Download gene cloning and identification of the Circumsporozoite protein of

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Community fingerprinting wikipedia , lookup

Non-coding DNA wikipedia , lookup

Genomic library wikipedia , lookup

Magnesium transporter wikipedia , lookup

Interactome wikipedia , lookup

Amino acid synthesis wikipedia , lookup

Peptide synthesis wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Gene wikipedia , lookup

Protein wikipedia , lookup

RNA-Seq wikipedia , lookup

Metalloprotein wikipedia , lookup

Ancestral sequence reconstruction wikipedia , lookup

Expression vector wikipedia , lookup

Deoxyribozyme wikipedia , lookup

Biochemistry wikipedia , lookup

Protein–protein interaction wikipedia , lookup

Nucleic acid analogue wikipedia , lookup

Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup

Monoclonal antibody wikipedia , lookup

Gene expression wikipedia , lookup

Western blot wikipedia , lookup

Genetic code wikipedia , lookup

Biosynthesis wikipedia , lookup

Silencer (genetics) wikipedia , lookup

Protein structure prediction wikipedia , lookup

Two-hybrid screening wikipedia , lookup

Point mutation wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

Proteolysis wikipedia , lookup

Transcript
Circumsporozoite protein of Plasmodium
berghei: gene cloning and identification of the
immunodominant epitopes.
D J Eichinger, D E Arnot, J P Tam, V Nussenzweig and V Enea
Mol. Cell. Biol. 1986, 6(11):3965. DOI: 10.1128/MCB.6.11.3965.
These include:
CONTENT ALERTS
Receive: RSS Feeds, eTOCs, free email alerts (when new articles
cite this article), more»
Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml
To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
Updated information and services can be found at:
http://mcb.asm.org/content/6/11/3965
MOLECULAR AND CELLULAR BIOLOGY, Nov. 1986, p. 3%5-3972
0270-7306/86/113965-08$02.00/0
Copyright © 1986, American Society for Microbiology
Vol. 6, No. 11
Circumsporozoite Protein of Plasmodium berghei: Gene Cloning and
Identification of the Immunodominant Epitopes
DANIEL J. EICHINGER,lt* DAVID E. ARNOT,2 JAMES P. TAM,3 VICTOR NUSSENZWEIG,'
AND VINCENZO ENEA2
Department of Pathology and Kaplan Cancer Center,' and Department of Medical and Molecular Parasitology,2 New
York University Medical Center, New York, New York 10016, and Rockefeller University, New York, New York 100213
Received 14 May 1986/Accepted 30 June 1986
The sporozoite stage of malaria parasites carries a protein
on its outer surface (1) which expresses a unique immunodominant epitope recognized by immunized or repeatedly infected hosts (33, 34). Sera from mice immunized with
Plasmodium berghei sporozoites immunoprecipitate a single
44,O000Mr protein, the circumsporozoite (CS) protein, from
extracts of surface-labeled sporozoites (33). Immunoprecipitation of extracts of metabolically labeled sporozoites with a
monoclonal antibody (3D11) directed to the CS protein
demonstrated that the 44,000Mr membrane form is derived
from a 54,000-Mr intracellular precursor (33). The CS protein
from monkey and human malaria parasites contains aminoand carboxy-terminal regions of relatively low immunogenicity which flank a central region of highly immunogenic, tandemly repeated amino acid units, the sequences
of which differ from species to species (2, 3, 6, 22). Monoclonal antibodies to the repeated amino acid units neutralize
parasite infectivity (20, 21), suggesting that CS proteins
might be useful as sporozoite-stage vaccines. The recent
isolation of the CS protein genes from species of malaria
parasites which infect humans has made possible the production of the large amounts of antigen necessary for testing
its immunoprophylactic value (10, 17, 35). However, an
easily manipulated animal model system is required for
studying the mechanisms of protective immunity and the
role of the CS protein during the initial stage of parasite
[35S]methionine, and 32P-labeled deoxynucleoside triphosphates were from Amersham Corp., Arlington Heights, Ill.
Antibodies. Monoclonal antibodies 3D11 and 4G1 and goat
anti-mouse immunoglobulin were used as purified immunoglobulin G fractions. Precipitation of immune complexes (13)
was performed with protein A-Sephadex (Pharmacia Fine
Chemicals, Piscataway, N.J.). Antibodies were radiolabeled
with 125I by using lodogen (Pierce Chemical Co., Rockford,
Ill.) according to the instructions of the manufacturers.
Immunoassays. A two-site immunoradiometric assay
(IRMA) was performed as described in reference 34. Flexible microtiter plates (Becton Dickinson Labware, Oxnard,
Calif.) were coated with monoclonal antibody 3D11 to P.
berghei and then blocked with phosphate-buffered saline
containing 1% bovine serum albumin (PBS-BSA). In vitro
translation reactions or lysates (4) of Escherichia coli DH-5
harboring various plasmids were diluted in PBS-BSA and
incubated in 3D11-coated wells for 2 h at room temperature.
The wells were washed with PBS-BSA, incubated with
50,000 cpm of 1251I-labeled 3D11 (2 x 107 cpm/,ug) for 2 h at
room temperature, washed, and counted in a gamma
counter.
An inhibition assay (used in Figs. 2B and 3) was performed
by mixing 10 ,ul (50,000 cpm; 2 x 107 cpm/,ug) of 251I-labeled
