Vol. 59, No. 11

Site-Specific Mutagenesis of the Catalytic Subunit of Cholera Toxin:Substituting Lysine for Arginine 7 Causes Loss of Activity

W. NEAL BURNETTE,1* VERNON L. MAR,' BARBARA W. PLATLER,2 JOHN D. SCHLOTTERBECK,2MICHAEL D. McGINLEY,1 KENDALL S. STONEY,' MICHAEL F. ROHDE,' AND HARVEY R. KASLOW2

Amgen Inc., Thousand Oaks, California 91320,' and Department of Physiology and Biophysics,School of Medicine, University of Southern California, Los Angeles, California 900332

Received 19 June 1991/Accepted 20 August 1991

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a regionof nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown thatsubstitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates itsenzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxinsubunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structurebetween the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of avaccine containing an enzymatically inert analog of cholera toxin.

Vibrio cholerae and Bordetella pertussis are the respectiveetiologic agents of cholera and whooping cough. Thesebacteria each produce a multimeric protein exotoxin, termedcholera toxin (CTX) and pertussis toxin (PTX), capable ofADP-ribosylating distinct classes of guanine nucleotide reg-ulatory (G) proteins (22, 30). The catalytic A subunits ofthese toxins, CTXA of CTX and Si of PTX, contain discreteregions with nearly identical amino acid sequences (8, 9, 15,19, 25). One such region at the NH2 terminus consists of asequence of eight amino acids with only one mismatch. Ithas been previously shown that substitutions in this region ofPTX Si decrease its ADP-ribosyltransferase activity (4, 18,27). In particular, substituting a lysine for Arg-9 in Siessentially abolishes ADP-ribosyltransferase and cytotoxicactivities without destroying a potentially important neutral-izing epitope (4). This observation has led to the inclusion ofPTX analogs with the Si subunit Lys-9 mutation in acellularpertussis vaccines (2, 3).The rationale for use of inactive analogs of PTX in

vaccines protecting against B. pertussis infection is based, inpart, on the recognition that the toxin contributes to thevirulence of this organism (30). The observation that CTX isa colonization factor for V. cholerae (26) suggests thatinclusion of an inactive form of this toxin in a choleravaccine may also be desirable. Vaccines containing an

analog of CTX inactivated by removal of the catalytic Asubunit are currently under study (12). In this report, wepresent data that should contribute towards development ofan alternative toxoid that contains an inactive analog of theA subunit.CTXA is synthesized in V. cholerae as a preprotein that is

proteolytically cleaved to remove a signal peptide sequenceof 2,463 Da (20, 21). Further posttranslational processing ofCTXA by V. cholerae yields an amino-terminal CTXA1polypeptide (approximately 22 kDa) and a carboxyl-terminalCTXA2 polypeptide (approximately 5 kDa). The two poly-peptides are linked via a disulfide bridge; cleavage of thisbond is thought to be necessary for full ADP-ribosyltrans-ferase activity (10, 13, 20). The literature provides inconclu-sive evidence for the location of the cleavage site between

* Corresponding author.

CTXA1 and CTXA2. One report (21) suggests that thisoccurs at Ser-194; a second indicates that cleavage occurs atArg-192, resulting in the loss of a dipeptide (33). Thisdisparity in cleavage sites may arise from strain differencesin the V. cholerae used in these studies. For purposes of ourwork, CTXA1 was defined as terminating at Ser-194, and ourmolecular constructs provided for termination after thiscodon. Subsequent to the molecular cloning described be-low, we evaluated the mass of commercial-grade CTXA1(reduced CTXA from Inaba strain 569B; List Biologicals) ona Sciex API III mass spectrometer. By this means we foundthat CTXA1 terminates at Arg-192, which is consistent withthe finding of Yamamoto et al. (33).Recombinant plasmids containing the genes for the CTXA

