.toA'cular and Biochem'ical Para~itolog i. 53 (192) 45-52
C~ 1992 Elsc~icr Sicince Publishers B.V. All rights reserved
SEPI 5 19
NIOL RIO 0 1742
Characterization of the gene encoding sporozoite surface protein 2,
a protective Plasmiodiwn )'oelii sporozoite antigen
William 0. Rogers, Miriam D. Rogers, Richard C. 1Iedstrom* and Stephen L. Hoffman
,tafaria Prograni. Naral Mrdkal Research In~titiiue. Bethe.sda. YtD, USA
(Received 30 December 1991; accepted 6 February 1992)
Sporozoitc surface protein 2 (SSP2) is a 140-kDa, protective sporozoite surfaec protein from Plasynodium Yoelil distinct
from the circumsporomoite protein (CSP). A gcnomic clone containing the SSP2 gene was isolated and sequenced to determine
its size. structural organization and deduced primary amino acid sequence. The coding sequence consists orfa single, long open
reading frame encoding 826 amino acids. The overall structure or SSP2 is similar to that of the CSP. consisting or a central
region of immunogenic amino acid repeats flanked by non-repetitive sequence. SSP2 has one copy of a thrombospondin
repcat motif in common Avith '%c~cral cell adhesion molecules as Aceli as with the CSP and the thrombospondin related
anon~mous protein (TRAP) of P.foiiiparurr. Additionally. SSP2 shares substantial sequence similarity to TRAP. suggesting
that TRAP is the analogue of ssP2 in P. fulciparum.
Key -Aords: %falaria. Pla-iniodiumn: Sporozoite: Antigen
to the CSP [2-6). Monoclonal antibodies
(mAbs) [7,8,10] and cytotoxic T cells 
directed against the CSP are protective in
passive transfer. Nonetheless, it has not been
possible to induce active immunity with
recombinant or synthetic vaccines based on
the CSP alone [8,11-17] comparable to that
achieved by immunization with irradiated
sporozoites. We have therefore attempted to
idc.-,tify additional sporozoite surface antigens
which might be combined with the CSP in a
multicomponent vaccine. We recently descri-
bed a new sporozoite surface antigen, sporo-
zoite surface protein
Plasmnodison yoelii [5,18). Monoclonal antibo-
dies directed against SSP2 recognize a 140-kDa
protein in sporozoite extracts. Sequence ana-
lysis of a 1.5-kb genomic DNA fragment
encoding part of SSP2 revealed an immuno-
genie series of repeating amino acids and a
region of similarity to the region 11 domain or
the CSP I I]. Mice immunized with P8 15 mouse
mastocytoma transrectants expressing the
partial SSP2 sequence and the CSP were
Efforts to develop a pre-erythrocytic stage
malaria vaccine have focused almost entirely
on the circurnsporozoite protein (CSP)[lj. The
CSP is the predominant protein on the surface
of the infective malaria sporozoite. It is well
known that immunization of humans or
animals with radiation attenuated sporozoites
induces solid sterile immunity to malaria and
both humoral and cellular immune responses
gram Naval Medical Research Institute Bethesda, MD 20999-
5055. USA. Tel.! (301) 295-1776; Fax: (301) 295-6171.
. Pesnt ddess
U S.Naal edcalReearh
William 0. Rogers. Malaria Pro-
Note: Nucleotide sequence data reported in this paper have
been submitted to the Gcn~ankTrm data base with the accession
numbers M84732 and M84733.
Abbreviations: CSP. circumsporozoite protein. TRAP. throm-
botpondi n.related anonymous protein: mAb. monoclonal
92 9 14 021
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Characterization of the gene encoding sporozoite surface protein 2, a protective Plasmodium yoelii sporozoite
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Molecular and Biochemical Parasitology 1992 Vol.
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Malaria; Plasmodium; Sporozoite; Antigen
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protected against challenge with infective
,porozoites . We report here the character-
i/ation of a genomic clone containing the
complete SSP2 gene.
deletions "ere prepared using exonucleaselII
122] and the complete sequence determined by
the Sanger dideoy method 1231.
Fig. I shows the sequence of the 4.7-kb
insert of /PySSP2.10.
reading frame is present and includes the
previously described sequence of 2gMSY4
. The AT content of the coding and
noncoding regions, 63.2% and 80.7% respec-
tively, are similar to those found in other
Plasmodium genes . The sequence encodes a
polypeptide containing 826 amino acids with a
calculated molecular weight of 91 300. Several
possibilities may account for the discrepancy
between this calculated molecular weight and
the observed molecular weight, 140000 [5,18].
First, the gene might contain additional exons.
