The 9th Korea-Japan Parasitologists’ Seminar 2003:20-26

Review: Identification of Plasmodium yoelii rhoph2 gene
Osamu Kanekoa,*, Thanaporn Rungruanga, Takafumi Tsuboia,b, Motomi ToriiaaDepartment of Molecular Parasitology, Ehime University School of Medicine, Toon, Ehime 791-0295, Japan
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Ehime 790-8577, Japan* Corresponding author: Tel.: +81-89-960-5286; fax: +81-89-960-5287; e-mail address:
okaneko@m.ehime-u.ac.jp (O. Kaneko)bIntroduction
Plasmodium spp. are obligate intracellular parasites throughout much of their life cycle and entry into hosterythrocytes is a prerequisite for asexual blood stage development.

Plasmodium merozoites invade

erythrocytes, discharging the contents of their apical organelles, the micronemes, rhoptries and dense granules.
Proteins located in these organelles, particularly erythrocyte binding proteins, are considered to be importantfor erythrocyte invasion and have been studied as vaccine targets, with the aim of inducing antibodies blocking
invasion.

For example, passive immunization with monoclonal antibodies (mAb) specific for a 235-kDa

rhoptry protein [1], or active immunization with this protein [2], protect mice against blood stage challengewith P. yoelii. A number of rhoptry proteins have been identified, including a complex of high molecularmass proteins containing three distinct polypeptides, RhopH1, RhopH2 and RhopH3 (the RhopH complex) [3– 6].

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 [7 – 9].

The genes forRhopH1, 2 and 3 have been cloned from P. falciparum and P. yoelii [10 – 13]. The PfRhopH3 gene appears
to be essential for asexual development, as judged from unsuccessful attempts to disrupt the gene locus [14].
The genes encoding RhopH1 in P. falciparum and P. yoelii are members of a gene family that was originallydefined by a cytoadherence-linked asexual gene on chromosome 9 (clag9) [15, 16]. clag9 was initially
proposed to encode a protein involved in the binding of infected erythrocytes to host endothelial cells, whereas
our recent studies indicate that clag9 gene product is involved in the RhopH complex, suggesting a function inthe remodeling of the infected erythrocyte following invasion by the merozoite [17].
Part of the work presented in the Forum Cheju-9 (Oct, 2003) is currently under consideration for thepublication, thus, in this proceeding we will present the identification of P. yoelii RhopH2 gene, which wasonly briefly described in the reference 13.

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Materials and methods
Monoclonal antibody production
P. yoelii 17X (lethal) was maintained in Crj:CD-1 mice (Charles River Japan Inc., Japan).

Mature P. yoelii

schizonts were enriched by differential centrifugation on 45% Percoll (Amersham Pharmacia Biotech Inc., U.
K.) and disrupted by nitrogen decompression to release organelles.

The organelles were purified by sucrose

density gradient centrifugation and the rhoptry-rich fraction was selected after identification by negative
staining electron microscopy.

This fraction, containing 100 µg of protein, was injected intraperitoneally into

an 8-week old female BALB/c mouse, together with Freund's complete adjuvant on the first occasion and with
Freund's incomplete adjuvant 3 weeks later.

Six weeks after the initial injection, an intravenous boost withthe same amount of rhoptry-rich fraction in phosphate buffered saline (PBS, pH 7.4) was administered, and thespleen was used to produce hybridoma cell lines.

After an initial screening by immunofluorescencemicroscopy using acetone-fixed smears of mature P. yoelii schizonts, hybridoma cell lines were cloned by tworounds of limiting dilution.

Cloned 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.).
Affinity purification of ~140-kDa P. yoelii proteins and peptide sequencing
Preparations of enriched mature P. yoelii schizonts were lysed in extraction buffer (50 mM Tris-HCl, 0.2
M NaCl, 5 mM EDTA, 0.2% NP40, pH 7.4, containing 1 µg ml-1 leupeptin, 1 µg ml-1 pepstatin A, and 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Wako)) for 1 h. The lysate was centrifuged at
15,000 × g for 10 min at 4°C, and then P. yoelii RhopH (PyRhopH) complex proteins were purified by affinity
chromatography using MAb #25 bound to Formyl-Cellulofine (Seikagaku-Kogyo, Japan) from theparasite-lysate. The purified protein complex was resolved by 7.5% SDS-PAGE under reducing conditions,and the bands around 135 – 140 kDa were excised.

The extracted proteins were then digested overnight withlysyl-endo-peptidase at 37ºC in 500 mM Tris-HCl buffer.