3D11 in PBS-BSA with 90 ,ul of dilutions of lysates of E. coli
DH-5 harboring various plasmids or with 90 RI of dilutions of
synthetic peptides for 1 h at room temperature. A 45-,i
sample of each mixture was added to a well coated with
extracts of P. berghei sporozoites, incubated for 2 h at room
temperature, washed, and counted. A second type of inhibition assay (used in Fig. 7) was performed by mixing 3D11
at 50 ng/ml with dilutions of synthetic peptides in PBS-BSA
and incubating for 1 h at room temperature. Samples of each
mixture were incubated in wells coated with P. berghei
sporozoite extracts for 1 h at room temperature and washed.
The amount of 3D11 bound to each well was determined by
adding 100,000 cpm of 125I-labeled goat anti-mouse immunoglobulin per well, incubating for 1 h at room temperature,
washing, and counting.
Parasites. P. berghei (NK65) was maintained by passage
of the blood stage in hamsters. Sporozoites were obtained
from infected Anopheles stephensi mosquitoes 17 to 20 days
after the infective blood meal (31). Sporozoites from dis-
infection.
Here we describe the isolation of the CS protein gene of
the rodent parasite P. berghei, the complete nucleotide
sequence, and the identification of the epitope-encoding
region.
MATERIALS AND METHODS
Enzymes and isotopes. Restriction enzymes were from
New England BioLabs, Beverly, Mass.; Bethesda Research
Laboratories, Gaithersburg, Md.; and Boehringer Mannheim Biochemicals, Indianapolis, Ind. DNA polymerase and
reverse transcriptase were from New England BioLabs. 1251,
*
Corresponding author.
t Present address: Department of Molecular Biology and Genetics, The Johns Hopkins University, School of Medicine, Baltimore,
MD 21205.
3965
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was
cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a
monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the
CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a
central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions
encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central
repeated amino acid unit types contains the immunodominant epitopes.
366
MOL. CELL. BIOL.
EICHINGER ET AL.
2
3
4
5
7
6
x,
946743-
.
30-
It
0
sected salivary glands were used for RNA extraction, and
infected erythrocytes were used for DNA extraction.
RNA and cDNA methods. RNA was extracted from sporozoites by homogenization in guanidinium thiocyanate and
pelleting through a cesium chloride cushion (29). Fractionation of total RNA on a 10 to 40% sucrose gradient containing 1 M NaCl, 20 mM Tris hydrochloride (pH 7.4), and 10
mM EDTA with a CsCl cushion in a Beckman SW50.1 rotor
at 20°C for 18 h at 114,000 x g served to pellet most
translating RNA away from rRNA. A second 10 to 30%
sucrose gradient containing 20 mM Tris (pH 7.4) and 10 mM
EDTA and centrifuged in a Beckman SW50.1 rotor at 20°C
for 16 h at 114,000 x g fractionated the messengers by
relative size. RNA from each fraction was translated in a
wheat germ system (23), and the translation products were
assayed for CS protein in a two-site IRMA with monoclonal
antibody 3D11 (34).
Random primers generated from hamster liver DNA (15)
were used to prime for first-strand cDNA synthesis by using
reverse transcriptase. The second strand was synthesized by
methods described previously (9), and double-stranded
cDNA was tailed with dCTP (19), annealed to dG-tailed
pBR322 (Bethesda Research Laboratories), and used to
transform E. coli LE392 (11). Immunoscreening of cDNAcontaining colonies with 125I-labeled 3D11 was performed as
described previously (6).
DNA methods. DNA extracted from P. berghei-infected
hamster erythrocytes (8) was digested with HindIII, and the
fragments were fractionated on a 10 to 30% sucrose gradient
containing 20 mM Tris hydrochloride (pH 7.4), 10 mM
EDTA, and 1 M NaCl. DNA in the fraction most enriched
for sequences hybridizing to cDNA clone p872 was ligated to
preannealed HindIII arms of lambda L47.1 (14). Ligated
RESULTS
RNA extracted from sporozoites from dissected salivary
glands contained a messenger encoding a protein specifically
immunoprecipitated by 3D11 (Fig. 1, lane 2). This 53,000-Mr
protein migrated on SDS-polyacrylamide gel electrophoresis
(PAGE) slightly faster than the precursor form of P. berghei
CS protein (Fig. 1, lanes 7 and 8). The total RNA was
fractionated on two sucrose gradients of differing salt concentrations, first to remove rRNA and second to fractionate
the messengers by size. The fraction most enriched for CS
protein messenger was identified by assaying the translation
products in a two-site IRMA with 3D11 (data not shown).
To evaluate the complexity of this enriched RNA fraction,
we synthesized high-specific-activity cDNA and used it to
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
FIG. 1. In vitro translation of sporozoite RNA. The products of
in vitro translation of P. berghei sporozoite RNA (lane 3) were
immunoprecipitated with 4G1, a nonspecific monoclonal antibody
(lane 1), and with 3D11, a monoclonal antibody which reacts with
Pb44 (4) (lane 2), and fractionated by SDS-PAGE. 3D11 specifically
precipitates a protein of approximate 50,000 Mr. To compare this in
