Proceed. 7th Korea - Japan Parasitologists'
Seminar (Forum Cheju-7) October 23-25, 2001: 76 - 85.

Characterization of the high molecular mass rhoptry protein,
RhopH, complex, in Plasmodium yoelii
Osamu Kaneko*, Takafumi Tsuboi, Thanaporn Rungruang and Motomi Torii
Department of Molecular Parasitology, Ehime University School of Medicine,
Shigenobu-cho, Ehime 791-0295, JapanINTRODUCTION
Plasmodium spp. are obligate intracellular parasites and entry into host erythrocytes is aprerequisite for asexual-stage development. Plasmodium merozoites invade erythrocytes, discharging the contents of their apical organelles, the micronemes, rhoptries, and dense granules.
The molecules located in these organelles, particularly erythrocyte binding proteins, are considered to be important for erythrocyte invasion and have been studied as vaccine targets, withthe aim of inducing antibodies to block invasion. For example, passive immunization with monoclonal antibodies (MAb) specific for a 235-kDa rhoptry protein (Freeman et al., 1980), or
active immunization with the protein (Holder and Freeman, 1981), protect mice against bloodstage challenge with P. yoelii. A number of rhoptry proteins have been identified, including acomplex of high molecular mass proteins (the RhopH complex) containing three distinct
polypeptides, RhopH1, RhopH2 and RhopH3 (Campbell et al., 1984; Holder et al., 1985;
Lustigman et al., 1988; Hienne et al., 1998). Antibodies against the P. falciparum RhopH(PfRhopH) complex partially inhibit growth of P. falciparum in vitro and in vivo, consistent
with their potential as vaccine targets (Siddiqui et al., 1987; Cooper et al., 1988; Doury et al.,1994). The PfRhopH complex is considered to have a critical role in erythrocyte invasion
because the complex binds the erythrocyte and distributes into the erythrocyte and parasitophorous vacuolar membranes (Sam-Yellowe et al., 1988; Sam-Yellowe and Perskins, 1991).
The gene for RhopH3 has been cloned from P. falciparum (Brown and Coppel, 1991), and theprotein appears to be essential because attempts to disrupt the PfRhopH3 gene locus were
unsuccessful (Cowman et al., 2000). The genes encoding RhopH1 and RhopH2 have not been

*Corresponding author: Tel. +81-89-960-5286; fax +81-89-960-5287; e-mail address:
okaneko@m.ehime-u.ac.jp (O. Kaneko)76Fig. 1. (A) Immunoelectron microscopy of a merozoite-stage Plasmodium yoelii
parasite with MAb #25. The gold-particles were seen on the body of the rhoptries,
and no gold particles were seen on the micronemes or dense granules. (B) Western
immunoblot analysis of the affinity purified P. yoelii rhoptry proteins under reducing conditions. The proteins were visualized with Coomassie Brilliant Blue G-250(lane 1) or immunostained with MAbs #25, #32, anti-PyRhopH1A serum, or control serum for PyRhopH1A (lanes 2-5, respectively). These antibodies and antiserum also reacted under non-reducing conditions. The sizes of 3 bands characteristic for the Plasmodium RhopH complex (bar) and 2 uncharacterized bands(arrowhead) are indicated.

identified yet in any Plasmodium species.
In this review article, we describe the generation of monoclonal antibodies against P. yoelii
rhoptry proteins, and identification of the genes encoding RhopH1 and RhopH3.

Monoclonal antibodies
MAbs against P. yoelii rhoptry proteins were generated as described elsewhere (Kaneko etal., 2001). Cloned hybridoma cell lines were expanded as ascites in mice primed with pristane(Wako, Japan), and immunoglobulins were purified from ascitic fluid using an Ampure PA kit(Amersham Pharmacia Biotech Inc., UK). By immunofluorescence microscopy, MAbs #25[IgG1 (λ)] and #32 [IgG2b (κ)] showed a punctate pattern in mature schizonts, typical for an
antibody reacting with the apical organelles of Plasmodium merozoites. MAb #25 reacted with77

the body of rhoptries by immunoelectron microscopy (Fig. 1A)Western immunoblot analysis against P. yoelii proteins extracted from mature schizonts
revealed that the 140-kDa protein reacted with MAb #25, whereas the 100-kDa protein reactedwith MAb #32. Because the molecular masses of these proteins are similar to those of the proteins forming the RhopH complex in P. falciparum (Holder et al., 1985), we checked whether
or not these proteins form a complex. Affinity purification using MAb #25 co-purified 5 bands
at 140, 135, 100, 77, and 75 kDa. The 3 bands around 140, 135, and 100 kDa are a characteristic feature of the RhopH complex in P. falciparum (Fig. 1B, lane 1). Immunostaining of theaffinity-purified proteins using MAb #25 revealed that the 100-kDa protein reacted with MAb#32 (Fig. 1B, lanes 2 and 3).
Identification of genes encoding RhopH complex in P. yoelii
To obtain the sequence of the genes encoding the P. yoelii 140-, 135- and 100-kDa rhoptry proteins, partial peptide sequences were determined. The protein complex was purified frommature schizonts by affinity chromatography using MAb #25 followed by SDS-PAGE. The
bands at 140, 135, and 100 kDa were excised from the gel, digested with lysyl-endopeptidase,and peptide fragments were fractionated by reverse phase high-performance liquid chromatography and analyzed on a protein sequencer (PSQ-1 system; Shimazu, Japan). Sequencing of
peptides from the purified 135- and 140-kDa proteins identified 10 distinct amino acidsequences and the purified 100-kDa proteins identified 5 distinct amino acid sequences (Table