Peptide fragments were fractionated by reverse

phase high-performance liquid chromatography and analyzed on a protein sequencer (PSQ-1 system; Shimazu,Japan).
DNA and RNA isolation and construction of the cDNA library for P. yoelii
Genomic DNA (gDNA) was isolated from P. yoelii using IsoQuick™ (Orca Research Inc., Bothell, WA).
Total RNA was isolated from schizont-enriched P. yoelii-infected erythrocytes using Isogen (Nippon Gene,Japan) and dissolved in DEPC-treated water. Complementary DNA (cDNA) was generated with random
hexamers using a GeneAmp® RNA PCR kit (Applied Biosystems, Foster City, CA) and the cDNA library wasconstructed with lambda Zap® II vector according to the manufacturer’s instruction (Stratagene, La Jolla, CA).

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PCR and sequencing strategy
The P. yoelii gene coding for RhopH2 was identified by TBLASTN analysis of preliminary sequence data in
the P. yoelii genome database obtained from The Institute for Genomic Research website (http://www.tigr.org)[18]. The amino acid sequences of peptides identified in lysyl-endopeptidase digestion products ofaffinity-purified protein were used in this search. The cDNA sequence was determined by direct sequencing(ABI PRISM® 310 Genetic Analyzer; Applied Biosystems) of the RT–PCR products of P. yoelii mRNA andthe locations of introns were identified by comparison of cDNA and gDNA sequences in the database.
Polyclonal antibody production to PyRhopH2
Based on the deduced amino acid sequences of PyRhopH2, two synthetic peptide dendrimers (peptide B,QNKKEYQRKQREEII (amino acid residues (aa) 986–1000) and peptide C, THKPQFNFPENSNYR (aa1296–1310)) were made using a tetravalent multiple antigenic peptide (MAP) system (ABI 432A
PeptideSynthesizer; Applied Biosystems, Foster City, CA).

A mouse (BALB/c, 8-week-old female; CharlesRiver Japan Inc.) was immunised intraperitoneally with 50 µg of peptide B in Freund’s complete adjuvant.
After 3 weeks the mouse was boosted with the same amount of peptide in Freund’s incomplete adjuvant onfour occasions at 2-week intervals, generating antiserum α-yRhH2B.

A rabbit (Japanese White) was also

immunised using 200 µg of peptide C in adjuvant, administered by subcutaneous injection, to produce
antiserum α-yRhH2C.
SDS-PAGE, Western blot analysis, and Indirect IFA microscopy
For Western blotting of P. yoelii proteins, affinity-purified proteins were dissolved in SDS-PAGE loading
buffer, incubated at 85°C for 10 min, and subjected for the electrophoresis on a 7.5% polyacrylamide gel(ATTO, Japan). Proteins were then transferred from the gel to a 0.22 µm PVDF membrane (Bio-Rad,Hercules, CA). The proteins were either visualized with SYPRO Ruby protein gel stain (Molecular Probes
Inc., Eugene, OR), or immunostained with mouse or rabbit serum followed by horseradish
peroxidase-conjugated goat second antibody (Biosource Int, Camarillo, CA) and visualized with ECL plus
(Amersham Pharmacia Biotech Inc.) on RX-U film (Fuji, Japan).
For Immunofluorescence microscopy, thin smears of schizont-rich P. yoelii-infected mouse erythrocytes
were prepared on glass slides and stored at –80ºC.

Slides were thawed, acetone-fixed, preincubated with PBS

containing 5% non-fat milk at 37ºC for 30 min, reacted with rabbit antiserum at 37ºC for 1 h, followed byFITC-conjugated goat anti-rabbit (IgG and IgM) antibody (Biosource Int.) at 37ºC for 30 min.

Thedifferential interference contrast and FITC-stained images were collected using a fluorescence microscope(BX50; Olympus, Japan) with charged coupled device camera (DC500; Leica Microsystems, Germany), andprocessed using Adobe PhotoShop.

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Results
Peptide sequences of 135- and 140-kDa protein in the RhopH complex from P. yoelii
The RhopH complex was affinity-purified from extracts of P. yoelii-infected erythrocytes using mAb #25,
which recognises a 140-kDa protein, as described previously [12], and used for peptide sequence analysis.
Among 10 peptide sequences obtained, four sequences were identified as a part of PyRhopH1A and reported
previously [12]. The gene coding for PyRhopH2 was identified using the remaining six peptide sequences asqueries in a TBLASTN search of the P. yoelii genome database and a DNA sequence coding for a protein(PyRhopH2) was identified (Table 1 and Fig. 1A). Oligonucleotides were designed using gDNA sequence
and used to obtain cDNA sequence for pyrhoph2 (the sequence data is available in theGenBank™/EMBL/DDBJ under accession number AB075605).

Figure 1A shows the positions of theexon/intron boundaries for P. yoelii and P. falciparum rhoph2, which was independently identified by Lingand Holder (U. K.).

