changes to reduce losses of activity.

Fig. 2 depicts the DEAE purification of the pooled phenyl- Sepharose fractions. A single peak of enzymatic and invasive activity was eluted between 250 and 300 mM NaCl. The invasive activity of these fractions was not modified by the addition of any of the fractions of the flow-through. The maximum specific enzymatic activity of the purified protein was 0.4 mmol/min/mg of protein in different preparations and ~0.5 pmol of cAMP/mg of CHO protein/mg of bacterial protein/min for the invasive activity. On SDS-polyacrylamide

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

10688 Bordetella Adenylyl Cyclase

FRACTION NUMBER

FIG. 1. Hydrophobic chromatography of an urea extract of B. pertus& cells on phenyl-Sepharose CL-4B. Chromatography was performed as described under “Experimental Procedures.” Frac- tions were assayed for protein content (U), adenylyl cyclase enzymatic activity (A-A), and invasive adenylyl cyclase activity (intracellular CAMP production in CHO cells) (M). Fraction volume, 2.5 ml. The dotted Line represents the urea concentration in the eluant at the top of the column.

FRACTION NUMBER

FIG. 2. Ion-exchange chromatography of pooled and di- alyzed phenyl-Sepharose fractions on DEAE-Sepharose Fast Flow. Fractions 78-109 from phenyl-Sepharose chromatography (de- picted in Fig. 1) were pooled and dialyzed as described under “Exper- imental Procedures” and then applied onto a DEAE-Sepharose Fast Flow column. Symbols are as in Fig. 1. Fraction volume, 2 ml (fractions l-50) and 0.5 ml (fractions 51-142). The dotted Lne rep- resents the NaCl concentration in the eluant at the top of the column.

gel electrophoresis, the purified invasive enzyme appeared as a single band whose apparent molecular mass was estimated to be ~220 kDa. No other bands were detectable in silver- stained gels (Fig. 3). Table I shows the recoveries, yields, and enrichments of enzymatic and invasive activities throughout the purification procedure. It should be pointed out that although recovery for the catalytic activity was =30%, no precise statement can be made for the invasive activity be- cause of the removal of a presumptive inhibitor of this func- tion. We have repeatedly observed decay of the enzymatic and invasive activities during the experimental manipulations at 4 “C. Losses of activities were also noticed upon freezing and thawing. Once frozen in liquid nitrogen, both activities in the purified material were stable for at least 1 month. The enzyme is rapidly inactivated by boiling. A summary of the purifica- tion procedure is provided in Table I.

116- ' ' 92- 66- iz

45-

31-

1234 123 FIG. 3. SDS-polyacrylamide gel electrophoresis of various

preparations of B. pertussis adenylyl cyclase. Panel A, electro- phoretic profile of different stages of purification (9% polyacrylamide gel stained with silver). Lune 1, urea extract (protein load, 3 fig); lane 2, pool of phenyl-Sepharose fractions (1.5 pg); lune 3, flow-through of DEAE-Sepharose chromatography (1.5 pg); Lczne 4, purified ade- nylyl cyclase (1 pg). The positions of molecular mass standards (in kDa) are indicated on the lejr side (200, myosin; 116, 6-galactosidase; 92, phosphorylase b; 66, bovine serum albumin; 45, ovalbumin; 31, carbonic anhydrase). Punel I3, estimation of the apparent molecular mass of B. pertussis adenylyl cyclase (5% gel stained with Coomassie Blue). Lane 1, purified adenylyl cyclase (3 pg); k-me 2, purified adenylyl cyclase (3 pg) digested with glycopeptidase F as described under “Experimental Procedures”; lune 3, molecular mass standards in kDa (330, thyroglobulin; 200, myosin; 181, a*-macroglobulin; 116, 6-galac- tosidase).

Physical Properties-Amino-terminal sequence analysis of the pure invasive adenylyl cyclase revealed the following sequence: Met-Gln-Gln-Ser-His-Gln-Ala-Gly-Tyr-Ala-Asn- Ala-Ala-Asp-Arg-. This sequence is precisely that deduced for the amino terminus from the nucleotide sequence of the cya locus of B. pertussis and thus validates the proposed sequence and its starting point (7).

