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Biochemical and Biophysical Research Communications 287, 542–549 (2001)

doi:10.1006/bbrc.2001.5613, available online at http://www.idealibrary.com on

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ydrophobic Residues Phe552, Phe554, Ile562, Leu566,nd Ile574 Are Required for Oligomerizationf Anthrax Protective Antigen

idhi Ahuja, Praveen Kumar, and Rakesh Bhatnagar1

entre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

eceived August 17, 2001

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Anthrax protective antigen (PA) plays a central rolen facilitating the entry of active toxin components,amely, lethal factor and edema factor, into the cells.A is also the main immunogen of both human andeterinary vaccine against anthrax. During host cellntoxication, protective antigen binds to the receptorsn cell surface, gets proteolytically activated, oli-omerizes to form a heptamer and binds to lethal fac-or or edema factor. The complex, formed by bindingf lethal factor or edema factor to oligomerized PA, isnternalized by receptor-mediated endocytosis. Acidi-cation of the endosome results in the insertion of theeptamer into the membrane, thereby forming a porehrough which lethal factor or edema factor can trans-ocate into the cytosol. In this study we have identifiedydrophobic residues, Phe552, Phe554, Ile562, Leu566,nd Ile574, which are required for oligomerization ofnthrax protective antigen. Mutation of these con-erved residues to alanine impaired the oligomeriza-ion of protective antigen. Consequently, these mu-ants became nontoxic in combination with lethalactor and edema factor. Therapeutic importance ofhese mutants and their potential as vaccine candi-ates is discussed. © 2001 Academic Press

Key Words: anthrax protective antigen; hydrophobicesidues; site-directed mutagenesis; alanine substitu-ion; oligomerization; heptamers; intoxication.

Anthrax toxin complex is primarily responsible forhe major physiopathological effects observed duringnfection with Bacillus anthracis. The complex (1) com-rises of three proteins, i.e., protective antigen (PA, 83Da), lethal factor (LF, 90 kDa) and edema factor (EF,9 kDa). Individually, each of the three proteins lacksny toxic activity. However, the binary combination ofA and LF, called the lethal toxin, causes death inxperimental animals (2) and lysis of primary macro-

1 To whom correspondence should be addressed. Fax: 1 91 (11)6198234/165886. E-mail: [email protected] or [email protected].

542006-291X/01 $35.00opyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

ther hand, the combination of PA and EF, called thedema toxin, causes edema in the skin of infected an-mals (8).

Both lethal factor and edema factor require protec-ive antigen for entry into the host cells. During intox-cation, protective antigen first binds to its receptors onhe surface of susceptible cells. The cleavage of theeceptor-bound PA by the cell surface proteases (9),uch as furin, results in the release of a 20-kDa frag-ent from the N-terminal of the protein. The 63-kDa

ragment of PA (PA63) oligomerizes to form ring-shapedeptamer (10). LF or EF bind competitively to the sitexposed on release of 20-kDa fragment of PA. Thisntire complex undergoes receptor-mediated endocyto-is (3, 11). The acidification of the endosome causesajor conformational changes in the PA molecule,

eading to the insertion of the heptamer into the endo-omal membrane (12, 13). LF and EF are translocatedcross the endosomal membrane to the cytosol throughhese pores (14). After reaching the cell cytosol, LF andF, exert their toxic effects. EF is a calcium/almodulin-dependent adenylate cyclase that causesn increase in the intracellular cAMP levels of the hostells (15). Whereas, LF is a metalloprotease whichleaves several isoforms of MAP kinase within mam-alian cells (16–18).The crystal structure of PA has been determined at

igh resolution (19). Anthrax protective antigen hasour domains organized predominantly into b-sheets.omain 1 (consisting of residues 1–249) contains the

urin cleavage site (20), which defines two subdomains,ne forming the PA20 fragment and the other that haseen hypothesized to be involved in binding to LF andF. Domain 2 (consisting of residues 250–487) is or-anized into a nine-stranded b barrel. Recently, weave shown that residues 346 and 352 of this domainre required for membrane insertion and/or the trans-ocation of LF/EF to the cytosol (21). Domain 3 (con-isting of residues 488–594) is the smallest domain of

anthrax protective antigen. It has an exposed hydro-pkdr

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Vol. 287, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hobic patch on its surface whose role in vivo is notnown. Domain 4 (consisting of residues 595–735) me-iates the binding of protective antigen to cell surfaceeceptors (22).