vitro product with the CS protein from sporozoites, we made a
Western blot. Nonidet P-40 extracts of P. berghei sporozoites (lanes
4 and 7), translation products of total P. berghei sporozoite RNA
(lanes 5 and 8), and translation products of globin mRNA (Bethesda
Research Laboratories) (lanes 6 and 9) were fractionated by SDSPAGE and transferred to nitrocellulose (28). The filter with lanes 4
to 6 was probed with 251I-labeled 4G1 and that with lanes 7 to 9 was
probed with 1251-labeled 3D11. The upper and lower bands in lane 7
are the CS protein 54,000-M, intracellular precusor and 44,000-M,
membrane form, respectively. Numbers on left show the Mr (x 103).
DNA was packaged by using Packagene (Promega Biotech,
Madison, Wis.), and the products were used to infect E. coli
LE392. The library was probed by hybridizing filters with
105 cpm of self-ligated, nick-translated insert of p872 per ml
(108 cpm/,ug) in 6x SSC (lx SSC is 0.15 M NaCl plus 0.015
M sodium citrate)-50 mM sodium phosphate-5x Denhardt
solution-100 ,ug of salmon sperm DNA per ml for 18 h at
55°C and washing in 0.2x SSC-0.1% sodium dodecyl sulfate
(SDS) for 3 h at 45°C. Enzyme digestion, gel fractionation,
and transfer of DNA to nitrocellulose followed standard
procedures (15). Hybridization of Southern blots of genomic
DNA was performed under identical conditions as the library screening, but with 106 cpm of nick-translated p872 per
ml (2 x 108 cpm/,ug).
Peptide synthesis. Peptides NG16 (NDPPPPNPNDPAPPQG), DD17(1) (DPPPPNPNDPPPPNPND), DD17(2)
(DPAPPNANDPAPPNAND), and PP15 (PQPQPQPQPQPQ
PQP) were synthesized by the stepwise solid-phase method
(18), using the gradative deprotection strategy on a pacyloxybenzhydrylamine copolystyrene-1% divinylbenzene
resin (27). Boc-aminoacyl-p-acyloxybenzhydrylamine resin
(0.4 mmol/g substitution), prepared as described previously
(27), was placed into the reaction vessel of a Beckman 990M
synthesizer, and a coupling protocol via dicyclohexylcarbodiimide was used to give an average efficiency of about
99.7% completion per step. The coupling yields were quantitated only at nonproline residues since proline did not give
good color yields by the quantitative ninhydrin test. Couplings at the N and Q residues were mediated by
dicyclohexylcarbodiimide-1-hydroxylbenzotriazole to reduce dehydration side reactions of amide side chains. Cleavage of the peptides from resin supports followed a simple
two-step procedure. The N-tert-butoxycarbonyl and the
side-chain benzyl groups were removed by treatment with a
solution of CF3SO3H-dimethylsulfide-CF3CO2H-p-cresol
(10:30:50:10, vol/vol) for 2 h, and the resulting resins were
thoroughly washed with CH2Cl2, dimethylformamide, and
ethanol-tetrahydrofuran (1:1, vol/vol). The desired peptides
were released into solution after treatment with a solution of
methylamine-ethanol-tetrahydrofuran (5:45:50, vol/vol) for
16 h. The peptides after purification from reverse-phase (C18)
chromatography were shown to be homogeneous by analytical high-pressure liquid chromatography and gave excellent
agreements with the expected amino acid ratio upon 6 N HCI
hydrolysis. Peptide DA18 (DGQPAGDRADGQPAGDRA),
composed of a dimer of the P. vivax CS protein repeat unit,
was synthesized as described previously (2).
Sequencing. DNA fragments subcloned into pUC8 and
pUC19 and into M13mplO and M13mpl9 were sequenced by
the methods described in reference 16 and 24, respectively.
P. BERGHEI CS PROTEIN GENE
VOL. 6, 1986
probe digests of P. berghei genomic DNA. A sinmple pattern
of four to five visible bands was generated with three
different enz3yme digests, indicating that the fraction was of
relatively lo%vcomplexity (data not shown). Random priming
for cDNA s)ynthesis was performed after several negative
screenings c f cDNA libraries constructed by standard
oligo(dT) priiming procedures. Of approximately 117,000
random-primled recombinant colonies, one (872) was found
to express a Iprotein recognized by 3D11. This colony gave a
strong signal in the in situ immunoassay, and cell extracts of
this clone itnhibited the binding of 3D11 to P. berghei
sporozoite ex(tracts (Fig. 2B). However, when these extracts
were assayedi for the presence of multiple identical epitopes
per molecule , only weak signals were obtained (Fig. 2A).
200 I
150
cpm
100
50
I
I
I
B
201
151
kcpm
10I
Nucleotide sequencing of the p872 insert revealed an open
reading frame of 69 nucleotides flanked by 21 G and 11 C
bases (Fig. 3). This sequence encoded two quasi-repeated
eight-amino acid units: NDPPPPNP followed by NDPAP
PQG. The amino acid composition of both is similar to that
of other CS protein repeats. A synthetic peptide, NG16,
composed of the two eight-amino-acid units (NDPPP
PNPNDPAPPQG), specifically inhibited the binding of 3D11
to P. berghei sporozoite extracts, showing significant inhibition at 10-8 M peptide concentration (Fig. 3).
When used to probe blots of digested P. berghei bloodstage DNA, p872 hybridized to genomic fragments yielding a
pattern consistent with a single-copy gene (Fig. 4). p872 did
not hybridize with mosquito, hamster, or Plasmodium
falciparum DNA (data not shown).
Genomic DNA digested with HindIII was fractionated on