The apparent molecular mass of the pure invasive cyclase is ~220 kDa in repeated assays in low density SDS gels using various large molecular mass standards (Fig. 3). Similar values for SDS gels have been reported by others (8, 15, 16). This value is substantially larger than the molecular mass calcu- lated from the deduced sequence (177 kDa) of the 1706-amino acid protein (7). In an effort to account for the mass differ- ence, we used a highly sensitive test for glycoproteins (glycan detection kit, Boehringer Mannheim) but were unable to demonstrate significant amounts of carbohydrate. Moreover, digestion of the enzyme with glycopeptidase F did not result in any change in electrophoretic mobility (Fig. 3).

Analytical ultracentrifugation was performed in order to define some properties of the cyclase in solution. The first step was to determine the minimum (monomer) molecular mass. Preliminary experiments demonstrated that 6 M gua- nidine hydrochloride is an effective denaturant of the protein, as indicated by a shift in the tryptophan fluorescence emission maximum from 335 nm in buffer B to 348 nm in 6 M guanidine (data not shown). Accordingly, the cyclase was centrifuged to equilibrium in 6 M guanidine HCl. The resulting pattern of optical density at 280 nm versus radial position is shown as dots in Fig. 4B Nonlinear least square fitting of the data revealed a single component of molecular mass (175 ? 2 kDa). The predicted equilibrium distribution for a molecule of this size is shown as a solid line in Fig. 4B. The difference between

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

Bordetella Adenylyl Cyclase 10689

TABLE I Purification of the inuasive adenylyl cyclase of B. pertussis

Preparation Protein Total

Enzymatic activity

% Specific -Fold enrichment

Invasive activity (intracellular CAMP production in CHO cells)

Total % Specific -Fold enrichment

Urea extract 13.3 179 100 13 1 37 100 3 1

Phenyl-Sepharose pool 0.79 66 37 84 6 126 337 159 57

Purified cyclase 0.13 53 30 408 30 64 173 496 177

7.02

O.l- 0

1 0,05- ..-. A --,

a02 .: . . .,

-.%32 .- -. . . . -’

I 1 6.69 6.95 7.01

RADIUS WnJ

FIG. 4. Sedimentation equilibrium of purified adenylyl cy- clase. Solutions of cyclase were concentrated, dialyzed against the indicated solvents, and centrifuged to equilibrium as described under “Experimental Procedures.” In both panels A and B, the upper part presents the ohserved data (optical density uer.sus radial position) as dots and the best fit as a solid line. There are about 200 data points across each pattern. The Lower part of panels A and B shows the difference between data and fit (note difference in scale). A, native enzyme. The cyclase in buffer B was centrifuged to equilibrium at 3.7 ‘C. The indicated fit to the data is for a two-component mix, one of molecular mass 180 kDa and the other of molecular mass 810 & 8 kDa. The determination coefficient, r’, for this fit was 0.999, and the root mean square deviation error was 0.001. B, denatured enzyme. The cyclase was dialyzed versus 6 M guanidine hydrochloride and centrifuged at 14.2 OC. The indicated fit is for a single component of molecular mass 175 k 2 kDa. The determination coefficient, r*, for this fit was 0.996, and the root mean square deviation error was 0.001.

the data and the fit is shown in the plot of residuals at the bottom of the figure. Similar data were obtained when the solvent was 8 M urea rather than 6 M guanidine; a single

component of molecular mass 177 * 1.7 kDa was determined (data not shown).

In the undenatured form, the pattern is different. Cyclase in buffer B was centrifuged to equilibrium, and the resulting pattern of optical density versus radial position is shown as dots in Fig. 4A. Attempts to account for this distribution by a single component failed, yielding large and highly nonrandom residuals. A two-component fit, with one component having the molecular weight found above, yielded a good fit. This is shown as the solid line in Fig. 4A, with the residuals plotted below it. The second component in this case has a molecular mass of 810 ? 8 kDa. Other runs in buffer B yielded the same general picture: two components, one of which (the major one) had the “monomeric” size observed in guanidine or urea whereas the second component was much larger. The calcu- lated molecular mass of this component varied between 800 and 1000 kDa, and its relative abundance also varied. The large species is probably related to the large forms reported previously from sizing columns (11, 13, 14).

Cc&&tic Actiuity-Specific catalytic activities of 0.4 mmol of cAMP/min/mg of cyclase were attained in various prepa- rations in the presence of excess (lo-’ M) bovine brain cal- modulin. These values are comparable to those obtained by others (8, 15) as well as those found in the small noninvasive form of the enzyme (9, 10) especially when compared on a molar basis. Thus, the turnover number of our pure prepara- tions is calculated to be 71,000 mol/min/mol of enzyme. This is comparable to the noninvasive small forms (10) but about lo-fold greater than the highest turnover number for any membrane-derived adenylyl cyclase so far reported (27).

