Among the 3
proteases found in the excretory/
secretory proteins
(ESP) of Toxoplasma gondii, a 42 kDa
protease was
immunoprecipitated by a monoclonal
antibody (mAb),
Tg786, which detected the protein in
the rhoptry
organelles of T.
gondii by
immunofluorescence
assay (Ahn et al.,
2001). The secreted protease
targeted to the
plasma membrane of host cells, which
was suggested to
favor the appropriate environment
for the entry of
the parasite into host cells. Using the
mAb, a T. gondii cDNA expression library was
screened in order
to obtain the genetic information of
the 42 kDa rhoptry
protease.
A T. gondii ë ZAPII cDNA expression library
obtained through
the AIDS Research and Reference
Reagent Program,
Division of AIDS, NIAID, National
Institutes of
Health (McKesson Biosciences, Rockville,
Maryland, USA) was
screened in Escherichia
coli XL1-
Blue MRF’ (Stratagene,
using mAb Tg786 in
PBS/Tween-20 containing 1%
(w/v) BSA.
Positive plaques were detected using an
ECL Detection
System (Amersham Phamacia Biotech,
Uppsala, Sweden).
pBluescript SK phagemids were
isolated by co-infection of the ë ZAPII phage and the
ExAssist helper
phage (Stratagene). The excised
phagemids were
further propagated in the E. coli
SOLR host strain (Stratagene)
to purify the phagemid
DNA. All DNA
sequencing was performed using a
dye terminator
fluorescent-based sequence analysis
on an Applied
Biosystems 373 automated sequencer
using primers
directed to the vector T7 and T3 promoter
sequences. The
longest cDNA clone containing
a poly (A+) tail was selected for the design of the
�
Brief Communication �
Molecular
cloning of a rhoptry protein (ROP6)
secreted
from Toxoplasma gondii
Hye-Jin AHN, Sehra
KIM and Ho-Woo NAM*
Department
of Parasitology and the Catholic Institute of Parasitic Diseases,
College
of Medicine, Catholic University of Korea, Seoul 137-701, Korea
Abstract: Monoclonal
antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect
a 42-kDa rhoptry
protein (ROP6) which showed protease activity and host
cell binding characteristics after secretion. Using the mAb, a
colony containing a 3’-UTR was probed in a T.
gondii cDNA expression library. A full length cDNA sequence of the
rhoptry protein was completed after 5’-RACE, which
consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino
acid sequence of ROP6 consisted of a polypeptide of
480 amino acids without significant homology to any other
known proteins. This sequence contains an amino
terminal stop transfer sequence downstream of a short neutral
sequence, hydrophilic middle sequence, and hydrophobic
carboxy terminus. It is suggested that the ROP6 is inserted
into the rhoptry membrane with both N- and C-termini.
Key words: Toxoplasma
gondii, excretory/secretory proteins, ROP6, cDNA sequence,
hydrophilic domain
Korean Journal of Parasitology
Vol. 44, No. 3: 251-254, September 2006
�Received 14 July 2006, accepted after
revision 17 August
2006.
�This work was supported by the Korea Research
Foundation Grant
funded by the Korean Government
(MOEHRD)
(KRF-2005-041-E00128).
*Corresponding
author (e-mail: [email protected])
internal
gene-specific primers (Bioneer Co., Daejeon,
Korea).
The RH strain of T. gondii was maintained via peritoneal
passage in BALB/c
mice. Prior to use, tachyzoites
were purified by
centrifugation over 40%
Percoll (Amersham
Phamacia Biotech) in PBS solution.
Total T. gondii tachyzoite RNA was extracted
using Tri reagent
(Sigma Chemical Co., St. Louis,
Missouri, USA) for
the 5’-RACE procedure (Frohman
et al., 1998).
First strand cDNA was synthesized from
252 Korean
J. Parasitol. Vol. 44,
No. 3: 251-254, September 2006
Fig.
1. The cDNA and
deduced amino acid sequences of the ROP6 of Toxoplasma gondii. Nucleotide sequence: the
sequence of ROP6
contains an open reading frame of 1,443 bp downstream of the Tg consensus
translation initiation
sequence of
gtcaaa, as indicated by stars. The nucleotide numbers are shown on the right. Nucleotide
sequence is available
in the GenBank
under the accession number of AY792971, and the deduced amino acid sequence:
the ORF of 1,443
bp encodes a
polypeptide of 480 amino acids.
1 ìg of total RNA by using a Superscript
Preamplification
System (Life
Technologies, Gaithersburg, Maryland,
USA). DeoxyCTPs
were added to the
non-coding cDNA
using terminal deoxynucleotide
transferase (Life
Technologies). PCR amplification of
C-tailed cDNA was
performed with an anchor primer
(5’-CTA ATA CGA CTC ACT ATA GGG CAA GCA
GTG GTA TCA ACG
CAG AGT-
primer (5’-CGT TCG
AGA CTT GAG TCC CAG
GCT-
further with the
abridged universal anchor primer (5’-
AAG CAG TGG TAT
CAA CGC AGA GT-
with an internal
gene-specific primer (5’-GCG AAA
ACC GAA TTT TGC
ACC GAG-
PCR product was
cloned into the pGEM-T
EASY vector
(Promega, Madison, Wisconsin, USA) to
sequence using T7
and SP6 primers. The complete
1,908 bp sequence
was constructed by the 5’-RACE
method containing
an 1,443 bp open reading frame
(Fig. 1). A search
for homologous sequences in the
Toxoplasma
dbEST (Database of
Expressed Sequence
Tags) using the
BLASTn algorithm with default settings
resulted in the
match of a contig assembly containing
20 ESTs with high
BLAST scores. A full cDNA
sequence was
registered in the GenBank (Accession
No. AY792971) as a
42 kDa rhoptry protein (ROP6) of
T.