a sucrose gradient, and a fraction enriched in the 7,000-basepair fragment hybridizing to p872 (Fig. 4) was used to
construct a library in lambda L47. 1. A total of 50,000 plaques
were screened with the p872 insert, and 12 positive clones
were identified. Figure 5 indicates the subcloning and sequencing strategy of one of these, lambda 9/5-9. The sequence of p9-54 close to the region hybridizing with the p872
insert revealed an open reading frame of 1,017 bases (Fig. 6).
This encoded a 37,159-dalton CS proteinlike product, with
an amino-terminal hydrophobic signal sequence, a charged
region preceding a central region with two types of tandemly
repeated amino acid units, a charged region containing two
pairs of cysteines, a hydrophobic anchor sequence, and a
carboxy-terminal asparagine residue.
The insert of cDNA clone p872, when aligned with the
genomic sequence, bridges the two repeat types, starting at
base 651 and ending within bases 719 to 721.
Peptides DD17(1) (DPPPPNPNDPPPPNPND) and
DD17(2) (DPAPPNANDPAPPNAND), composed of dimers
of defined repeat units from the first repeat region, both
bound to monoclonal antibody 3D11, but differed in their
ability to inhibit 3D11 binding to P. berghei sporozoites (Fig.
7). Peptide PP15 (PQPQPQPQPQPQPQP), derived from the
second repeat region, did not inhibit 3D11 binding to P.
berghei sporozoites (Fig. 7).
p9-5 hybridizes to Southern blots of digested blood-stage
DNA from Plasmodium yoeli yoeli, another rodent-infecting
species, producing a banding pattern similar to that obtained
with P. berghei (data not shown).
DISCUSSION
5
The cloning of the CS protein
7.2
s
s
s
|
3.6
1.8
.90
.45
genes
from human malaria
parasites has made possible the development of candidate
vaccines (2, 3, 17, 35). However, the lack of an easily
manipulated animal model system with which to study the
immune response to a CS protein and protective immunity to
progress in this
challenge in a susceptible host has hindered
106
direction.
bacteria
bacteria
FIG. 2. Imrmunoassay of bacterial extracts. (A) Two-site IRMA.
Wells were co ated with 3D11, blocked with BSA, incubated with
dilutions of exitracts of E. coli DH-5 harboring p872 (*) or pBR322
(O), washed, iincubated with 100,000 cpm of 125I-labeled 3D11 per
well, washed, and counted in a gamma counter. (B) Inhibition of
binding of anttibodies to sporozoites by bacterial extracts. 1251
E.
labeled 3D11 ('50,000 cpm) was added to dilutions of lysates of
lesaes oE.
to- dilutionshf
P.
which expresses
p872 (*), p277-19
coli DH-5 ha;rboring cpm)7was
falciparum CS
and incubated. Samples of each
CS epitopes ( A), or pBR322
mixture were added to a well coated with extracts of P. berghei
sporozoites, in cubated, washed, and counted in a gamma counter.
(O(G)
development
To
tal model,
we
cloned and characterized the
experimen-
gene
encoding
protein from the rodent parasite P. berghei.
The in vitro translation product of sporozoite RNA im-
the CS
munoprecipitated by 3D11 migrated in SDS-PAGE ahead of
the largest
precursor
form of the protein (Fig. 1), suggesting
that in vivo the protein undergoes
fication(s) which does not
CS
occur
a
posttranslational modi-
in the wheat germ system.
protein precursor forms display charge heterogeneity on
two-dimensional PAGE (25), but carbohydrate residues have
not been detected on the intracellular or membrane forms of
the protein (A. Cochrane and E. Nardin, personal communication).
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
A
3967
3968
EICHINGER ET AL.
MOL. CELL. BIOL.
p872
NG16
G,,AMTGACCCACCACCACCAMCCCAATGACCCAGCACCACCACMGGAATAACMTCCACAACCACAGC,,
N D P P P P N P N D P A P P Q G N N N P Q P Q
N D P P P P N P N D P A P P Q G
12
10
kcpm
8
4
2
0.002
0.02
uq%mI
0.2
2.0
20
peptide
FIG. 3. Nucleotide sequence of p872 insert and immunoassay of a derived peptide. The insert of p872 was sequenced (top line), and
peptide NG16, corresponding to the first 16 predicted amino acids, was synthesized and tested in an inhibition assay. Dilutions of peptide
NG16 (*) and peptide DA18 (P. vivax repeats [2]) (K) were incubated with 1251I-labeled 3D11, added to wells coated with P. berghei sporozoite
extracts, and incubated. Wells were washed and counted in a gamma counter.
The difficulties encountered in isolating a cDNA clone for
this CS gene stemmed from two sources. (i) CS protein
mRNA appears to lack a poly(A) tail. In comparison with the
rabbit globin mRNA included as an internal standard, the
retention of CS protein-translating RNA on oligo(dT)cellulose was low, and the eluted CS protein mRNA was not
retained more efficiently upon a second cycle of oligo(dT)cellulose fractionation (data not shown). (ii) Although DNA
complementary to the CS protein mRNA could be synthesized by random priming of a size-selected RNA fraction,
the overall cloning efficiency of the CS protein cDNA
sequences was extremely low. This is inferred from the
observation that radiolabeled, random-primed cDNA made
with the size-selected RNA fraction yielded a simple hybridization pattern on Southern blots of genomic DNA that
included the hybridization bands obtained when p872 was
used as a probe. Yet a library of over 105 clones yielded only
one positive. The possibility that the under-representation of