Domain 3 of anthrax protective antigen consists pri-arily of four-stranded b sheet and four helices. Helixis part of the triangular strand-helix-loop structure

hat permits five hydrophobic residues Phe552,he554, Ile562, Leu566, and Ile574 to form a solvent-xposed ‘hydrophobic patch’. In the current study, weave shown that mutation of any of the five residues ofhe hydrophobic patch to alanine impaired the oli-omerization of PA. The mutant proteins, when addedn combination with LF or EF were considerably lessoxic to J774A.1 macrophage-like cells and CHO cells,espectively. These mutated molecules may be consid-red better candidates to be used as anthrax vaccine.

ATERIALS AND METHODS

Site-directed mutagenesis. For site directed mutagenesis of PAene, PCR was done using pMS1 (23) as the template. Briefly, twoomplementary mutagenic primers spanning the site, at which mu-ation has to be introduced, were used in the first round of PCR tomplify two segments of the gene. This was followed by a secondound of PCR using the purified products of first round of PCR asemplate with primers that introduced HindIII and BamHI sites at9 and 39 ends of the PCR product. The amplified product, obtainedfter the second round of PCR, was digested with HindIII andamHI and ligated to the backbone obtained upon digestion of pMS1ith the same enzymes. The ligation mix was transformed into E.

oli DH5a competent cells. Colonies obtained were screened for re-ombinant plasmid. Constructs selected after restriction analysisere sequenced to ensure that the desired mutation has been incor-orated.

Expression and purification. The recombinant constructs wereransformed into Escherichia coli BL21(DE3) competent cells. Theells carrying the plasmid were grown at 37°C in LB broth with 100g/ml ampicillin and 0.01% MgSO4 at 250 rpm. When A600 reached.8, IPTG was added to a final concentration of 0.5 mM, to induce thexpression of recombinant protein. Following induction, the cellsere allowed to grow at 30°C for three hours and then harvested by

entrifugation at 6000g for 15 min at 4°C. The periplasmic proteinsere isolated by osmotic shock and PA was purified to homogeneitysing ion exchange and hydrophobic interaction chromatography.

Mammalian cell culture. Macrophage-like cell line J774A.1 wasaintained in RPMI 1640 medium containing 10% heat-inactivatedCS, penicillin (100 U/ml) and streptomycin (100 mg/ml). Chineseamster ovary (CHO) cell line was maintained in EMEM mediumupplemented with non-essential amino acids, 25 mM Hepes (pH.4), penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% heat-nactivated FCS.

Cytotoxicity assay. J774A.1 cells were plated at a density of 105

ells/ml in 96-well tissue culture plates and were grown to 80 to 90%onfluence. At the start of the experiment, spent medium and de-ached cells were removed by aspiration and replaced with RPMIontaining 1 mg/ml LF and varying concentration of wild-type orutant PA. The cells were incubated for 3 h at 37°C in a humidifiedO2 incubator. After 3 h, cell viability was determined with 3-(4,5-imethylthiazol-2-yl),-5-diphenyltetrazolium bromide (MTT) dye, asescribed previously (7). All experiments were done in triplicate.

543

ell plates and grown to confluence. To begin the experiment, oldedia was replaced with H199 medium containing 0.5 mg/ml each ofF and PA (wild-type or mutant protein). After incubation for 2 h at7°C, the cells were examined under the microscope for elongationesponse.