and CTXB subunits (21), the latter encoding the homopen-tameric B oligomer, were obtained from the American TypeCulture Collection (ATCC 39051 and 39053, respectively).The CTXA and CTXB subunit genes were individuallysubcloned into pUC19 vectors; the Arg-7 codon of CTXAwas substituted with that for lysine by linker mutagenesis, aspreviously described for Arg-9 of the PTX S1 subunit (4).Utilizing appropriate synthetic oligonucleotide linkers, weindividually subcloned the genes for CTXB, CTXA(CTXA1+A2), CTXA1, and both CTXA and CTXA1 withan Arg-7--*Lys codon substitution (CGG to AAG) into theEscherichia coli vector previously used for the direct, non-fusion expression of PTX subunits (5). The presence of thecodon mutations were confirmed by partial DNA sequenceanalysis.Recombinant CTX subunit proteins were produced essen-

tially as described for PTX subunits (5). Briefly, competentE. coli were transformed with each of the expression vectorconstructs, selectants were fermented at 37°C and induced at42°C, and cell pastes were recovered. After lysis of the cellsin a French press, recombinant CTX subunits were recov-ered as insoluble inclusion bodies, washed by centrifugationin 1 mM dithiothreitol, and stored at -70°C.The protein concentrations of these preparations were

determined by use of the Bio-Rad protein assay. The per-centage of CTX-related protein contributing to the totalprotein of these samples was subsequently determined bysubmitting each sample to sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) (14), staining the

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INFECTION AND IMMUNITY, Nov. 1991, p. 4266-42700019-9567/91/114266-05$02.00/0Copyright ©3 1991, American Society for Microbiology

VOL. 59, 1991

1 2 3 4 5 6 7 8

CTXA1+ A2

CTXA1 --

CTXB --

FIG. 1. Recombinant CTX subunits and subunit analogs pro-duced in E. coli. Genes encoding the CTXA and CTXA1 forms ofthe A subunit, with and without site-specific codon substitutions,and the CTXB subunit gene were individually cloned into anexpression plasmid optimized for high-level production of heterolo-gous proteins in E. coli (5). The gene constructs possessed amethionine initiation codon in place of their signal sequences. Aftertransformation of competent bacteria and isolation of transformants,each of the recombinant proteins was produced following inductionand fermentation in E. coli. Insoluble inclusion bodies containingthe recombinant subunits were recovered by cell lysis and differen-tial centrifugation (5) and subjected to SDS-PAGE, and the gel wasstained with Coomassie blue R250. Lanes: 1, native CTX; 2,recombinant CTXA; 3, recombinant CTXA Arg-7--*Lys analog; 4,recombinant CTXA1; 5, recombinant CTXA1 Arg-7--*Lys analog; 6,recombinant CTXB; 7, native CTXB; 8, native CTXA1.

gels with Coomassie blue, and subjecting the stained gels tointegrative densitometry. Before the assay for ADP-ribosyl-transferase activity, naturally occurring CTX (purchasedfrom Sigma Chemical Co. and List Biologicals) and recom-binant subunits were incubated in 8 M urea-25 mM NaXPO4(pH 7.0)-10 mM dithiothreitol for 1 h at 37°C. Samples werethen centrifuged at 10,000 x g for 15 min at room tempera-ture, and the supernatants were recovered and adjusted to 1,ug of CTX-related protein per 4 ,ul (assuming completesolubilization) by using the 8 M urea buffer. In later exper-iments, the concentration of CTXA-related protein wasdirectly measured in the postcentrifugation supernatant frac-tions; there was no significant difference between the even-tual results obtained with samples for which the proteinconcentration was evaluated prior to centrifugation (see Fig.2) and those for which it was determined after centrifugation.For purposes of recombinant expression in E. coli, we

deleted from all of our molecular constructs that portion ofthe CTXA gene (and the CTXB gene) encoding its signalpeptide, substituting instead a methionine initiation codon.Each of these native and analog CTXA subunit species wassubsequently produced at high levels in E. coli (Fig. 1).CTXA does not appear to be processed to CTXA1 andCTXA2, probably because of the absence of the specificprotease in E. coli. Amino acid sequence analysis of recom-binant CTXA1 and its Arg-7-*Lys analog was performedwith an Applied Biosystems model 477A protein sequenator(20 cycles each). The results of this evaluation indicated thatthe amino-terminal methionine is not processed by E. colimethionyl aminopeptidase; thus, the recombinant CTXAand CTXA1 proteins are likely to be methionyl analogs.