CS protein cDNA clones was due to the killing of bacteria
was ruled out when rescreening of the library with p872
insert failed to detect additional clones.
The insert of cDNA clone p872 appeared to encode more
than one epitope recognized by monoclonal 3D11, since a
low but reproducible signal was obtained in the two-site
IRMA (Fig. 2A). The sequence of the insert, however,
reveals no perfectly repeated unit of amino acids. The first 14
amino acids contain one 8-amino acid unit of NDPPPPNP
followed by NDPAPP, similar in sequence to positions one
through six of the preceding unit. Synthetic peptide NG16
(NDPPPPNPNDPAPPQG), representing the first 16 amino
acids of the p872 insert, specifically bound 3D11 and inhibited 3D11 binding to P. berghei sporozoite extracts (Fig. 3),
confirming that this sequence contains the CS epitope(s).
1
2 3.1-
2
3
*
9.46.54.3-
3
2.32.0-
.56-
FIG. 4. Southern blot of P. berghei blood-stage DNA probed
with p872. P. berghei genomic DNA was digested with HindIII (lane
1), Sau3A (lane 2), or RsaI (lane 3), fractionated in a 1% agarose gel,
blotted to nitrocellulose (26), and probed with nick-translated p872
as described in the text. Numbers on the side indicate the positions
of markers in kilobases.
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
6
P. BERGHEI CS PROTEIN GENE
VOL. 6, 1986
3969
1000
49
p9-54
T T
I
I.
1
I
Tf
I
pAPPNAND
"*lU
I
Po-I
*!
I
13
TI
I
2
°
T
3
1
<~~~I
FIG. 5. Subcloning and sequencing strategy of the genomic fragment isolated with cDNA clone p872. The XbaI-HindIII fragment from the
6,800-base-pair insert in lambda 9/5-9 (top line) hybridized with p872 was subcloned into pUC19 (to create p9-54, middle line) and further
mapped. The bottom line contains a restriction map and the sequencing strategy for the region of p9-54 to which p872 hybridized. AUG and
TAA indicate the boundaries of a 1,017-base open reading frame.
However, this synthetic peptide did not produce a positive
signal in the two-site IRMA, suggesting that the weak signal
obtained in the two-site IRMA with bacterial extracts was
due to aggregation of the p872-encoded protein product.
The blood-stage genomic fragment isolated with p872
contains an open reading frame encoding a typical CS
protein (Fig. 6). The amino-terminal 23 amino acids contain
a hydrophobic signal sequence, the exact cleavage site of
which is not known. Bases 312 to 353 encode a highly
charged area, analogous to region I of Dame et al. (3), which
ends with amino acids NKLKQP, part of a peptide used by
Vergara et al. (32) to elicit antibodies that cross-reacted with
P. berghei sporozoites and several simian and human malaria sporozoites.
The central part of the protein is composed of two regions
of tandemly repeated amino acid units. The largest region
contains 13 complete eight-amino acid units and one half
unit, which vary slightly in sequence owing to position 1
C-to-G changes in specific proline codons, yielding alanine
substitutions. The first four units are of uniform sequence,
the fifth and sixth each have one alanine substitution, the
seventh through the twelfth each have two substitutions, and
the last complete unit returns to the original form without
substitutions. In all cases the proline-to-alanine substitutions
(CCA to GCA) occur in the second or sixth residue or both
of the unit. This stepwise accumulation (or loss) of single
base changes suggests that variation is introduced gradually
throughout the repeated region, and in a directional manner.
It would be of interest to determine whether the repeats of
other antigenically related rodent CS proteins (such as those
of Plasmodium chabaudi and Plasmodium vinkei [30]) exhibit the same or a similar pattern of change.
The units are composed of the restricted group of amino
acids found in all CS repeats (2, 6, 7; M. Galinski, et al.,
manuscript in preparation). These two repeat units seem to
present slightly different epitopes to monoclonal antibody
3D11, since the PPPPNPND unit peptide was 30 to 50 times
better at inhibiting 3D11 binding to P. berghei sporozoites
than was the PAPPNAND unit peptide (Fig. 7). Two other
aspects of this repeated region are noteworthy. First, one of
the proline-to-alanine changes produces the sequence
PNAN within eight of the units. This sequence constitutes
the tandem repeats of the CS protein in P. falciparum (3, 7).
The reported cross-reactivity (12) displayed by monoclonal
antibodies that react with P. falciparum and P. berghei
sporozoites may be due to these PNAN units.
Second, the position 1 changes in the proline codons are
the only base substitutions in this region. There are no
position 3 changes for the proline, alanine, and aspartic acid
codons inside this repeat region, while outside the repeats
three of the four codons for proline and alanine and both
codons for aspartic acid are used. Both codons for asparagine are used in the repeats, but the relative position of the
AAC and AAT codons in each eight-codon unit is constant.
Selection at the protein level for retention of these epitopes
cannot account for this degree of restricted codon usage and
suggests, as mentioned in previous papers (5, 7a), that a
mechanism operates which maintains the repeated DNA