Table 1. Amino acid sequences of peptides derived from PyRhopH1A and PyRhopH3, which wereobtained from the purified Plasmodium yoelii 135-kDa, or 100-kDa rhoptry proteins
Fraction name

Peptide amino acid sequence

PyRhopH1A (135-kDa proteins)aR49
YQLSLISRTYGDEFQR52
DDNVLFEDVDKEEEF
R66#2
NEYNEIXNARXSDTNFKR67NLIELHDTFNIXXK
PyRhopH3 (100-kDa protein)
R51
IDDFDFKR52
YNEEWLPKR57
FLTTNELSFTKR61
SSNIFVDAFQKR62IFSIFK

Corresponding sequence and location

NLIELHDTFNISSKYQLSLISRTYGDEFQDDNVLFEDVDKEEEF
NEYNEIINARLSDTNFK(335-349)b(1271-1285)(1224-1240)(757-770)

IDDFDFKYNEEWLPKFLTTNELSFTKSSNIFVDAFQKIFSIFK(407-413)(725-732)(296-306)(373-383)(384-389)

aOnly amino acid sequences of peptides derived from PyRhopH1A are shown.
bLocations are indicated with amino acid number.

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Fig. 2. Gene structure of PyRhopH1A, 1A-P, and 3. Cysteine residues are indicated withthe vertical bar above the schematics.

1).
For 135-kDa and 100-kDa bands, DNA fragments were PCR-amplified from cDNA libraryof P. yoelii 17X (lethal) schizonts with combinations of degenerate oligonucleotides, sequenced,
and the full sequence of the putative open reading frame of the cDNA was completed. Genomic
DNA (gDNA) sequence was obtained from PCR products amplified from splinkerette genomic libraries of P. yoelii using gene-specific and splinkerette-specific primers. For 140-kDa band,
gDNA sequence was obtained by BLASTN analysis using partial peptide sequences and gDNA
and cDNA sequence was confirmed (manuscript in preparation).
For 135-kDa protein, we completed the cDNA and gDNA sequences encoding an open
reading frame of 1292 amino acids in 8 exons (Fig. 2, PyRhopH1A). The deduced amino acidsequence has a putative secretory signal peptide at amino acid (aa) residues 1-24. The four peptide sequences (R49, R52, R66#2, and R67) were located in this protein sequence. Several of
the other peptide sequences were identified by BLAST analysis of the P. yoelii genome database (TIGR) in another gene homologous to that encoding P. falciparum RhopH2 (Holder AA,
personal communication).
For 100-kDa protein, comparison of the 4018 nucleotides of the gDNA to the corresponding cDNA revealed a 2649 nucleotide open reading frame in 7 exons, which is consistent withthe structure of pfrhoph3. SignalP software predicted a putative signal peptide sequence at aa 124 (Fig. 2, PyRhopH3). Based on ClustalW analysis of the PyRhopH3 with PfRhopH3 aminoacid similarity followed by manual adjustment for best fit, the RhopH3 protein was divided into
3 types of block: conserved blocks (70.0-86.4% similarity and 42.3-65.2% identity; aa 24-220,
231-262, 274-299, 398-457, 475-540, and 560-651 of PyRhopH3), semi-conserved blocks(45.6-57.5% similarity and 21.5-39.7% identity; aa 300-397, 652-730, and 749-821), and vari-79

Fig. 3. Air-dried segmented schizont-stage parasites fixed with ice-cold acetone and reactedwith anti-PyRhopH1A serum (row A), showing a typical punctate pattern for the apicalorganelle, similar to the images with MAbs #25 and #32 (rows B and C, respectively).
Control serum gave no signal (row D). Phase contrast and FITC images were merged in theright panel (Overlay). The bar indicates 10 µm.

able blocks (5.3-27.8% similarity and 5.3-22.2% identity; aa 221-230, 263-273, 458-474, 541559, 731-748, and 822-882). The conserved blocks are clustered at the N-terminal and central
region, whereas the C-terminal region is variable. Twelve cysteine residues located in the conserved blocks are conserved between PyRhopH3 and PfRhopH3, suggesting a conformationalrole of these residues and a potential functional importance for these blocks (Shirano et al.,2001).
Antisera generated based on the deduced amino acid sequences of PyRhopH1 andPyRhopH3 reacted to the 135- and 100-kDa band of the purified PyRhopH complex, respectively (Fig. 1B, lane 4 for PyRhopH1A). Immunofluorescence microscopy revealed that these
antisera reacted at the apical end of the merozoites (Fig. 3). These findings confirmed that80