The recombinant CTXA, CTXA1, and analog subunitswere then assayed for enzyme activity. Four microliters ofeach supernatant fraction was added to individual assaytubes. For the analysis of samples in the presence of humanerythrocyte membranes, 6 ,u of water was added to eachsample and then 28 ,u1 of a solution containing 10 mMNaXPO4 (pH 7.5), 1 mM EDTA, and 100 ,ug of membraneprotein was also added. For analysis of samples in thepresence of bovine rod outer segment (ROS) membranes, 22,ul of water was added to each sample and then 12 RIl of asolution containing 20 mM Tris-HCl (pH 7.5), 1 mM CaCl2,2 mM dithiothreitol, 0.3 ,ug of aprotinin, and 100 ,ug of ROSmembrane protein prepared as previously described (31) wasalso added. For analysis of samples in the absence ofexogenous G protein substrates, 34 pu1 of water was added toeach sample. To every sample was then added 62 ,u1 of areaction mixture to yield a final concentration of 250 mMNaXPO4 (pH 7.0), 300 puM ATP, 100 p.M GTP, 10 mMthymidine, and 30 p.M [32P]NAD (167 mCi/mol). Sampleswere left on ice for 1 h and then incubated for 30 min at 30°Cwith mixing at 10-min intervals. The reactions were eachterminated by adding 50 RI of 5 mM NAD in 0.3% sodiumdeoxycholate, vortexing the samples, and quenching on icefor 10 min. Fifty microliters of 40% trichloroacetic acid wasthen added to each sample, and the samples were thenincubated on ice for at least 15 min; in some experiments,samples were stored at -70°C before further processing.After the addition of 2 ml of water, each sample wasvortexed and centrifuged at 2,500 x g for 30 min at 4°C. Thesupernatants were aspirated, and the pellets were subjectedto SDS-PAGE. The gels were evaluated by protein stain andby autoradiography. As a control, the ADP-ribosyltrans-ferase activity was determined for native CTX treated in thesame manner as the recombinant subunits, except that ureawas omitted from the first incubation step and the incubationtime was shortened from 1 h to 15 min.The ADP-ribosyltransferase activities of recombinant

CTXA and CTXA1, and their Lys-7 analogs, were comparedwith that of naturally occurring CTX under three assayconditions (Fig. 2). All assays contained [32P]NAD as thesubstrate. In one set of assays, there was no intentionaladdition of an exogenous source of G protein. In the secondassay set, human erythrocyte membranes containing theCTX substrate Gsa were added. In the third set, ROSmembranes containing transducin were added. Since therecombinant CTX constructs were treated with urea beforeassay, the native CTX was assayed both with and withouturea treatment.The effect of substituting lysine for Arg-7 in both CTXA

and CTXA1 was identical and unambiguous when evaluatedunder all three assay conditions, namely, complete abolitionof detectable ADP-ribosyltransferase activity. This was ob-served by loss of autocatalysis when no G protein substratewas added (Fig. 2A) and loss of ability to ADP-ribosylate theGsa substrate contained in the human erythrocyte mem-brane preparation (Fig. 2B). Although the ROS membranepreparation displayed a complex pattern of proteins that ismodified by CTXA catalysis (Fig. 2C), this activity was alsoentirely abrogated by substitution at Arg-7. The striking lossof enzymatic activity is similar to that observed for the PTXS1 analog having the equivalent mutation (Arg-9---Lys) (4).This result indicates that the Arg-7 residue of CTXA1 andthe Arg-9 residue of PTX S1 share functional homology.Further examination of the data reveals other noteworthyfindings. First, combining the Lys-7 analogs with nativeCTX did not prevent ADP-ribosylation of Gsot or target