sequences in this region of the CS gene. In P. berghei this
mechanism would appear to tolerate only small divergences
from the basic DNA repeat unit, and those changes that do
occur are position 1 substitutions which yield productive
changes at the protein level. The repeats of all CS protein
genes display various levels of base changes, which may
indicate that this mechanism works with different efficiencies
in different species.
Following this first region of repeats is a second, seemingly unrelated region, composed predominantly of alternat-
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
I
I
3970
M OL. CELL. BIOL.
EICHINGER ET AL.
*50
TG ATA ACC CTC ACA TTT GAC AAT CCT TAT AAA AGG AGT TMMAAA AAA ACA TTA ACA AAA MC
* 100
MA ATC TAT ATA TAC ACG CAT ATA TTT AM ATG MG MAG TGT ACC ATT TTA GTT GTA GCG
M K K C T I L V V A
*150
TCA CTT TTA TTA GTT MAT TCT CTA CTT CCA GGA TAT GGA CAM MAT AAA ATC ATC CM GCC
S L L L V N S L L P G Y G Q N K I I Q A
*200
CAM AGG MAC TTA MAC GAG CTA TGT TAC MAT GAM GGA MAT GAT MAT AAA TTG TAT CAC GTG
Q R N L N E L C Y N E G N D N K L Y H V
*250
CTT MC TCT MAG AAT GGA AM ATA TAC MAT CGA MT ACA GTC MAC AGA TTA TTG CCG ATG
L N S K N G K I Y N R N T V N R L L P M
*550
CCA CCA AAC GCA MT GAC CCA GCA
P P N A N D I P A
MAT GAC CCA GCA CCA CCA MC GCA
N D IP A P P N A
CCA CCA AAC CCA AAT GAC CCA GCA
P P N P N D P A
*350
CGT MT MT AAA TTG AAA
R N N K L K
MAC CCA MAT GAC CCA CCA
N P N D P
CCA CCA CCA CCA MC GCA
P P P P N A
MC GCA MT GAC CCA GCA
N A N D P A
CCA CCA MAC GCA MT GAC CCA GCA CCA CCA AAC GCA
P P N A N D P A P P N A
*650
MAT GAC CCA GCA CCA CCA MAC GCA MT GAC CCA CCA
N D IP A P P N A N D I PP
*700
CCA CCA CAA GGA MT MC MT CCA CAM CCA CAG CCA
P P Q G N N N P Q P Q P
*750
CAG CCA CAA CCA CAG CCA CAG CCA CAM CCA CAG CCA
Q P Q P Q P Q P Q P Q P
CGG CCG CAG CCA CAA CCA CAG CCA
R P Q P Q P Q P
*800
CGA CCA CAG CCA CAA CCA CAG CCA GGT GGT MAT MAC MAT MAC AAA MT MT MAT AAT GAC
R P Q P Q P 0 = G G N N N N K N N N N D
*850
GAT TCT TAC ATC CCA AGC GCA GAM AAA ATA CTA GAM TTC GTT AAA CAG ATC AGG GAT AGT
D S Y I P S A E K I L E F V K Q I R D S
*950
ATC ACA GAG GAA TGG TCT CAA TGT MAC GTA ACA TGT GGT TCT GGT ATA AGA GTT AGA AAM
I T E E W S Q C N V T C G S G I R V R K
*1000
CGA AAA GGT TCA MAT MAG AAA GCA GAA GAT TTG ACC TTA GAM GAT ATT GAT ACT GM ATT
R K G S N K K A E D L T L E D I D T E I
*1050
TGT AAA ATG GAT AAA TGT TCA AGT ATA TTT MAT ATT GTA AGC AAT TCA TTA GGA TTT GTA
S S I F N I V S N S L G F V
*1100
ATA TTA TTA GTA TTA GTA TTC TTT MAT TAM ATA MAC
I L L V L V F F N FIG. 6. Nucleotide sequence of the P. berghei CS gene. An open reading frame in the region of p9-54 to which p872 hybridized encodes
a product with the typical features of other CS proteins, such as: an amino-terminal signal sequence (here encoded by bases 93 to 161), a
charged region ending with KLKQP (here encoded by bases 306 to 368), a central region of tandemly repeated amino acid units (eight-amino
acid units are in short boxes and another region of repeated PQ pairs is in one long box), followed by a carboxy-terminal region with two pairs
of cysteines, and a hydrophobic anchor sequence with a terminal asparagine residue (here encoded by bases 1014 to 1109).
C
K
M
D
K
C
ing proline-glutamine pairs, with two arginine substitutions
for glutamine via position 2 A-to-G changes. The proline
codon usage appears to be restricted, since 16 of 17 codons
for this amino acid are CCA, while the usage for glutamine
and arginine is not restricted and follows no obvious pattern.
Because of its smaller size, and since 3D11 and three other
anti-P. berghei monoclonal antibodies do not bind to a
peptide derived from this repeat region (Fig. 7 and data not
shown), it seems unlikely that this PQ repeat region contains
epitopes of high immunogenicity. Whether this region is
naturally immunogenic is being tested. CS proteins with two
sets of repeats have also been found in some strains of
Plasmodium cynomolgi (M. Galinski et al., in preparation),
but, as with P. berghei, the role of these extra repeats is
unknown.
Except for the potential co- and posttranslational processing sites, the KLKQP sequence just amino terminal to the
first repeat region, and the PNAN units within this repeat
region, there is no extensive conservation of amino acids
between P. berghei and P. knowlesi (22), P. cynomolgi (6;
M. Galinski et al., in preparation), P, falciparum (3, 7), or P.
vivax (2) in the amino-terminal and repeat regions of the CS
proteins. In contrast, the carboxy-terminal region shows
extensive conservation of amino acids among all five species, especially through the last 70 to 77 residues of each CS
protein. By introducing no more than one gap per compari-
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
CTC CGA AGA AAM AAA MT GAG AAA AAA MC GAM AAA ATA GAG
L R R K K N E K K N E K I E
*400
CAG CCA CCA CCA CCA CCA MC CCA MT GAC CCA CCA CCA CCA
Q PI P P P P N P N D
=P P P
*450
CCA CCA AAC CCA AAT GAC CCA CCA CCA CCA MAC CCA MT GAC
P P N P N D P P P P N P N D
*500
MAT GAC CCA CCA CCA CCA MC GCA AAT GAC CCA GCA CCA CCA
N D P P P P N A N D P A P P
P. BERGHEI CS PROTEIN GENE
VOL. 6, 1986
son, P. berghei shows amino acid conservation at 47 of the