gondii. Using the second
in-frame ATG as a starting
site downstream of
the typical Tg consensus translation
initiation
sequence of GTCAAA (Seeber, 1997),
the ROP6 gene
encoded a polypeptide of 480 amino
acids with a
molecular mass of 42 kDa. The protein
sequence was then
compared to entries in the
GenBank database
using BLASTp search, which
resulted in no matches
to any previously known proteins.
The hydropathicity
of the amino acids sequence
was obtained from
ExPASy using the Kyte and
Doolittle (1982)
calculation. The sequence contains
amino terminal
stop transfer sequence downstream of
a short neutral
sequence, a long middle hydrophilic
sequence, and a
hydrophobic carboxy terminus (Fig.
2), thereby
suggesting that the ROP6 is inserted into
the rhoptry
membrane with both the N- and C-termini
in a similar
fashion to that of other ROP proteins
except for ROP1.
The membrane
insertion of ROP6 was confirmed by
differential
centrifugation and the Triton X-114 phase
partitioning
(Bordier, 1981) of the protein. When the
supernatants of
the tachyzoite extracts in PBS at 2,500
Ahn et al.: ROP6 gene of T. gondii 253
Fig.
2. Hydropathicity of
ROP6 sequence and the N’-terminal
amino acid
sequences of ROP proteins. The internal
hydrophilic
sequence is lodged in the rhoptry membrane
in both the N’-
and C’-termini. Potential stop transfer
sequences (the
signal sequence in case of the ROP1) are
indicated with
boxes.
Fig.
3. Physical
sedimentation of RH tachyzoite extracts
(A) and Triton X-114 phase partitioning of the extracts
(B).
S: supernatant of
RH tachyzoite extracts after centrifugation,
and P:
precipitant. Total parasite proteins (lane 1), A:
proteins recovered
in the aqueous phase (lane 2), and D:
detergent (lane 3)
phase after partitioning were blotted
with by mAb Tg786
and mAb to SAG1 as a control.
g centrifugation
was sequentially centrifuged at
mAb Tg786 in the
precipitant with a smudge in the
supernatant (Fig.
3A). The tachyzoite extracts in the
extraction buffer
(
NaCl,
0.5% (v/v) precondensed Triton
X-114 (Pierce,
Rockford, Illinois,
USA) and a 1 : 100 (v/v) dilution of
aqueous and DMSO
protease inhibitor stocks) were
centrifuged at
detergent phase
and not in the aqueous phase (Fig.
3B). A major
surface antigen of T. gondii (SAG1)
behaved as a
control of the typical membrane inserting
protein in both
centrifugations.
Until now, 9
rhoptry proteins (ROP1 - ROP9) have
been found to
contribute to the formation of the parasitophorous
vacuole (Ngo et
al., 2004). We have added
the genetic information
of ROP6 to the genes of ROP1,
2, 4, 8, and 9,
but still no clues to the properties from
the gene sequences
or deduced amino acid sequences,
even protease
moiety of ROP6. These are similar to
other secretory
proteins from secretory organelles,
such as, the MIC
proteins of micronemes and the GRA
proteins of dense
granules. ROP proteins are potent
antigens that can
induce strong parasite-directed Tcell
and B-cell
responses (Reichman et al., 2002).
Therefore, ROPs
deserve to be considered as promising
candidates
for vaccine development. ROP6
appears to
constitute an excellent candidate for a
potential vaccine,
because it is secreted as ESP and
exhibits protease
activity essential for the entry of the
parasite into host
cells.
REFERENCES
Ahn HJ, Song KJ,
Son ES, Shin JC, Nam HW (2001) Protease
activity and host
cell binding of the 42-kDa rhoptry protein
from Toxoplasma gondii after secretion. Biochem
Biophys
Res Commun 287: 630-635.
Bordier C (1981)
Phase separation of integral membrane
proteins in Triton
X-114 solution. J
Biol Chem 256: 1604-
1607.
Frohman MA, Dush
MK, Martin GR (1998) Rapid production
of full-length
cDNAs from rare transcripts: amplification
using a single
gene-specific oligonucleotide
primer.
Proc
Natl Acad Sci USA 85: 8998-9002.
Kyte J, Doolittle
RF (1982) A simple method for displaying
the hydropathic
character of a protein. J Mol Biol 157:
105-132.
Ngo HM, Yang M,
Joiner KA (2004) Are rhoptries in
Apicomplexan
parasites secretory granules or secretory
lysosomal
granules? Mol Microbiol 52: 1531-1541.
Reichmann G,
Dlugonska H, Fischer HG (2002)
Characterization
of TgROP9 (p36), a novel rhoptry protein
of Toxoplasma gondii tachyzoites identified by T cell
clone. Mol Biochem Parasitol 119: 43-54.
Seeber F (1997)
Consensus sequence of translational initiation
sites from Toxoplasma gondii genes. Parasitol
Res 83:
309-311.
254 Korean
J. Parasitol. Vol. 44,
No. 3: 251-254, September 2006