Binding of PA to cell surface receptors and its proteolytic cleavage.774A.1 cells were plated in 24-well plates and were incubated at°C with 400 ng of wild-type or mutant PA. After 15 min, the cellsere washed with cold PBS. They were then scraped off and lysed.ne hundred micrograms of cell protein was heated for 3 min at 95°Cnd subjected to 10% SDS–PAGE. PA was identified by immunoblotnalysis with anti-PA antiserum. To study the proteolytic cleavage ofA on cell surface, the same procedure was followed except that PAas incubated with cells for 2 h at 4°C.To study the binding of radiolabelled mutant proteins to CHO

ells, the mutant proteins were radioiodinated, as detailed before (4),nd were allowed to bind to CHO cells at 4°C. After 20 min ofncubation, the cells were washed to remove unbound protein andere solubilized in 100 mM NaOH. Counts associated with the cellsere measured, using a gamma counter, to determine the amount of

adiolabelled protein binding to the cells.

In vitro cleavage of PA and its binding to LF. To study theinding of LF to PA in solution, PA was cleaved with trypsin (1 nger mg of PA) for 30 min at 30°C in 25 mM Hepes, 1 mM CaCl2 and.5 mM EDTA. Trypsin was inactivated with 1 mM PMSF and theicked PA was incubated with LF (1 mg/ml) for 15 min in 20 mM Tris,H 9.0 containing 2 mg/ml Chaps. The samples were analyzed on aondenaturing 5–10% gradient gel.

Binding of EF to receptor-bound PA. CHO cells plated in six-welllates, were cooled and were allowed to incubate with mutant pro-eins at 4°C. The cells were later washed and radioiodinated EF wasdded. After 1 h of incubation at 4°C, unbound protein was removedy washing the cells with cold PBS. The cells were then solubilizedith 100 mM NaOH. Radioactivity associated with the cells waseasured to determine the amount of radioiodinated EF binding to

eceptor-bound mutant proteins.

Oligomerization of PA on cells. J774A.1 cells were plated in 24-ell tissue culture plates and were allowed to grow to 90% conflu-nce. The cells were cooled and 1 mg of wild-type or mutant PA wasdded to the cells at 4°C. After 4 h of incubation the cells wereashed and scraped off. Cells were lysed with SDS–lysis buffer. Oneundred micrograms of cell lysate was heated at 95°C for 3 minefore loading on 5% SDS–PAGE. Immunoblot analysis was doneith anti-PA antibodies to detect SDS- and heat-resistant oligomers.

ESULTS

Site-directed mutagenesis. To study the role of hy-rophobic residues, Phe552, Phe554, Ile562, Leu566,nd Ile574 of anthrax protective antigen, the residuesere individually substituted with alanine in five dif-

erent constructs (F552A, F554A, I562A, L566A, and574A). Three constructs with double mutations werelso made (F552A 1 F554A; I562A 1 I574A and556A 1 I574A). Construct with mutation at all theve residues was also used for the study (F552A 1554A 1 I562S 1 L566A 1 I574A).

Expression and purification of the mutant proteins.he plasmid pMS1, carrying PA gene under the controlf bacteriophage T7 promoter, has an Omp A signalequence attached to the 59 end of the gene. The signalequence, that delivers the proteins to periplasm, is

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leaved by E. coli signal peptidases. Proteins were re-eased from the periplasmic space by osmotic shock andA was purified to homogeneity by ion exchange andydrophobic interaction chromatography. The purifiedutant proteins were analyzed by SDS–PAGE (Fig. 1).

FIG. 1. Purified PA mutants from E. coli. All the mutants werexpressed in E. coli BL21(DE3) cells. The proteins were purified fromeriplasm of cells by ion-exchange and hydrophobic interaction chro-atography. The purified proteins were analysed on 12% SDS–AGE and stained with Coomassie blue. Lane M, molecular weighttandard; Lane 1, F552A; lane 2, F554A; lane 3, I562A; lane 4,566A; lane 5, I574A; lane 6, F552A 1 F554A; lane 7, I562A 1574A; lane 8, L566A 1 I574A; lane 9, F552A 1 F554A 1 I562S 1566A 1 I574A.