last 74 positions (63%) for P. knowlesi, 44 of 74 (59%) for P.
cynomolgi, 41 of 77 (53%) for P. falciparum, and 43 of 70
(61%) for P. vivax. The conserved portion of the protein
extends from the anchorlike sequence through the so-called
region II of Dame et al. (3), a 13-amino acid sequence
conserved in all CS proteins (2, 3; M. Galinski et al., in
preparation). Also within this conserved portion are four
cysteines whose positions relative to the carboxy terminus
are maintained in all CS proteins. The entire carboxyterminal region of P. berghei has 49 exact matches with the
terminal 97 amino acids of P. falciparum (with three gaps
inperteti in the
3971
ACKNOWLEDGMENTS
This work received financial support from the Agency for International Development (DPE0453-A-00-5012-00), the National Institutes of Health (Public Health Service grant 5P01-A121642), the
McArthur Foundation, a National Institutes of Health Predoctoral
Training grant (CA-09161) to D.J.E., and an Irma T. Hirschl Career
Development Award to V.E. This investigation also received financial support from the UNDP/World Bank/WHO Special Programme
for Research and Training in Tropical Diseases.
We thank Fidel Zavala for antibodies and sporozoite-coated
plates, Arturo Ferreira for antibody 4G1, and Lynda Caiati, Lauri
Weinstein, and David Keeney for excellent technical assistance.
fnlrinnr,im eniioence)t
P
fmtannot
dhseftie
LITERATURE CITED
1. Aikawa,
M., N. Yoshida, R. Nussenzweig,
and V. Nussenzweig.
rodent
1981.
(Plasmodiumprotective
berghei) antigen
is a differentiation antigen. J.sporozoites
Immunol.
I
I
sol.
ow.
i.o
I
|
10
100
ug/ml poptide
FIG. 7. Inhibiition of binding of antibody to sporozoites by synthetic peptides. IVlonoclonal antibody 3D11 was mixed with dilutions
of synthetic pept ides and incubated. Samples of each mixture were
added to P. be?rghei sporozoite-coated wells, incubated, and
washed. The am()unt of 3D11 bound to each well was detected with
I'll-labeled goat anti-mouse immunoglobulin. Symbols: A, peptide
NG16 (NDPPPIPNPNDPAPPQG); A, peptide DD17(1) (DPPP
PNPNDPPPPNPIND); O, peptide DD17(2) (DPAPPNANDPAP
PNAND); *, peiptide PP15 (PQPQPQPQPQPQPQP).
malarial
126:2494-2495.
2. Arnot, D. E., J. W. Barnwell, J. P. Tam, V. Nussenzweig, R.
Nussenzweig, and V. Enea. 1985. Circumsporozoite protein of
Plasmodium vivax; gene cloning and characterization of the
immunodominant epitope. Science 230:815-818.
3. Dame, J. B., J. L. Williams, T. F. McCutchan, J. L. Weber,
R. A. Wirtz, W. T. Hochmeyer, G. S. Sanders, E. P. Reddy, W.
L. Maloy, J. D. Haynes, I. Schneider, D. Roberts, C. L. Diggs,
gene encoding the
and L. H. Miller. 1984. Structure of the
surface antigen on the sporozoite of the
immunodominant
human malaria parasite Plasmodium falciparum. Science
225:593-599.
4. Ellis, J., L. S. Ozaki, R. W. Gwadz, A. H. Cochrane, V.
Nussenzweig, R. S. Nussenzweig, and G. N. Godson. 1983.
Cloning and expression in E. coli of the malarial sporozoite
surface antigen gene from Plasmodium knowlesi. Nature (London) 302:536-538.
5. Enea, V. 1985. The circumsporozoite antigen: the gene and the
antigen. Ann. Ist. Super. Sanita 21:291-298.
6. Enea, V., D. E. Arnot, E. Schmidt, A. H. Cochrane, R. W.
Gwadz, and R. S. Nussenzweig. 1984. Circumsporozoite gene of
Plasmodium cynomolgi (Gombak): cDNA cloning and expression of the repetitive circumsporozoite epitope. Proc. Natl.
Acad. Sci. USA 81:7520-7524.
7. Enea, V., J. Ellis, F. Zavala, D. E. Arnot, A. Asanavich, A.
__Masuda,
cpm
of
The
I.
Quakyi, and R. Nussenzweig. 1984. DNA cloning of
Plasmodium falciparum circumsporozoite gene: amino acid
sequence of repetitive epitope. Science 225:628-629.
7a.Enea, V., M. Galinski, E. Schmidt, R. Gwadz, and R. S.
Nussenzweig. 1986. Evolutionary profile of the circumsporozoite
gene of the Plasmodium cynomolgy complex. J. Mol. Biol.
188:721-726.
8. Ferreira, A., V. Enea, T. Morimoto, and V. Nussenzweig. 1986.
Malaria sporozoite infectivity measured with a DNA probe.
Mol. Biochem. Parasit. 19:103-109.
9. Gubler, U., and B. J. Hoffman. 1983. A simple and very efficient
method for generating cDNA libraries. Gene 25:263-269.
10. Gysin, J., J. Barnwell, D. H. Schlesinger, V. Nussenzweig, and R.
Nussenzweig. 1984. Neutralization of the infectivity of sporozoites of Plasmodium knowlesi by antibodies to a synthetic
peptide. J. Exp. Med. 160:935-940.
11. Hanahan, D. 1983. Studies on transformation of Escherichia coli
with plasmids. J. Mol. Biol. 166:557-580.
12. Hockmeyer, W. T., and J. B. Dame. 1985. Recent efforts in the
development of a sporozoite vaccine against human malaria, p.
233-245. In M. Z. Atassi (ed.), Proceedings of the Third
International Symposium on Immunobiology of Proteins and
Peptides. Plenum Publishing Corp., New York.
13. Kessler, S. W. 1975. Rapid isolation of antigens from cells with
staphylococcal protein A antibody absorbant: parameters of the
interaction of antibody complexes with protein A. J. Immunol.
115:1617-1624.
14. Loenen, W. A., and W. J. Brammar. 1980. A lambda vector for
cloning large DNA fragments made with several restriction
enzymes. Gene 10:249-259.
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
On the basis of amino acid composition and sequence
ie CS protein can be divided into three parts.
