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Antimicrobial Agents and Chemotherapy, August 2001, p. 2397-2399, Vol. 45, No. 8
Department of Epidemiology, University of Michigan
School of Public Health, Ann Arbor, Michigan 48109
Received 2 April 2001/Returned for modification 24 April
2001/Accepted 21 May 2001
Artemisinin and its derivatives are endoperoxide-containing
antimalarial drugs that appear to form adducts in situ with the Plasmodium falciparum translationally controlled tumor
protein (TCTP) homolog. Immunoprecipitation with antibody to
recombinant TCTP suggests that adducts may form with both monomeric and
dimeric TCTP.
The artemisinin derivatives are an
important new class of antimalarials derived from an ancient Chinese
herbal remedy. Artemisinin derivatives, such as artesunate, artemether
and arteether, are now widely used in Southeast Asia and other areas
where multidrug-resistant parasites are found (22).
Artemisinin derivatives appear to act by a two-step mechanism. First,
intraparasitic heme and/or free iron catalyze the decomposition of the
endoperoxide bridge to eventually form carbon-centered free radicals.
Second, these free radicals then act as alkylating agents, reacting
with both intraparasitic heme and proteins. Since this mechanism was
first proposed in 1991 (21), supporting evidence for it
has been contributed by at least 17 research groups in 12 countries
(1, 4-9, 12, 14, 15, 17, 18, 23-28, 30, 32, 34, 35).
When Plasmodium falciparum-infected erythrocytes are
incubated with radiolabeled dihydroartemisinin, the radioactivity
associates covalently with both parasite protein and heme (3, 16,
21). When lysates of the labeled parasites are analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), several radiolabeled protein bands can be seen on
autoradiograms. These bands are seen even when low therapeutic
concentrations of the drug are used, but they are not seen when labeled
drug is incubated with uninfected erythrocytes or when a radiolabeled
but inactive congener,
[3H]deoxydihydroartemisinin, is incubated with
infected erythrocytes (3).
One of the alkylated proteins has been identified as the malarial
translationally controlled tumor protein (TCTP) homolog (11). TCTPs have been identified in dozens of other
organisms and have been shown to bind calcium (19). In
vitro, recombinant P. falciparum TCTP has been shown to bind
calcium and heme and to react covalently with
[3H]dihydroartemisinin in the presence of heme
(10, 11). In P. falciparum, some TCTP appears
to be associated with food vacuolar membranes (10). Since
the food vacuole is rich in heme (29), it is possible that
artemisinin might react with the TCTP molecules associated with this
structure. Artemisinin-resistant strains of Plasmodium
yoelii express higher levels of TCTP than do artemisinin-sensitive strains (33). However, there is no direct evidence that
the alkylation of TCTP by artemisinin is responsible for the
antimalarial effects of the drug.
TCTP was identified as a target for artemisinin by a process involving
cutting out a labeled band from a gel and obtaining its N-terminal
sequence (11). Thus, it is possible that the wrong band
was sequenced and that the true target might be another protein with
similar electrophoretic mobility. In order to rule out this
possibility, we attempted to immunoprecipitate a
[3H]dihydroartemisinin-TCTP complex with
antibodies made to recombinant TCTP.
P. falciparum strain FCR3 was cultured by the method of
Trager and Jensen (31) and synchronized by sorbitol
lysis (20). Red cells infected with late rings and
trophozoites (25 to 30% parasitemia) were incubated in culture medium
in the presence of 1.5 µCi of
[3H]dihydroartemisinin (1.4 Ci/mmol/ml; Moravek
Biochemical, Brea, Calif.) per ml or 0.1 to 0.2 mCi of
[35S]methionine (Amersham Life Science Inc.,
Arlington, Heights, Ill.) per ml for 3 h. Cells were pelleted by
centrifugation at 1,500 × g for 5 min and washed three
times with RPMI 1640 without serum. The parasites were isolated from
red blood cells by saponin lysis (13) and stored at
One milliliter of the supernatant was precleared by incubation with 50 µl of 50% protein A-Sepharose slurry (Pharmacia Biotech) in dilution
buffer (10 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 0.1% Triton
X-100, 0.025% sodium azide, and 0.1% bovine serum albumin) on a
rocking-platform shaker overnight at 4°C and centrifuged at 200 × g for 1 min. The pellet was discarded.