However, no additional long open reading
frames were found either 700 bp 5' to the
initiation codon or 1500 bp 3' to the first in
frame stop codon. No Plasmodiun consensus
intron boundary sequences  were found in
the flanking regions. A large intron could
extend beyond the region we have sequenced,
but previously described Plasniodium introns
have been less than 600 bp long. Second, SSP2
may be a glycoprotein. and indeed, there are
several consensus N-glycosylation sites in the
sequence (Fig. I). Finally, the protein may
migrate anomalously in SDS-PAGE gels,
perhaps as a result of its very high proline
The deduced amino acid sequence of SSP2 is
shown in Fig. I and a map of the sequence in
Fig. 2. Like a number of other Plasmodium
surface antigens , the deduced amino acid
sequence contains tandem repeats of simple
amino acid repeats and is particularly rich in
proline (18.0%) and asparagine (21.2%). The
general structure of SSP2 is similar to that of
the CSP (Fig. 2). There is a central region of
short, repeated peptide sequences flanked on
both sides by non-repetitive sequence. Hydro-
phobicity analysis  identified a putative N-
terminal hydrophobic leader  as well as
putative transmembrane and cytoplasmic do-
mains  near the carboxy terminus (Figs. I
and 2). It is interesting, however, that while
SSP2 bli both a transmembrane domain and a
A single long open
.Materials and Methods
Parasites and DNA isolation. P. y'oelii 17X
(NL) parasites were maintained and DNA
isolation performed as described previously
Genomic library construciion and screening. A
P. yoelii genomic library was constructed using
2.0-7.0-kb fragments generated by partial
DNAse I digestion as previously described
. Purified insert DNA from ;.gMSY4 was
nick translated and used to screen the library
under standard high stringency conditions .
Five positive clones were isolated, and one,
/.PySSP2.10 was found to contain a 4.7-kb
insert which overlapped both ends of,;.gMSY4.
D..4 sequencing. Phage DNA of *PySSP2.10
%%as prepared from liquid lysates by standard
methods. Insert DNA was released by EcoRl
digestion and the inserts were cloned into
M13mpl8 and pUCI8. Overlapping clones
spanning the insert were generated in pUCI8
and M13 using exonuclease Ill digestion .
Single and double stranded templates were
sequenced using the dideoxy method  and
Sequenase (U.S. Biochemical Corp., Cleve-
land, 01). Sequence analysis was carried out
using Genepro 4.2 and DNASIS software.
A ).gtil DNase I genomic library was
screened with the 1.5-kb fragment of the
SSP2 gene contained in AgMSY4. Five posi-
tive clones were obtained. One, APySSP2.10,
contained a 4.7-kb insert which included
within it the complete sequence of the
2gMSY4 sequence. The 4.7-kb insert was
subcloned into pUCI8 and Ml3mpl8. Nested
CAT TAAACC Al TAMAAAA.7,AAAT7TTATA)A~TITT1TT7AATTI TCTTTATAT'AT A0ATATATATATAC AT TTATATATACTCTTGTIC1 11T TTATCCATTA)AAAA- A ATNAT
ATC CAT A 'AT T.AT TTI T-MA'ArT7A.AAA~A"ATA'AA ATGACCCCTTGTCTT GAAGCAACAT IflT-7A TA1TTAACTGdT TCATCTTflT TTAC AT ATATTTGTTCACAl
AAA.AC ATI AAT I ItCTC 0ýACALC TAT GATAATTAJAC TATATATCAGTITA TAT I'A~C-00GAACACACTCTC TC C TAT ATATATATAATTGCAAACC TGTACACATT11TA
-- - - -- - - - lydrophobic leader D.e
L L G N 5
K Y I
O C*-- -- -- -- --
C I S V F 1. N
E T L D E
F V V L
K Y S Z E V C T E 0
1 a I
H V I
L V D N
AATGTATC TI TGACACT TTTT-TCAACAAATTCACCI CAArTAATTAAACT
T L F S T N
Y G 3 T S K
N T X
V V 0
A I I
GATTrTACC TACATCTAC TGCG(.flCTGCATCAATThAAAACGAAACATGTKAATCTAGCAAT7tATAGGTGrTCCTGCACGTCTTAATAACCAkATATAATAGAATTTTAGTTGGATGTCA7
V V 14 0
L K at R
V N N
AGATAC GC ACCAT Gr CCAT A-7AC TCTTCTGGTAGITCGCAATGAAGCCCAkMTATCATAAACC TTT TC TTACTAAAGTTT7CT CAGGAAGI ACAAACAATTGcTCA77TT ccA~AATGG
A F C
Y Y S S
I -- thrmebompondin
Z It I A
1 ----------------3-maer repeats -------------------
A D C P
S N P
X P N
N P N N P
- - -
N N N
M N P N
PSN PH N
N P N KP
K Pp Nr
K P S I
N K P
K P S
P K K P S4951
- - -------------------
P KN K S
z P K K P
N e P K
cATMCCAAA CCAT AAACCCAAATC.CATATCAATACCAAcATAAC CATCAAACCCAcAA
P I N
K N P L
P K C
N P v
I I 81
P K 9
5 K V K T
P K P
Z 9 K S K K 8
I P 5 P
F X 9 P
I --------------------- 1-----trepats~mra
C L A I I P
S A A 6
A K P A P N
Fig. 1. Sequence of the 4.7-kb insert of APySSP2.lO. The inrerred amino acid sequence of SSP2 is shown below the open
rading rrame. T1c location of the conserved thrombospondin motif. of the repeated peptide motifs, and or the putative
hydrophobic leader and transmcmbrane domain is indicated. Potential N-glycosylation sites are underlined.