Two characteristics of the catalytic activity of I?. pertussis adenylyl cyclase are its high sensitivity to calmodulin and the absence of a Ca’+ requirement for activation at higher con- centrations of calmodulin. As can be seen in Fig. 5, the calmodulin activation curve for the pure enzyme is superim- posable on that of the urea extract over the entire range of calmodulin concentrations from lo-‘* to 10e6 M, This suggests that significant amounts of altered forms of the enzyme with different calmodulin sensitivities are not present in our crude preparations. Half-maximal activation occurred at 3-4 X lo-” M calmodulin in different preparations, and X-fold stimula- tion could be observed at 2-3 X lo-” M. This is in general agreement with previous observations for various states of purification but differs from severai reports (8, 13) in which a substantial loss of calmodulin sensitivity appears to have occurred during purification. The extent of stimulation was as great as 1500-3000-fold, depending on the basal activity.

When calmodulin was identified as the main regulator of B. pertussis adenylyl cyclase, it was also found that activation was not arrested by addition of EGTA (1). This was later shown to be the result of Ca’+-independent stimulation of the

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

10690 Bordetella Adenylyl Cyclase

*,‘a UREA EXTkACT ’ ’ 0, 0 PURIFIED CYCLASE a

LEJ, , ,I 9 8 7 6

-LOG CALMODULIN IMI

FIG. 5. Adenylyl cyclase activity of urea extracts or puri- fied enzyme from B. pertussis. Incubations were for 12 min at 30 “C with 1 mM ATP labeled with 0.33 &i of [w~*P]ATP, 2 mM MgCl*, total Ca’+ of 24 +M or 2 mM EGTA, and 60 mM Tris-HCl buffer, pH 7.9. Urea extract, 0.8 pg/tube; pure cyclase, 19.4 rig/tube in a final volume of 60 ~1. Enzyme and bovine brain calmodulin were diluted in low calmodulin bovine serum albumin (0.1 mg/ml Pentex) to yield a final albumin concentration of 0.05 mg/ml.

FREE ‘.X+ (Ml

FIG. 6. Calcium requirement of adenylyl cyclase activity at 1 x lo-’ and 1 X lOwE M calmodulin (CAM). Incubations were

for 12 min at 30 ‘C with 5 mM ATP, 10 mM MgClz, 60 mM Tris buffer, pH 7.9, and 1 mM EGTA. Pure enzyme was diluted in 0.1 mg/ ml serum albumin as was the calmodulin to yield final concentrations of 12.4 ng of enzyme/tube and 0.05 mg/ml of albumin. Free Ca*+ concentrations were calculated according to (19) based on contami- nating total Ca*+ of 14 FM plus varying concentrations of added CaCL

cyclase at 50-loo-fold higher concentrations of calmodulin (28-31). As shown in Fig. 5, such Ca*+-independent stimula- tion is a property of the pure enzyme, characterized by an 80- fold shift to higher calmodulin concentrations. The two-phase activation curve is not always obtained but has been observed by us (29) and others (8). We have also been able to show that the pure invasive cyclase is sti-mulated by the carboxyl- terminal half of bovine calmodulin (residues 78148) with a potency that is identical to that found previously for urea extract, i.e. 3-4 X lo-’ M half-maximal stimulation (18) (data not shown).

Because of the absence of a Ca*+ requirement at higher calmodulin concentrations, a Ca*+ response curve can be demonstrated only at low calmodulin levels. At 1 X lo-’ M

calmodulin, adenylyl cyclase activity shows a rather narrow maximal plateau at 60-100 pM free Ca*+ (calculated by the Fabiato Cafree program (19)) (Fig. 6). These Ca*+ curves are

similar to those obtained previously with crude enzyme prep- arations (28). At free Ca2+ concentrations >125 FM, a sharp, concentration-dependent inhibition of cyclase activity oc- curred independent of calmodulin concentration (Fig. 6) as noted earlier for crude systems (28-31). It seems probable that this inhibition occurs by a mechanism different from the calmodulin activation since it can also be demonstrated with basal activity (data not shown) and appears to be associated with structural changes in the enzyme (32).