FIG. 2. Cytotoxicity of PA mutants in combination with LF. J77ions of mutant PA along with 1 mg/ml of LF. After 3 h of incubatio

544

ate the biological activity of the mutant proteins, cy-otoxicity assays were performed on J774A.1 cells. Theells were treated with 1 mg/ml of LF along with thendicated concentration of mutant PA for 3 h and thenell viability was determined (Fig. 2). It was found thatubstitution of Ala at residues 552 and 574 resulted inomplete loss of activity. However, Ala-substitution atosition 554 decreased the toxicity of the mutantF554A) fourfold as compared to the wild-type protein.

utants I562A and L566A were slightly toxic at highoncentrations. The double mutants (F552A 1 F554A;562A 1 I574A, and L556A 1 I574A) and the mutantrotein harboring mutations at all five positions wereompletely non-toxic to J774A.1 cells, when addedlong with LF.Similar results were obtained when CHO cells were

reated with EF in combination with mutant proteins.F is an adenylate cyclase that elicits elongation re-ponse in CHO cells, when added in combination withild-type PA. CHO cells were treated with 0.5 mg/mlach of EF and mutant PA for 3 h before the cells wereicroscopically examined. Except for mutant F554A,

1 cells plated in 96-well plate were treated with various concentra-ell viability was determined with MTT dye.

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Loss or decrease in toxicity of mutant PA proteinsndicates that the mutant proteins were defective inheir ability to mediate the entry of the catalytic sub-nits of anthrax toxin, namely, LF and EF, into theusceptible cells. This suggests that the hydrophobicide chain of the residues that make up the ‘hydropho-ic patch’ or the overall structure of this region isssential for the biological activity of anthrax protec-ive antigen. Further studies were conducted with mu-ant proteins to identify the defects that affected theoxicity of these proteins.

Binding of mutants to cell-surface receptors. Therst step of intoxication by anthrax toxin is the bindingf PA to the receptors on the surface of host cells. Totudy the binding of mutant proteins to cell-surfaceeceptors, the mutant proteins were incubated with774A.1 cells at 4°C. After fifteen minutes of incuba-ion, the cells were washed (to remove unbound pro-ein) and lysed. Cell lysate was subjected to SDS–AGE and protective antigen bound to the cells was

dentified by immunoblot analysis with anti-PA anti-erum. Results in Fig. 3 and Table 1 show that theutant proteins were unimpaired in their ability to

ind cell-surface receptors.

Proteolytic cleavage of mutant proteins. Once boundo cell surface receptors, PA gets cleaved by cell surfaceroteases to release 20-kDa fragment, thereby, expos-ng site for binding of LF and EF on the 63-kDa pro-ein. To study the proteolytic cleavage of mutant pro-eins on the cell surface, the proteins were allowed toind to cell surface receptors at 4°C. After 2 h of incu-ation, the cells were washed and lysed. The lysate wasubjected to SDS–PAGE and PA was identified by im-unoblot analysis with anti-PA antibodies. The blot

Fig. 4a) shows that all the mutants were proteoly-ically activated on the surface of J774A.1 cells.

This cleavage of PA can be mimicked in solution byrypsin. As shown in Fig. 4b, the mutant proteins

FIG. 3. Binding of PA mutants to cell surface receptors. J774A.1ells were plated in 24-well plates. The cells were cooled and 400 ngf wild-type or mutant PA was then allowed to incubate with cells at°C for 15 min. The cells were washed with cold PBS. They were thencraped off and lysed with SDS–lysis buffer. The samples were boiledor 3 min and loaded on SDS–PAGE. PA bound to the cells wasetected by immunoblotting with anti-PA antibodies.

545

ielded 63 kDa protein, in vitro. On the other hand,imited digestion of the mutant proteins with chymo-rypsin yielded 37- and 47-kDa fragments, just likehe wild-type protein (data not shown). The unal-ered cleavage pattern of the mutant proteins sug-ests that alanine substitution of the solvent ex-osed residues (Phe552, Phe554, Ile562, Leu566, andle574) did not affect the conformation of protectiventigen.