cytoplasmic domain, the CSP has only a
membrane anchor, suggesting that these 2
proteins interact differently with the memn-
The arrangement or short amino acid repeats
in SSP2 is complex. There is first a region of 23
perfect repeats of the tripeptide PNN, followed
by 3 degenerate copir"s, PND PSN PNN. There
PR! epa (QGPGAP) (QQf'P)
Resew I Reg$= 13
Fig. 2. Structure of SSP2 and CSP. Shown are schematic diagrams or the sequence of SSP2 (A) and the P. yodifi CSP (B) 133).
The diagrams are drawn approximately to scale.
is then a short segment of non-repetitive
sequence, followed by 3 tandem copies of the
pentamer PNKP(K/N), 4 copies of PNKPN
alternating with 4 copies of PNEPSN, and 18
tandem degenerate copies of PNEPSN. These
tandem repeats are followed by a short region
SSP2 MXLLGNSKYIFVVLLLCISVFL-NGQE---- TLDEIKYSEEVCTEQIDIIIILLDGSGSIG
tINHLGNV1(YLVlIVFLI FFDLFLVNGk1DVONN IVDEIKYSEEVCNDQVDLYLIAMCSGS IR
RHiNWVNHAVPLA1-I(XQLNLNDNA1 NLYNVFSNNAKE I I RLHSDASKNKEKALI I IRS
LONNYS PNGNTN LTSAL LVVDT L INERllYRPDA ZOLA Z I LTDG I PDLPRS TAVVM OLXR
LLSTNLPYGRTNLTDALLQVRKHLNtFlRINRENPJ4QLVVI LTDGIPDS ZODS LKESRKLSD
TRAP RGVKIAVFGIGQG INVAFNRFLVGCHPSDGKCNLYADSAWENVIKNVIGPFKK'AVCVEVEK
SNNGYhIAGGI IGGLA L4GCAGVGYNF IAGSSAAGLAGAEPAPFEDVIPDDDKDIVNEQ
Fig. 3. Alignment of the N-erminal (A) an'd C-terminal (B) regions of P. yo'effi SSP2 and P. fakiparumn TRAP. :'indicates
identical amino acids, '. indicates conservative substitutions. The thrombospondin repeat motif is underlined.
in which short repeat motifs, PEE and PSN,
are interspersed "i1h non-repetitive sequence.
FInally, there is a tandem duplication of the
I I-mer, PI-I-.SNPKI- PIN. All of the repeat
sequences in SSP2 are clearly distinct from the
repeats in the P. yoe/ii CSP, QGPGAP and
QQPP. The only common feature which the
SSP2 repeats share with the CSP repeats is the
general structure PXXPXX, which might be
expected to impart to the repeat domains of
both proteins a structure rich in ,l-bends [28).
SSP2 shares sequence motifs with several
plasmodial proteins and molecules involved in
cell adhesion. Thrombospondin,
region !1, properdin, the terminal complement
components and the thrombospondin related
anonymous protein share similarities based
[29,30]. This sequence is found in 3 copies in
thrombospondin, 6 copies in properdin and
one copy in all CS proteins sequenced to date.
A similar sequence, underlined in Fig. I, is also
found in SSP2. In SSP2 this thrombospondin
motif is found amino terminal to the central
repeat region, while the analogous sequence in
the CSP is found in Region 11, carboxy
terminal to the repeats.