Inuasiue Actiuity in CHO CeUs-The invasive properties of B pertussis adenylyl cyclase were studied in CHO cells by measuring intracellular CAMP accumulation following the addition of the cyclase to the culture medium. Nanogram levels of the cyclase were able to raise the intracellular CAMP levels of CHO cells. For incubation periods of 40 min, a linear dose-response dependence was observed up to 450 ng of ade- nylyl cyclase added, with intracellular CAMP levels rising in excess of 3000-fold of the basal values (Fig. 7, punel A). When the incubation was prolonged for 60 min, a plateau was reached at about 300 ng of added adenylyl cyclase (not shown). At the dose of 450 rig/well, a linear time dependence of intracellular CAMP accumulation was observed between 15 and 45 min (Fig. 7, punel B) followed by a plateau. At a lower dose the same general trend was observed, but a plateau was reached at 60 min (not shown). Although the rate of CAMP increase was low in the first minutes, the stimulation was almost immediate, with CAMP levels reaching 5 times the basal levels in 1 min.

A requirement for calcium has been observed for invasion by crude extracts of B. pertussis adenylyl cyclase of various eukaryotic cell types (6, 11). We therefore tested the effect of calcium concentration in the cell medium on the ability of the purified adenylyl cyclase to raise the intracellular CAMP levels in CHO cells. As shown in Fig. 8, punel A, an absolute requirement for extracellular calcium can be shown to exist for the production of intracellular CAMP by the pure adenylyl cyclase. In a calcium-free medium, the CAMP production in response to the addition of the cyclase was negligible. Fur- thermore, the concomitant addition of EGTA together with calcium ions prevented intracellular CAMP production by the pure adenylyl cyclase of B. pertussis.

We have described previously a marked effect of the incu- bation temperature on the intracellular CAMP production in response to urea extract of B. pertussis adenylyl cyclase in

0 %I 180 270 360 433 0 IO 20 31 40 5n 60 70 a0 -32 ,N”AS,“E ADENYLATE CYCLASE ,“g, TIME ,rnl”,

FIG. 7. Dose-response curve and time course of intracellu- lar CAMP production by B. pertussis invasive adenylyl cy- clase in CHO cells. Panel A, CHO cells were incubated with the indicated amounts of purified invasive adenylyl cyclase for 45 min at 37 ‘C, and intracellular CAMP was then extracted and measured as described under “Experimental Procedures.” Panel f3, cells were incubated with 450 ng of purified invasive adenylyl cyclase for the indicated times, and then intracellular CAMP was measured.

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

Bordetelh Adenylyl Cyclase 10691

4 I 0 0.5 1.0 1.5 2.0 0 10 2fl 30 40

[CM+] ImMb TEMPERATURE I’W

FIG. 8. Modulation of the invasive adenylyl cyclase of B. pertussis by calcium and temperature. Panel A, cells were washed once with Ca’+-free EMEM containing 0.3 mM isobutylmethylxan- thine and then preincubated with the same medium for 30 min at 37 ‘C; after another change of the same medium, cells were incubated with 220 ng of purified invasive adenylyl cyclase in the presence of the indicated concentrations of CaC&; the arrow indicates CAMP levels observed where cells were incubated with the adenylyl cyclase in the presence of 2 mM cells and 2 mM EGTA (0). Panel II, cells were washed once with HMEM containing 0.3 PM isobutylmethyl- xanthine and preequilibrated with the same medium for 30 min at the indicated temperatures in normal atmosphere; after another change of medium, preequilibrated at the desired temperatures, cells were incubated with 180 ng of purified invasive adenylyl cyclase for 60 min at the temperatures shown.

CHO cells (6). As shown in Fig. 8, punel B, the same effect was demonstrated with the purified cyclase. The optimal temperature range is from 20 to 29 ‘C, and the intracellular CAMP level at 37 ‘C is 58% of the maximum.

There have been reports (33) of the inhibitory effect of calmodulin on the increase of intracellular CAMP levels by B. perrusk adenylyl cyclase. In a previous study (6), we reported that susceptibility to this inhibition varied among different cell types. In the present study, we have observed that the stimulation of intracellular CAMP production in CHO cells during 60 min of incubation with 270 ng of purified adenylyl cyclase was not significantly different when the cyclase was preincubated 10 min at room temperature with lO-‘j M cal- modulin (7.3 & 1.3 nmol of cAMP/mg of protein) than in the absence of calmodulin (7.0 * 0.28 nmol of cAMP/mg of protein). This confirms our earlier observation (6) that the nature of the host cell is an important determinant in this blocking effect of calmodulin.