Anti-recombinant TCTP antibody and prebleed control antibody were
prepared as previously described (10). The precleared lysates, approximately 105 to
107 cpm, were incubated with anti-TCTP or various
amounts of control antibody (between 7.5 and 180 µg) for 3 to 4 h on a rocking-platform shaker at 4°C. Amounts of 20 to 40 µl of a
50% protein A-Sepharose slurry were added to 200 µl of precleared
clarified lysate and incubated for an additional 2 h on the
platform shaker at 4°C. The mixtures were centrifuged at 200 × g for 1 min, and the pellets were saved. The pellets were
washed twice with 1 ml of dilution buffer: once with 1 ml of buffer A
(10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.025% sodium azide) and once
with 1 ml of buffer B (50 mM Tris-HCl, pH 6.8). The supernatants
were discarded. Amounts of 20 to 50 µl of SDS and sample
buffer (Novex, San Diego, Calif.) were added to each pellet, incubated
at 80°C for 10 min, and then microcentrifuged for 5 s. The
resulting supernatants were then electrophoresed using 10% NuPAGE gels
(Novex) and either stained with Coomassie blue or exposed to XAR2
autoradiography film (Eastman Kodak, Rochester, N.Y.).
In order to evaluate our immunoprecipitation protocol,
[35S]methionine-labeled parasites were first
studied. Previously, when anti-TCTP antibodies were used for
immunoblotting, only a single band at 22 to 23 kDa reacted, suggesting
that the antibody is specific for TCTP (10). After
immunoprecipitation by the same antibody, a band at 22 kDa,
corresponding to monomeric TCTP, was seen, as expected, on SDS-PAGE
gels (Fig. 1). This band was not seen
after immunoprecipitation by control antibody. Anti-TCTP also
precipitated proteins at higher molecular masses, including 45, 52, 67, and 71 kDa (Fig. 1). These bands were either absent or faint on
the lane immunoprecipitated with control antibody. In light of the
recent observation that recombinant rat TCTP self-aggregates (36), the bands at 45 and 67 kDa could represent dimeric
and trimeric TCTPs. Alternately, they may represent aggregates of TCTP
with other proteins.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2397-2399.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Immunoprecipitation of
[3H]Dihydroartemisinin Translationally Controlled Tumor
Protein (TCTP) Adducts from Plasmodium
falciparum-Infected Erythrocytes by Using Anti-TCTP
Antibodies
and
ABSTRACT
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70°C. Pelleted parasites were lysed either by incubating with 1 ml
of lysis buffer (12.5 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1%
sodium deoxycholate, 1% bovine serum albumin, 1 mM
phenylmethylsulfonyl fluoride, 0.4 U of aprotinin per ml, pH 7.5 or 8)
for 1 h on ice or by sonicating three times in lysis buffer
without detergent (each time for 10 s followed by 2 min at 4°C).
The parasite lysate was centrifuged at 3,000 × g for
10 min at 4°C, and the supernatant was saved. Then, the supernatant
was recentrifuged at 10,000 × g for 30 min at 4°C. The clarified supernatant was stored at 70°C or used immediately.
View larger version (35K):
[in a new window]
FIG. 1.
Autoradiogram of [35S]methionine-labeled
parasite. The parasite pellets were lysed by sonication in lysis buffer
without detergent and immunoprecipitated with 90 µg of anti-TCTP and
prebleed control antibody (lanes A and B, respectively). The film was
exposed for 9 h.
Immunoprecipitation was then carried out on lysates of
[3H]dihydroartemisinin-labeled
parasites (Fig. 2). After SDS-PAGE and autoradiography, the anti-TCTP immunoprecipitate contained labeled bands at 22 and 45 kDa and at a high molecular mass (lane D) which were
not seen after immunoprecipitation by prebleed antisera (lane E).
Recombinant TCTP, which contains a 12-amino-acid expression tag,
migrated at approximately 25 kDa (lane C), as described previously (11).
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Thus, both 22- and 45-kDa bands in the anti-TCTP immunoprecipitate, which probably represent monomeric and dimeric TCTPs, were labeled after whole parasites were incubated with [3H]dihydroartemisinin. Drug-derived radioactivity was also found in the high-molecular-mass region of the gel and may have several causes. First, it could be due to high-molecular-weight oligomers of TCTP or to the association of TCTP with other proteins. Second, it could be an artifact caused by artemisinin-induced cross-linking of membrane proteins previously reported (2).
These data confirm that [3H]dihydroartemisinin reacts with both monomers and dimers of TCTP in situ. This is the first evidence that TCTP self-aggregates in a normal cell. Interestingly, P. falciparum TCTP oligomers appear to be stable in SDS, while recombinant rat TCTP oligomers were found to dissociate in SDS (36). This might be due to structural differences between rat and malarial TCTPs or between native and recombinant TCTPs. Nevertheless, the formation of stable TCTP dimers might provide a clue to its biological function and to the mechanism of action of artemisinin.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant R21 AI 26848 (S.R.M.) and the Royal Thai Government (J.B.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109. Phone: (734) 647-2406. Fax: (734) 764-3192. E-mail: meshnick{at}umich.edu.
Present address: Department of Parasitology, Pramongkutklao College
of Medicine, Bangkok 10400, Thailand.
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2397-2399, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2397-2399.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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