Fig. 4. Plasmodium RhopH1/Clag family. (A) The deduced amino acid sequence ofClag 2 was based on the exon/intron structure predicted by PfAGSS from P. falciparum
chromosome 2 (Gardner et al., 1998; Huestis et al., 2001). The sequences for
PfRhopH1/Clag 3.1, 3.2, and Clag 9 were obtained from CAB10571-2 and AF055476,
respectively. The sequence for Clag 7 is after Holt et al. (2001). Conserved Cys residues
in all members (solid vertical line) and among PfRhopH1/Clag 2, 3.1, 3.2, and/or 7(broken lines) are indicated in the schematics. (B) Phylogenetic tree based on the aminoacid sequences of the RhopH1/Clag multigene family. Bootstrap values per 100 are indicated.

cloned genes were encoding proteins of PyRhopH complex.
PyRhopH1 is a member of a multigene family (RhopH1/Clag family)
TBLASTN analysis of the P. yoelii genome database (TIGR) revealed the existence of aparalogue of the pyrhoph1 gene we had cloned (identified as c2m3539 in the TIGR database).
Thus we designated the gene identified from the peptide sequences as pyrhoph1a and the second gene found in the TIGR database as pyrhoph1a-paralogue (pyrhoph1a-p). Using oligonucleotides designed on the gDNA sequence of pyrhoph1a-p, the cDNA sequence was determined(Accession number AB060735). The deduced amino acid sequence showed that PyRhopH1AP was a 1350-amino acid residue protein with a signal peptide (aa 1-22) similar to PyRhopH1A.

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Fig. 5. Schematic of the early stages of erythrocyte invasion by the malaria merozoite, and theputative roles of the protein encoded in the multigene family.

A comparison of the cDNA sequence and the gDNA sequence in the database revealed that the
open reading frame of pyrhoph1a-p was encoded in 9 exons. The 5th intron in pyrhoph1a-p ispresent at a location corresponding to the middle of the 5th exon of pyrhoph1a (Fig. 2,
PyRhopH1A-P).
TBLASTN analysis of the P. falciparum genome databases revealed that PyRhopH1 washomologous to members of the P. falciparum cytoadherence linked asexual gene (clag) family,
which was originally defined by a cytoadherence linked asexual gene on chromosome 9 (clag9) (Holt et al., 1999; Trenholme et al., 2000), proposed to encode a protein involved in the binding of infected erythrocytes to host endothelial cells. However Holder's group in UK, independently identified a member of the clag family, clag3.1 or 3.2, encoding RhopH1 in P. falciparum by MALDI-ToF analysis. Thus, we propose to rename the genes encoding the P. falciparum 155-kDa rhoptry proteins (PfRhopH1) pfrhoph1/clag 3.1 and 3.2. Therefore the clag
gene family is renamed as the rhoph1/clag gene family. To compare the sequences, all membersof RhopH1/Clag family described in the literature were aligned. Ten Cys residues, including
one at residue 24 in the putative signal sequence of PyRhopH1A, are conserved in all members,
while 1 Cys residue in the central part are only found among Clag 2 and PfRhopH1/Clag 3.1
and 3.2 and 4 Cys residues at the C-terminus are only found among Clag 2, Clag 7, and
PfRhopH1/Clag 3.1 and 3.2 (Fig. 4A). Among 5 rhoph1/clag members identified so far in P. falciparum, Clag 9 has phylogenetically distant from other members (Fig. 4B).
The multigene families localized in the microorganelles of the merozoites may play a critical role in the redundancy of the invasion of the host erythrocyte. At the neck of the rhoptry, afamily called reticulocyte binding-like (rbl) has been identified, including 5 members in P. falciparum (pfrh1, pfrh2a, pfrh2b, pfrh3, and pfrh4) (Rayner et al., 2000 & 2001; Taylor et al.,
2001; Triglia et al., 2001; Kaneko et al., 2002). In the microneme, another family called ery-82

throcyte binding-like (ebl) has been identified, including 5 members in P. falciparum (jesebl,
pebl, eba-175, baebl, and ebl-1) (Michon et al., 2002). In addition to these families, we identified a new family (rhoph1/clag) localized at the body of the rhoptry (Fig. 5). Using multiple
multigene families at a different step during the invasion, malaria parasites have a potential toinvade into a variety of host erythrocytes. Further investigation is currently conducted to clear
the role of the multigene family in the alternative invasion pathway of the malaria parasites.

ACKNOWLEDGEMENTS
Sequence data for P. falciparum chromosomes 6-8 was obtained from The Sanger Centre
website (www.sanger.ac.uk/Projects/P_falciparum/), which was accomplished as part of the
Malaria Genome Project with support by The Wellcome Trust. Preliminary sequence data for P.
yoelii genome was obtained from The Institute for Genomic Research website (www.tigr.org),
which is carried on in collaboration with the Naval Medical Research Center and is supportedby the U.S. Department of Defense.

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