NOTES 4267

4268 NOTES

A 1 2 3

CTXA1 + A2

CTXA1

B 1 2 3

CTXAIC'I+A2TXAI

C I 2 3*,;

CTXA +tA2CTXA1 1

FIG. 2. ADP-ribosyltransferasCTX and recombinant subunit analand subunit analogs were incutwithout the addition of exogenousin the text. Incorporation of [3subjecting the samples to SDS-Praphy. (A) No G protein substratmembranes added; (C) ROS mem

experimental samples were as fc

(blank); lane 2, native CTX withcCTX with urea treatment; lanerecombinant CTXA Arg-7--*Lys a]

Arg-7-Lys analog plus native Cllane 8, recombinant CTXA1 Arg-nant CTXA1 Arg-7*Lys analog p

substrates in the ROS preparation by the toxin; thus, it isunlikely that an inhibitor in the recombinant extract caused

4 5 6 7 8 9 the loss in enzyme activity of the Lys-7 analogs. Second,recombinant CTXA ADP-ribosylated G protein substrates toa lesser extent than did recombinant CTXA1; this finding isconsistent with the previous observation that removal of thecarboxyl terminus is essential for efficient ADP-ribosylation(20).

Incubation of CTX with [32P]NAD can result in theautoincorporation of [32P]ADP-ribose into the CTXA1 sub-

iS unit (23). The addition of certain synthetic substrates, such40W as arginine methyl ester, can inhibit this autoribosylation,

presumably because the ester competes with CTXA1 as asubstrate (23). In contrast, the simultaneous addition of bothlipids and certain protein cofactors known as ADP-ribosyla-tion factors (ARFs) can substantially promote ADP-ribosyl-ation of CTXA1 (29). In the experiments reported herein,there was no intentional addition of exogenous ARFs. Figure2A clearly shows the presence of 32p in the recombinant

4 5 6 7 8 9 CTXA and CTXA1 subunits but none in naturally occurringCTXA1. The addition of human erythrocyte membranes(Fig. 2B) diminished the autoincorporation of 32P by recom-binant CTXA1 but not by CTXA. An interpretation of this

tV u S S Z X * latter result is that Gsa acts as a competitive inhibitor of theautoribosylation of CTXA1; on the other hand, because the

1. _u recombinant CTXA polypeptide does not efficiently recog-nize Gsot as a substrate, Gsa fails to inhibit autoribosylation.Unlike the addition of human erythrocyte membranes, theaddition of ROS membranes failed to inhibit autoincorpora-tion of [32P]ADP-ribose by recombinant CTXA1, despite thefact that ROS provides G protein substrates. The contrastingeffects caused by the addition of membranes from differentsources might partially result from the presence of certainfactors (e.g., ARFs) in the ROS membrane preparation,absent in the erythrocyte membranes, that strongly promoteADP-autoribosylation of the recombinant CTXA1.

4 5 6 7 8 9 The quantitative difference in autoribosylation betweenthe recombinant and native CTXA1 subunits may haveresulted from interactions with components in the recombi-nant extracts or from anomalies in ordered protein structure;

* s ^the significance of this difference is not clear. In separateexperiments (not shown), performed under the same condi-

w _ w ~~~~~tions described in the legend to Fig. 2A, the extent ofADP-autoribosylation was determined to be <0.1 mol of 32p

W _ _ per mol of recombinant CTXA1. Studies utilizing purifiedrecombinant and naturally occurring CTXA and CTXA1with purified G proteins, in the presence and absence ofpurified ARF, should help resolve issues regarding ADP-