To test whether or not a proteolytic step might be involved in the expression of the invasive properties of the adenylyl cyclase, we examined the effect on intoxication of several protease inhibitors and found no effect for the addition of 300 nM aprotinin and 1 pM leupeptin on the intracellular CAMP production in CHO cells in response to a full range of concen- trations of purified adenylyl cyclase during 60 min of incu- bation.

DISCUSSION

We describe the isolation in good yield of the cell-associated invasive adenylyl cyclase of B. perrms& It is a single poly- peptide possessing b5th highly potent enzymatic activity (0.4 mmol/mg/min) and highly potent invasive activity (0.5 pmol/ mg of enzyme/mg of cell protein/min). It retains the high calmodulin sensitivity of the crude system. Ca*+ is not abso- lutely required for catalytic activity but i.s required for inva- siveness. The apparent SDS gel molecular mass of ~220 kDa is anomalous since the value obtained by equilibrium centrif- ugation (175-177 kDa) is in good agreement with the deduced

TABLE II Molecular properties of B. pertussis invasive adenylyl cyclase

CaM. Cai+, calcium-saturated calmodulin; CaM. Cai+, calcium- free calmodulin; CaM (78-148), tryptic carboxyl-terminal fragment of calmodulin (18).

Molecular mass Sequence (7) 177 kDa SDS-polyacrylamide gel elec- ~220 kDa

trophoresis Sedimentation equilibrium 177 kDa

Specific enzymatic activity Specific invasive activity

0.4 mmol/mg/min 0.5 amol/mg cell protein/mg

bacterial protein/min

Turnover number

K,, for (CaM. Cd+) K a for (CaM.C&+)

7l,OOO/min

3-4. lo-” M 2.10-‘M

K,, for (CaM 78-148) 3-4.16’ M

sequence molecular mass (Table II). Its amino-terminal se- quence has been determined for the first 15 amino acids and shows that it is the product of the cyu gene, which has been recently cloned and expressed in E. coli. Our sequence data also provide confirmation to the proposed start of translation of the cyu gene (7).

Our data show that ~11 the invasive adenylyl cyclase activity found in dialyzed urea extracts of B. pertussis can be ac- counted for by a single polypeptide of large molecular mass which is able to raise intracellular CAMP levels of mammalian cells when added in its purified form to cell media.

The present evidence and that of other investigators thus suggest that the 177-kDa adenylyl cyclase of B. perhssis is the invasive or toxic form. It has two clearly identifiable domains: the catalytic domain (and calmodulin-binding site) in the amino-terminal portion, and a domain presumably downstream from this which is required for penetration into host cells, as shown by the absence of invasive activity of the catalytically active 45-47-kDa fragment (g-12) and by the finding that invasiveness can be dissociated from catalytic activity with appropriate blocking antibodies (34). The inva- sive activity is very labile, which may account for the produc- tion of catalytically active but noninvasive fragments, partic- ularly in the culture medium. Although it has been suggested (16) that the large form of the enzyme is a precursor for a small invasive form, the present evidence and that of Rogel et ul. (8) and Hewlett et al. (15) are not consistent with that view. Nevertheless, it is conceivable that very limited prote- olysis may yield a more or less intact carboxyl-terminal frag- ment that may promote penetration of the amino-terminal catalytic domain when no longer linked covalently to the catalytic domain. This possibility is currently under investi- gation. Such proteolysis occurring either in the organism or in solution is not, however, a requirement for toxic activity although it may occur in the cell membrane of the host cell. Regardless of proteolysis, the functional studies demonstrate that the 177-kDa B. pern.&s invasive adenylyl cyclase pos- sesses all the properties that characterize the crude systems. Therefore, there appears at present no need to postulate additional components necessary for expression of virulence by this protein.

Acknowledgments-We thank Dr. Anastassios Raptis for showing us preliminary antibody and proteolysis data which also indicate that the 220-kDa enzyme is invasive. We also thank Julie Hannah and Dr. C. R. Manclark for their belp and the use of their P-3 facility and V. R. de Grange for manuscript typing and editorial assistance.

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from

Bordetella Adenylyl Cyclase

1.

2.

3.

4. 5.

6.

7.

8.

9.

10.

11. 12.

13. 14.

15.

16.

17.