The overall structure of the gene in P. yoelii and P. falciparum is very similar, each genecontaining nine introns in similar locations. PyRhopH2 are predicted to have a signal sequence at theN-terminal end and encodes multiple Cys residues at the conserved location between PfRhopH2, but otherwiseno structural or sequence similarity to known proteins in the public databases was identified.
pyrhoph2 encodes a 140-kDa protein located at the merozoite apical end
The identity of the pyrhoph2 was confirmed by Western blot analysis of sera raised against synthetic
peptides based on the deduced amino acid sequence of PyRhopH2. As is the case with mAb #25, a 140-kDa
protein was recognised (Fig. 1B).

The rabbit serum raised against peptide C from PyRhopH2 gave thepunctate pattern of fluorescence with P. yoelii schizonts, typical of antibodies reacting with apical organelles,
similar to that obtained with mAb #25 (Fig. 1C).
Table 1. Peptide amino acid sequences obtained from Plasmodium yoelii (17XL) affinity-purified rhoptry
proteins
Fraction name
Amino acid sequence determined
Corresponding translated sequence
from P. yoelii 17XL bby peptide sequencing a
R77
R56
R65
R68
R60
R66#1

DFHIYGLYFDLE c
LIHEVVPEDGNIEE cSNXXRHVLVSRNXILKELXENVXXIYSXDFLERTVYVFIHSSFIHNLTK c

188

DFHIYGLYFDLELIHEVVPEDGNIEE557SNFIRHVLVSRNFILK
1004
ELDENVTEI1145
YSRDFLERRVY1243
VFIHSSFIHNLTK
306

aP. yoelii RhopH2 was affinity-purified, digested with lysyl-endopeptidase and the peptide fragments werefractionated by reverse-phase high-performance liquid chromatography and analysed on a protein sequencer.
X, undetermined amino acid.
b
The number at the start of a peptide gives the position of the leading amino acid in the translated sequencesof P. yoelii RhopH2.
cThese sequences were used in TBLASTN search of the P. yoelii database and gave a strong match with asingle sequence in a contig (TIGR c5m1675)

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Fig 1. (A) The positions of the exon/intron boundaries for P. yoelii and P. falciparum rhoph2. Start (atg)and stop (taa) codons, predicted signal sequence (sig) are indicated. Cys residues are indicated with verticalbars under the scheme, longer bar indicate conserved Cys residues between two orthologs. Regions used togenerate antisera and locations of the identified peptide sequences are also indicated. (B) Affinity-purified
proteins were subjected for the electrophoresis on a 7.5% polyacrylamide gel and visualized with SYPRO
Ruby protein gel stain (lane 1), or transferred to a PVDF membrane and immunostained with mouseanti-yRhH2B (lane 2) or rabbit anti-yRhH2C (lane 3) serum and visualized with ECL plus. A 140-kDa
protein was recognised by these sera. (C) Immunofluorescence microscopy. Thin smears of schizont-rich P.
yoelii-infected mouse erythrocytes were reacted with rabbit anti-yRhH2C serum and visualized with FITC.
The punctate pattern of fluorescence, typical of antibodies reacting with apical organelles was observed.
Control rabbit preimmune serum detect no signal. DIC, differential interference contrast.

Discussion
By compared with the increasing knowledge of the microneme function in the parasite gliding motility(reviewed by [19]), little is known about the rhoptry function. It appears that P. falciparum RhopH3 istransferred on invasion to the parasitophorous vacuole membrane (reviewed by [20]) and to persist throughout
the development of ring stage parasites.

Recently RhopH2 was detected on the erythrocyte cytosol side ofthe parasitophorous vacuole membrane in P. falciparum [21].
suggesting that its integrity is important for function.

The complex is not disrupted during transfer

Furthermore the transfer of the PfRhopH complex

during invasion and its location in newly invaded erythrocytes would suggest that its role is post-invasion.
We have now identified the gene coding for the last member of the RhopH complex.

Therefore, we havethe basic tools for studying each protein within the complex, both singly and in combination with each other.
Recently we identified PyRhopH complex specifically bound GPI-anchored molecules on the mouseerythrocyte surface using newly developed semi-quantitative erythrocyte binding assay based on the flow
cytometry [Rungruang et al, under submission].

It will be important to determine the regions within theseparate molecules that bind with each other and host erythrocyte surface molecules.

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Acknowledgements
We thank K. Okugawa in the Integrated Center for Science, Ehime University for their expertise.

Animalexperiments in this study were carried out in compliance with the Guide for Animal Experimentation at Ehime
University School of Medicine. This work was supported in part by Grants-in-Aid for Scientific Research14370084 and 15406015 (to M. T.) and Encouragement of Young Scientists 15790215 (to O. K.) from theMinistry of Education, Culture, Sports, Science and Technology, Japan.
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** Note. This PDF file was created by O. Kaneko to represent the original article in the “Proceedings of the9th Korea-Japan Parasitologist’s Seminar October 28-30, 2003 (Convention Center of Grand Hilton Hotel,Seoul, Korea)”. I used color figure instead of W&B image.

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