Am autoribosylation of CTXA1.The argument could be made that the Arg-7--+Lys substi-

tutions result in molecular lesions that prevent acquisition ofe reactions catalyzed by native a higher-order structure consistent with enzymatic activity.logs. CTX, recombinantsubunits, However, both CTX and PTX have been shown to retainGated withs32P]NAD, with and such activity after treatment with a variety of denaturantsG protein substrates, as described2P]ADP-ribose was assessed by (4-6). Further, our previous work with PTX S1 has demon-AGE and subsequent autoradiog- strated that this conservative amino acid substitution pre-es added; (B) human erythrocyte serves the competence of this subunit to form an importantbranes added. In each panel, the neutralizing antigenic determinant (4, 6), after careful refold-llows: lane 1, no sample added ing subsequent to treatment with chaotropic and reducing)ut urea treatment; lane 3, native agents, and to acquire a conformation consistent with in4, recombinant CTXA; lane 5, vitro association with B oligomer to assemble a holotoxinlikenalog; lane 6, recombinant CTXA macromolecular species (1). Studies in progress will deter-rX; lane 7, recombinant CTXA1; mine whether such assembly can be accomplished with*7--Lys analog; lane 9, recombi- recombinant CTX subunits and subunit analogs; they will

also ascertain whether various subunit analogs, introducedby homologous recombination of gene segments, can sup-

INFECT. IMMUN.

AN~

NOTES 4269

port toxin assembly and secretion from V. cholerae. Never-theless, we undertook a preliminary comparative examina-tion of the conformation of the recombinant CTXA speciesused in these studies as a function of their relative sensitivityto proteolysis. Briefly, recombinant CTXA1, its Lys-7 ana-log, and naturally occurring CTXA1 were each solubilized inurea and renatured as described for the experiment shown inFig. 2; these samples, and native CTXA1 not exposed tourea, were then treated with trypsin and chymotrypsin andsubjected to SDS-PAGE. The rates of proteolytic degrada-tion for each of the urea-treated proteins and the electropho-retic patterns of their degradation products were remarkablysimilar (data not shown); little proteolysis of native CTXA1untreated with urea was observed. Thus, our analysis failedto detect conformational differences resulting from substitu-tion of lysine for Arg-7 but indicated that renaturation ofnaturally occurring and recombinant proteins into a formidentical to that of native CTXA1 was not achieved bysimple treatment with 8 M urea followed by dilution.

E. coli heat-labile enterotoxin (HLTX) and CTX catalyzethe same ADP-ribosyltransferase reactions (24, 32). Thecatalytic subunits of HLTX and CTX are extraordinarilysimilar, being >77% homologous in both amino acid andnucleic acid sequences (33). Recent results of Lobet et al.(17) indicate that substitutions for Arg-7 in the catalyticsubunit of HLTX also substantially eliminate ADP-ribosyl-transferase activity. Together with the results from previousinvestigations concerning the importance of Arg-9 in theactivities of PTX Si (1, 4, 7, 16), the findings presentedherein suggest a crucial role for this conserved arginineresidue in the ADP-ribosyltransferase reactions catalyzed byeach of these toxins. The observation that they all use NADas a substrate, but modify distinct G proteins, suggests thatthe arginine residue may interact with the common NADsubstrate. These data, combined with the sequence similar-ities, strongly imply an evolutionary relationship amongthese three bacterial toxins. Definitive structural evaluationof shared function must await the eventual determination oftheir individual crystal structures; such a structure hasrecently been reported for HLTX at a resolution of 0.23 nm(28).The observation that Arg-9 is a critical residue in the

catalytic subunit of PTX was made during the search for arecombinant DNA-derived analog that would serve as acomponent of a safe and effective whooping cough toxoidvaccine which was substantially devoid of the toxicity at-tributed to its ADP-ribosyltransferase activity yet whichretained full immunogenic potential (4). Cholera, like pertus-sis, is a disease of global dimensions; considerable effort hasbeen devoted to the development of a vaccine containing liverecombinant V. cholerae producing an enzymatically inac-tive CTX analog by eliminating its A subunit (12, 13).Abrogation of the activity by selective site-specific mutation,as we have described, affords an alternative approach thatpermits the retention of this subunit immunogen in a genetictoxoid. The ability to produce subunit components of thetoxin molecule in E. coli, and perhaps achieve assembly ofthe multimer in vitro (1, 11), also provides a novel andefficient means by which to manufacture a toxoid vaccine.

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