REFERENCES

Wolff, J., Cook, G. H., Goldhammer, A. R., and Berkowitz, S. A. (1980) Proc. N&l. Acad. Sci. U. 8’. A. 77,3841-3844

Hewlett, E. L., Urban, M. A., Manclark, C. R., and Wolff, J. (1976) Proc. X&. Acad. Sci. U. S. A. 73.1926-1930

Weiss, A. A., Myers, G. A., Crane, J. K., and Hewlett, E. L. (1986) in Microbiology-1986 (Leive, L., ed) pp. 70-74, American Soci- ety for Microbiology, Wash. D. C.

Confer, D. L., and Eaton, J. W. (1982) Science 217,948-950 Pearson, R. D., Symes, P., Conboy, M., Weiss, A. A., and Hewlett,

E. L. (1987) J. Immunol. 139. 2749-2754 Gentile, F., Raptis, A., Kniplingi L. G., and Wolff, J. (1988) Eur.

J. Biochem. 175,447-453 Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullman, A., and

Dandrin, A. (1988) Mol. Microbial. 2, 19-30 Rogel, A., Schultz, J. E., Brownlie, R. M., Cooke, J. G., Parton,

R., and Hanski, E. (1989) EMBO J. 8, 2755-2760 Wolff, J., Cook, G. H., Goldhammer, A. R., Londos, C., and

Hewlett, E. L. (1984) Ado Cychc Nucleotide Protein Phos- phorykttion Res. 17,161-172

Ladant, D., Brezin, C., Alonso, J.-M., Crenon, I., and Guiso, N. (1986) J. Biol. Chem, 261, 16264-16269

Hanski, E., and Farfel, Z. (1985) J. Biol. Chem. 260,5526-5532 Shattuck, R. L., Oldenburg, D. J., and Storm, D. R. (1985)

Biochemistry 24, 6356-6362 Kessin, R. H., and Franke, J. (1986) J. Bucteriol. 166, 290-296 Weiss, A. A., and Hewlett, E. L. (1986) Annu Reu. Microbial. 40,

661-668 Hewlett, E. L., Gordon, V. M., McCaffery, J. D., Sutherland, W.

M., and Gray, M. C. (1989) J. Biol. Chem. 264, 19379-19384 Masure, H. R., and Storm, D. R. (1989) Biochemistry 28, 438-

442 Rogel, A., Farfel, Z., Goldschmidt, S., Shiloach, J., and Hanski,

18.

19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

E. (1988) J. Biol. C&m. 263, 13310-13316 Wolff. J.. Newton. D. L.. and Klee, C. B. (1986) Biochemistry 25,

795lii955 -

Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Pctris) 75, 463- 505

Gentile, F., Raptis, A., Knipling, L. G., and Wolff, J. (1988) Biochim. Biophys. Actu 971,63-71

Sackett, D. L., Linnoldt, R. E.. Gibson. C., and Lewis, M. S. (1989) Anal. Bio&m. i80, 3i9-325

Cohn. E. J.. and Edsall. J. (1943) in Proteins. Amino Acids and Pebtides, pp. 370-381; Rheinhold Publishing Corp., New York

Sackett, D. L. (1989) Anut. Biochem. 179, 242-244 Wessel, D., and Flugge, V. I. (1984) Anal. Biochem. 138, 141-

143 Wrav. W.. Boulikas, T.. Wrav. V. P., and Hancock, R. (1981)

Anal. Biochem. ilk, i97-2C% Tarentino. A. L.. Gomez. C. M.. and Plummer. T. H.. Jr. (1985)

Biochemistry 24,466514671 Pfeuffer, E., Molluer, S., Lancet, D., and Pfeuffer, T. (1989) J.

Biol. Chem. 264.18803-18807 Greenlee, D. V., Andreasen, T. J., and Storm, D. R. (1983)

Biochemistry 21,2759-2764 Kilhoffer, M. C., Cook, G. H., and Wolff, J. (1983) Eur. J.

Biochem. 133, 11-15 Haiech, J., Predeleanu, R., Watterson, D. M., Ladant, D., Bel-

lalou, J., Ullmann, A., and Barzu, 0. (1988) J. Biol. Chem. 263, 4259-4262

Hewlett, E., and Wolff, J. (1976) J. Bacterial. 127, 890-898 Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R.

L., and Storm, D. R. (1988) J. Biol. Chem. 263,6933-6940 Shattuck, R. L., and Storm, D. R. (1987) Biochemistry 24,6323-

6328 Raptis, A., Knipling, L., and Wolff, J. (1989) Infect. Immun. 57,

1725-1730

by guest, on April 15, 2012

ww

w.jbc.org

Dow

nloaded from