Oligomerization of PA and binding to LF. Trypsin-icked PA when allowed to interact with LF forms higholecular weight complex that has very low mobility

n nondenaturing PAGE. This complex results fromF-induced oligomerization of PA63. To study the abil-

ty of mutant proteins to oligomerise and bind to LF,utant proteins were cleaved with trypsin and allowed

o incubate with 1 mg LF, before loading the samples onondenaturing PAGE. Figure 5 shows that the wild-ype protein and mutant F554A were able to formigh-molecular weight complex that has low mobilityn native PAGE. Other mutants (F552A, I562A, I574A,552A 1 F554A, I562A 1 I574A, L556A 1 I574A, and552A 1 F554A 1 I562S 1 L566A 1 I574A) failed to

orm this complex. A reduced amount of low mobilityand was observed when trypsin-nicked L566A wasllowed to bind to LF.

Binding of EF to receptor-bound PA mutants. Totudy the ability of the mutant proteins to bind ra-iolabelled EF, the mutant proteins were allowed toind to cell-surface receptors at 4°C. The cells wereater washed and incubated with radiolabelled EF inold medium. After 1 h of incubation, the cells weregain washed to remove unbound protein andolubilised in 100 mM NaOH. Counts associatedith the cells were measured to determine themount of radiolabelled EF bound to the mutantroteins. It was observed that the binding of EF tohe mutant (F554A) was only slightly affected by

TABLE 1

Specific Binding of Mutant Proteins to Cell Surface Receptors

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la-substitution at Phe554. However, binding to thether mutants was considerably reduced (approxi-ately 50%), in comparison to the wild-type PA.

Oligomerization of mutant proteins on cells. Totudy the ability of mutant proteins to form oligomers,he mutant proteins were incubated with J774A.1 cellsor 4 h at 4°C. After washing, the cells were lysed withDS–lysis buffer. The samples were boiled for 3 minefore loading on SDS–PAGE. Immunoblot analysisas done with anti-PA antiserum to detect formationf SDS and heat-resistant oligomers. In comparison tohe wild-type protein, reduced amount of oligomeriza-ion of mutant proteins (F552A, F554A, I562A, L566A,

FIG. 4. Proteolytic cleavage of mutant proteins. (a) The purifiedhe cells were then washed and lysed. Cell lysate was loaded on SDnti-PA antibodies. (b) The purified mutant proteins were digested wnd 0.5 mM EDTA. After 30 min, trypsin was inactivated and theoomassie blue. Lane WT, wild-type protective antigen; Lane 1, F55552A 1 F554A; lane 7, I562A 1 I574A; lane 8, L566A 1 I574A; larotective antigen; and lane M, molecular weight standard.

FIG. 5. Binding of LF to PA mutants in solution. PA or itsutants were treated with trypsin before incubating with LF (1g/ml) for 15 min in 20 mM Tris, pH 9.0 containing 2 mg/ml Chaps.he samples were then loaded on a nondenaturing 5–10% gradientel. The gel was stained with Coomassie blue.

546

nd I574A) was detected (Fig. 6). This indicated thathe mutant proteins were defective in their ability toorm SDS- and heat-resistant oligomers.

tant proteins were allowed to bind to J774A.1 cells at 4°C, for 2 h.AGE and protective antigen was detected by immunoblotting withtrypsin (1 ng per mg of PA) at 30°C in 25 mM Hepes, 1 mM CaCl2

ples were analysed on 12% SDS–PAGE. The gel was stained with; lane 2, F554A; lane 3, I562A; lane 4, L566A; lane 5, I574A; lane 6,9, F552A 1 F554A 1 I562S 1 L566A 1 I574A; lane U, undigested

FIG. 6. Oligomerization of PA on cell surface. J774A.1 cells werelated in 24-well plates and were cooled before the start of experi-ent. One microgram of wild-type or mutant PA was then allowed to

ind to cell surface receptors at 4°C. The cells were later washed toemove unbound protein, scraped off and lysed with SDS lysis buffer.he samples were boiled for 3 min before loading on 5% SDS–PAGE.mmunoblot analysis was done with anti-PA antiserum to detectormation of SDS and heat-resistance oligomers. Lane 1, F552A; lane, F554A; lane 3, I562A; lane 4, L566A; lane 5, I574A; and lane WT,ild-type protective antigen.