The N-terminal and C-terminal regions of
SSP2 bear a remarkable similarity to TRAP
which extends well beyond the similarity to the
thrombospondin motif. Fig. 3A shows an
alignment of the N-terminal regions of SSP2
and TRAP. Over a region of 281 amino acids,
there is 43% similarity at the amino acid level,
Ten of II cysteine residues are conserved, the
only exception being a single cysteine in the
putative hydrophobic leader of SSP2. Fig. 3B
show the alignment of the C-terminal regions
of the 2 proteins. Over a region of 71 amino
acids, there is 56% identity at the amino acid
level. SSP2 and TRAP may be members of a
protein family involved in interaction between
the sporozoite and erythrocytic stages of
Plasmodium and the cells of the host.
zoite surface antigen from P. yovlii 1181. We
have recently observed that immunization of
mice wkith a combination of P815 mouse
CSP and the original
SSP2 [181 are protected against challenge with
infective P. .oclii sporo/oites . SSP2 and its
presumed homologs in the human PhIsmodiun
species are therefore important vaccine candi-
dates. We have here reported the complete
sequence of the P. yoelii SSP2 gene.
The deduced amino acid sequence of SSP2
shares a number of characteristics with other
Plasnodium surface antigens in general and
with the CS protein in particular. First, it is
by a central repeat region
consisting of tandem repeats of several differ-
ent short peptide sequences. As in the case of
the CSP repeats from many Plasmodium
species, the amino acids used in the SSP2
repeats are chosen from a restricted set of
amino acids (P,N,E,Q,G,D,A,R,V for CSP
repeats and P,N,E,K,S,I for the SSP2 re-
peats). As in the P. yoelii CSP, there are
several different repeat units in the repeat
region, however, the organization of the
repeats in SSP2 is somewhat more complex
than that in the CSP. There are 2 major repeat
regions, one consisting of tandem repeats of
the tripeptide PNN, the second consisting of 2
basic repeat units, PNKPN and PNEPSN. The
units are intercalated in the general structure
AAAABABABABBBB, where A = PNKPN
and B = PNEPSN. This organization could
have arisen from an ancestral I I.mer repeat
unit AB = PNKPNPNEPSN by duplication
of the component 5- and 6-mers 3t the amino
and carboxy terminal ends of an ancestral I I-
mer repeat region. One would expect that over
the course of evolutionary time the central,
alternating AB repeats could be eliminated by
homologous recombination, resulting in a
which is in fact observed in a number of CSP
repeat regions 131-33]. The SSP2 repeat region
may, therefore, represent an intermediate step
in a general mechanism in Plasmodium antigen
genes by which an ancestral tandem duplica-
tion of a relatively long sequence evolves into a
1.5-kb fragment of
SSP2 is a new, non-CSP, 140-kDa sporo-
repeat region characteriied by tandem repeals
of 2 or more different short pcptide sequenceS.
Sporo/oites which have been inoculated into
the mammalian host progress rapidly from the
circulation to infect hepatoc)tes. It is likely
that rapid homing to the liver requires specific
cell-cell interaction between the sporozoite and
hepatocytes, Kupffer cells, or endothelial cells
in the hepatic circulation. It is thus interesting
to find that SSP2 shares a sequence with the
cell adhesion molecules,
components, as well as with the CSP Region
11 and another Plasmodium antigen, throm-
(TRAP). These thrombospondin motifs are
centered on the nonapeptide WSPCSVTCG,
and are found in 3 copies in thrombospondin.
6 copies in properdin, and one copy in all CS
proteins sequenced to date [29,30]. SSP2 may
also have a role in cell-cell interactions between
the sporozoite and the mammalian host.
SSP2 bears a striking similarity to TRAP
which extends considerably beyond the throm-
bospondin repeat motif. The first 281 amino
acids of SSP2 and TRAP have a
similarity at the amino acid level. Ten of 11
the NaaIl Medical Research and Development
Command Project No. 3M 16102BSI3AK III
and 3M 1 62787A870AN 121. The opinions and
assertions contained herein are the priate ones
of the authors and are not to be construed as
official or reflecting the views of the Navy
Department or the Naval Service at large.
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cysteines in the amino terminal sequence of
SSP2 are identically conserved in TRAP, the
only exception being a single cysteine in the
putative hydrophobic leader of SSP2. A region
of 56% identity extending over 71 amino acids
is found at the carboxy
similarity in overall structure, as well as the
striking amino acid sequence similarities at the
amino and carboxy termini strongly suggest
that TRAP is the P. falciparum analogue of
SSP2. It is interesting that has a large repeat
region, while TRAP, an9
related protein, has none. If SSP2 and TRAP
are indeed analogous proteins with the same
function, the absence of repeats in TRAP may
call into question the functional importance of
repeats in Plasmodium antigens generally.
We thank Stephen
discussions. This research was supported by
Merritt for helpful
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