Molecular cloning of a rhoptry protein (ROP6)

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

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 λ 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, La Jolla, California, USA)

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 λ 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 �

�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: howoo@catholic.ac.kr)

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 μg of total RNA by using a Superscript Preamplification

System (Life Technologies, Gaithersburg, Maryland,

USA). DeoxyCTPs were added to the 3’ end of 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-3’) and a gene-specific

primer (5’-CGT TCG AGA CTT GAG TCC CAG

GCT-3’). The first-round product was then amplified

further with the abridged universal anchor primer (5’-

AAG CAG TGG TAT CAA CGC AGA GT-3’) coupled

with an internal gene-specific primer (5’-GCG AAA

ACC GAA TTT TGC ACC GAG-3’). The secondround

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

100,000 g, major parts of the protein was blotted with

mAb Tg786 in the precipitant with a smudge in the

supernatant (Fig. 3A). The tachyzoite extracts in the

extraction buffer (10 mM Tris-HCl, pH 7.4, 150 mM

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 13,000 g, which blotted the ROP6 in the

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.

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