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ISCUSSION

According to the current model of host cell intoxica-ion, anthrax protective antigen binds to cells, getsroteolytically activated to oligomerise and form hep-amers. Lethal factor or edema factor the catalyticomponents of anthrax toxin competitively bind to thiseptamer (envisaged as prepore) that is internalizedy cells by receptor-mediated endocytosis. Acidificationf the endosome induces conformational changes lead-ng to the insertion of the prepore into the membrane.he pore thus formed serves as conduit for passage ofartially or completely unfolded activity moieties intohe cell cytosol. In this study we show that the resi-ues, Phe552, Phe554, Ile562, Leu566, and Ile574 areequired for oligomerization of anthrax protective an-igen. Site directed mutagenesis of these residues im-aired the ability of PA to facilitate the entry of LF orF. The mutants were therefore nontoxic.Crystal structure of protective antigen (19) shows

hat the scaffold for these residues is a strand-helix-oop structure that allows these residues to form aairly flat surface (roughly of the shape of an isosce-es triangle) on domain 3 of anthrax protective anti-en. Solvent-exposed ‘hydrophobic patch’ formed byhe hydrophobic side-chains of these residues, is suf-ciently accessible to participate in intermolecular

nteractions. In fact, in crystals of PA, this patch isccupied by phenylalanine residue from a neighbor-ng molecule. Three likely candidates on the neigh-oring subunit of the heptamer that could possiblyorm a hydrophobic contact with the patch on domainare Phe427, Phe313 and Phe314. To investigate theossibility that Phe427 might interact with thisatch, we made two constructs, one in which the Pheesidue was substituted with Ala (F427A) and an-ther in which this residue was deleted (F427del).oth these mutant proteins were expressed and pu-ified from E. coli, as detailed before. When added inombination with LF to J774A.1 cells, both the mu-ant proteins were found nontoxic. These mutantsould bind to cell surface receptors, get proteolyti-

Sequence Alignment of A

PA KDITE...F D F N F DADP IPIDES.CV E L I F DCDT IPIDES.CV E L I F DIOTA IPIDES.CV E L I F DSB MPIDES.CVE L I F DC2 LEISKNEKI Q V F L D

Note. PA, Anthrax protective antigen; ADP, ADP-ribosyltransferasoxin component Ib of Clostridium perfringens; SB, Sb component of Cotulinum.

547

ally cleaved, oligomerise and bind to LF. They wereefective in a step beyond oligomerization and bind-ng of LF. This suggested that Phe427 is not involvedn oligomerization of PA. Our studies are in completegreement with a recent report (24) that implicateshe427 in translocation process (and not oligomer-

zation). The other two candidates that could be in-olved in hydrophobic contact with patch on domain, Phe313 and Phe314, were studied by Singh et al.25). They have shown that a deletion mutant lack-ng the two hydrophobic residues (Phe313 andhe314), was indeed defective in its ability to formligomers. However, requirement of these residuesor making hydrophobic contact between the hep-amer subunits is not yet established.

All the mutations made during the course of thistudy were on solvent-exposed residues. Ala substitu-ions of these residues is likely to produce minor, if any,hanges in conformation of protective antigen. Thus,hese nontoxic mutants may be used as potential can-idates for vaccine development. Furthermore, thistudy shows that these mutants were unimpaired inheir ability to bind cell surface receptors. Thus, theyan compete with wild-type toxic protein and minimize/bolish its toxic effects when used as a therapeuticgent. Studies pertaining to therapeutic potential ofhese mutants are underway.