conservation, tihe
different amino-terminal sequences may
Apparently mainy dfeent
firstermin, provides may
fulfill the requiirements of this first region, provided they
begin with a sig;nal-like sequence and end with KLKQP. The
primary constrnaint on the second region, the repeats, is the
tandem repetitiion of a limited group of amino acids, the
particular unit sequence of which can vary. The third, or
carboxy-terminial, region is most restricted in actual amino
acid sequence, suggesting specific structural requirements
for the function of this region. Although it would appear that
each of these three regions is under different selective
as to the
make definitive statements asto
pressure, one ccannot make
regions with suchthea
relative rates oiIf mutation of these threesements
small number cAf cloned sequences to compare and without
an accurate e:stimate of the time of divergence of the
different specie:s of parasites.
The sequenc(e of the P. berghei CS protein should provide
the foundation for a mouse-rat model system with which to
study and optinnize the immune response to sporozoites, and
the cloned genme should allow for the isolation and comparison of the CS ;protein genes from other rodent parasite
species.
3972
EICHINGER ET AL.
216:160-162.
22. Ozaki, L. S., P. Svec, R. S. Nussenzweig, V. Nussenzweig, and
G. N. Godson. 1983. Structure of the Plasmodium knowlesi gene
coding for the circumsporozoite protein. Cell 34:815-822.
23. Roberts, B. E., and B. M. Paterson. 1973. Efficient translation of
tobacco mosaic virus RNA and rabbit globin 9S RNA in a
cell-free system from commercial wheat germ. Proc. Natl.
Acad. Sci. USA 70:2330-2334.
24. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci.
USA 74:5463-5467.
25. Santoro, F., A. H. Cochrane, V. Nussenzweig, E. H. Nardin,
R. S. Nussenzweig, R. W. Gwadz, and A. Ferreira. 1983.
Structural similarities among the protective antigens of sporozoites from different species of malaria parasites. J. Biol. Chem.
258:3341-3345.
26. Southern, E. 1975. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol.
98:503-517.
27. Tam, J. P. 1985. A gradative deprotection strategy for the
solid-phase synthesis of peptide amides using p-acyloxybenzhydrylamine resin and the sN2 deprotection method. J. Org.
Chem. 50:5291-5298.
28. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl. Acad. Sci.
USA 76:4350-4354.
29. Ulrich, A., J. Shine, J. Chirgwin, R. Pietet, E. Tischer, W. J.
Rutter, and H. M. Goodman. 1977. Rat insulin genes: construction of plasmids containing the coding sequences. Science
196:1313-1319.
30. Vanderberg, J., R. Nussenzweig, and H. Most. 1969. Protective
immunity produced by the injection of X-irradiated sporozoites
of Plasmodium berghei. V. In vitro effects of immune serum on
sporozoites. Mil. Med. 134:1183-1190.
31. Vanderberg, J. P., R. S. Nussenzweig, and H. Most. 1968.
Further studies on the Plasmodium berghei-Anopheles
stephensi rodent system of mammalian malaria. J. Parasitol.
54:1009-1016.
32. Vergara, U., A. Ruiz, A. Ferreira, R. Nussenzweig, and V.
Nussenzweig. 1985. Conserved group specific epitopes of the
circumsporozoite proteins revealed by antibodies to synthetic
peptides. J. Immunol. 134:3445-3448.
33. Yoshida, N., P. Potocjnak, V. Nussenzweig, and R. S. Nussenzweig. 1981. Biosynthesis of Pb44, the protective antigen of
sporozoites of Plasmodium berghei. J. Exp. Med. 154:
1225-1236.
34. Zavala, F., A. H. Cochrane, E. H. Nardin, R. S. Nussenzweig,
and V. Nussenzweig. 1983. Circumsporozoite protein of malaria
parasites contains a single immunodominant region with two or
more identical epitopes. J. Exp. Med. 157:1947-1957.
35. Zavala, F., J. P. Tam, M. R. Hollingdale, A. H. Cochrane, I.
Quakyi, R. S. Nussenzweig, and V. Nussenzweig. 1985. Rationale
for the development of a synthetic vaccine against Plasmodium
falciparum malaria. Science 228:1436-1440.
Downloaded from http://mcb.asm.org/ on June 4, 2013 by guest
15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
cloning, a laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
16. Maxam, A. M., and W. Gilbert. 1979. A new method for
sequencing DNA. Proc. Natl. Acad. Sci. USA 74:560-564.
17. Mazier, D., S. Mellouk, R. L. Beaudiou, B. Texier, P. Druilhe,
W. Hockmeyer, J. Trosper, C. Paul, Y. Charoenvit, J. Young, F.
Miltgen, L. Chedid, J. P. Chigot, B. Galley, 0. Brandicourt, and
M. Gentilini. 1986. Effect of antibodies to recombinant and
synthetic peptides on P. falciparum sporozoites in vitro. Science 231:156-159.
18. Merrifield, R. B. 1963. Solid phase peptide synthesis. I. The
synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-2154.
19. Nelson, T., and D. Brutlag. 1979. Addition of homoplymers to
the 3'-ends of duplex DNA with terminal transferase. Methods
Enzymol. 68:41-50.
20. Nussenzweig, R., J. Vanderberg, and H. Most. 1969. Protective
immunity produced by the injection of X-irradiated sporozoites
of Plasmodium berghei. IV. Dose response, specificity and
humoral immunity. Mil. Med. 134(Suppl.):1176-1182.
21. Nussenzweig, R., J. Vanderburg, H. Most, and C. Orton. 1967.
Protective immunity produced by the injection of X-irradiated
sporozoites of Plasmodium berghei. Nature (London)
MOL. CELL. BIOL.