Sequence analysis of anthrax protective antigen andomologous toxin proteins show that these five resi-ues are either identical or substituted with anotherydrophobic amino acid, in ADP-ribosyltransferase oflostridium difficile, CdtB of Clostridium difficile, iota

oxin component of Clostridium perfringens, Sb compo-ent of Clostridium spiroforme and C2 toxin (compo-ent II) of Clostridium botulnium (Table 2). Highlyonserved nature of these residues emphasizes theirunctional importance. Furthermore, the homology athe level of primary sequence is also reflected in aimilar pattern of host cell intoxication. For example, aew common features between anthrax protective an-igen and Clostridium botulinum C2 toxin (26) are (i)oth the toxins require proteolytic activation to formligomers that are predominantly heptamers; (ii) both

hrax Protective Antigen

TSQN I KNQ L AELNV TN I YTVLDKI KLNAKMNILITANK I KDS L KTLSD KK I YNV.... KLERGMNILITANK I KDS L KTLSD KK I YNV.... KLERGMNILITSEI I KEQ L KYLDD KK I YNV.... KLERGMNILITANL I KER L NALND KK I YNV.... QLERGMKILITNND F ENQ L KNTAD KD I MHC.... IIKRNMNILV

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ndocytosis; (iii) acidification of the endosomes triggershannel formation in these proteins, through whichatalytic toxin components are translocated. Moreover,hannels formed by both the toxins are cation-elective. Similar studies on Clostridium perfringensota b toxin (27) show that it forms oligomers uponroteolytic activation and that translocation of iota anto the cytosol requires acidification of the endosome.ow pH induced formation of ion channels has alsoeen reported for Clostridium difficile toxin B (28).hese reports strongly suggest that the conserved res-

dues (marked in Table 2) might be playing a similarole (possibly in oligomerization) in intoxication pro-ess of these homologous toxins. This possibility opensp new avenues for further study that will help usetter understand the structural features and molecu-ar interactions that govern self-assembly and oli-omerization of proteins to form transmembrane chan-els.To summarize, in this study, we show that residues

he552, Phe554, Ile562, Leu566, and Ile574 are re-uired for oligomerization of anthrax protective anti-en. Alanine-substitution of any of these residues re-ulted in mutants that were oligomerization-defectivend were therefore non-toxic in combination with LF orF. These non-toxic mutants may be considered as

andidates for development of vaccine against anthrax.oreover, since these mutant proteins can competeith wild-type PA for cell-surface receptors, they have

mmense therapeutic potential.

CKNOWLEDGMENTS

Financial support from DBT, Government of India, made this workossible. Both N.A. and P.K. receive fellowships from UGC, Govern-ent of India.

EFERENCES

1. Leppla, S. H. (1991) The anthrax toxin complex. In Sourcebook ofBacterial Protein Toxins (Alouf, J. E., and Freer, J. H., Eds.), pp.277–302, Academic Press, London.

2. Smith, H., and Keppie, J. (1954) Observations on experimentalanthrax: Demonstration of a specific lethal factor produced invivo by Bacillus anthracis. Nature 173, 869–870.

3. Friedlander, A. M. (1986) Macrophages are sensitive to anthraxtoxin through an acid dependent process. J. Biol. Chem. 261,7123–7126.

4. Bhatnagar, R., Singh, Y., Leppla, S. H., and Friedlander, A. M.(1989) Calcium is required for expression of anthrax lethal toxinactivity in the macrophage-like cell line J774A.1. Infect. Immun.57, 2107–2114.

5. Friedlander, A. M., Bhatnagar, R., Leppla, S. H., and Singh, Y.(1993) Characterization of macrophage sensitivity and resis-tance to anthrax lethal toxin. Infect. Immun. 61, 245–252.

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is required for expression of anthrax lethal toxin cytotoxicity.Infect. Immun. 62, 2958–2962.

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Hydrophobic Residues Phe552, Phe554, Ile562, Leu566, and Ile574 Are Required for Oligomerization of...

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