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Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells
Veterinary Parasitology 109 (2002) 191–201
Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells Yoshifumi Nishikawa a , Khajornsak Tragoolpua a,b , Levi Makala a , Xuenan Xuan a , Hideyuki Nagasawa a,∗ a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan b Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand Received 18 January 2002; received in revised form 24 July 2002; accepted 14 August 2002
Abstract We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; Surface protein; GPI; NcSRS2; Baculovirus; Insect cells
∗ Corresponding author. Tel.: +81-155-49-5644; fax: +81-155-49-5643. E-mail address: [email protected] (H. Nagasawa).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 2 5 6 - X
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1. Introduction Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii (Dubey et al., 1988). It is frequently diagnosed as the cause of bovine abortion of epidemic proportion worldwide (Dubey and Lindsay, 1996). Vertical transmission contributes significantly to the spread of N. caninum, with most congenital infections resulting in the birth of healthy calves (Bjorkman et al., 1996; Pare et al., 1996; Schares et al., 1998). Surface proteins of obligate intracellular parasites collectively serve a number of essential functions because they represent the initial point of contact and interaction with the host cell and components of the host immune system. The cloned N. caninum surface protein (NcSRS2) (Hemphill et al., 1997; Howe et al., 1998) could be found in dense granules and rhoptries and on the surface of tachyzoites (Hemphill, 1996; Hemphill and Gottstein, 1996; Hemphill et al., 1997; Howe et al., 1998). It functions in host cell adhesion and invasion (Hemphill et al., 1997; Nishikawa et al., 2000), and has been reported as an efficacious antigen component in enzyme-linked immunosorbent assay (Schares et al., 2000a; Nishikawa et al., 2001c) and is a potential target/candidate antigen in the development of vaccine against N. caninum infection (Nishikawa et al., 2001a,b). Approximately six surface protein molecules have been identified in tachyzoites of N. caninum (Schares et al., 1999), including the NcSRS2 that is anchored to the parasite cell surface by a glycosyl-phosphatidylinositol (GPI) molecule (Howe et al., 1998; Schares et al., 2000b). In earlier studies, we have detected expression of the full length NcSRS2 gene on the surface of N. caninum tachyzoites (Nishikawa et al., 2000), and in insect cells infected with recombinant baculovirus (Nishikawa et al., 2001c), indicating that NcSRS2 is exclusively in the cell surface. In the light of increasing evidence pointing to the importance of GPI anchored proteins in enhancing the function(s) of the major surface proteins of protozoan parasites, in this study, we examined the terminal location of the full length and three truncated versions of rNcSRS2, using the insect-baculovirus expression system that has both capabilities of performing posttranslational modifications and high yields of recombinant proteins.
2. Materials and methods 2.1. Parasites Neospora caninum tachyzoites (Nc-1 strain) were maintained in human foreskin fibroblast cells (Hs68) cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS). For the purification of tachyzoites, the parasites and host cell debris were washed in cold phosphate-buffered saline (PBS) and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0 m-pore filter (Millipore, Bedford, MA). 2.2. Cells and virus The Autographa californica nuclear polyhedrosis virus and its recombinant viruses were grown in Spodoptera frugiperda (Sf9) cells in a TC-100 insect medium (Gibco BRL, Grand
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Island, NY) supplemented with 10% FBS and 0.26% bacto tryptose broth (Difco, Detroit, MI). 2.3. Cloning of full length and truncated NcSRS2 genes To clone full length and truncated NcSRS2 genes into baculovirus, two primer sets summarized in Fig. 1A were used. The template used in the polymerase chain reaction (PCR) was N. caninum tachyzoite cDNA produced by ZAP-cDNA Synthesis kit (Toyobo, Osaka, Japan). The resulting PCR fragments were blunted by Klenow fragment, and were then ligated with the baculovirus transfer vector pBacPAK8 (Clontech, Palo Alto, CA), which had been previously digested with SmaI. 2.4. Construction of the recombinant baculoviruses, which express NcSRS2 Sf9 cells were co-transfected with a transfer vector and BaculoGold® Baculovirus DNA (PharMingen, San Diego, CA) using LipofectinTM reagent (Gibco BRL). After 4 days of incubation at 27 ◦ C, the culture supernatant containing recombinant viruses expressing NcSRS2 gene was harvested and subjected to plaque purification. After three cycles of purification, recombinant viruses-expressing NcSRS2 were obtained. 2.5. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot Sf9 cells (1 × 106 ) infected with recombinant baculovirus at 5 plaque forming units per cell (PFU/cell) were cultured with 1 ml medium. At the end of culture, the culture medium was harvested by centrifugation (5 min, 1450 × g, 4 ◦ C), and the cells were suspended in 1 ml PBS. Purified N. caninum tachyzoites (2 × 107 ) suspended in 100 l PBS, and samples of Sf9 cells (cells or culture supernatants) were mixed with 100 l, and 1 ml, 2× SDS gel-loading buffer (100 mM Tris–HCl (pH 6.8), 100 mM 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol), respectively, under reducing conditions. Uninfected Sf9 cells (1 × 106 ) were used as a negative control. The samples were heated at 95 ◦ C for 5 min and 10 l samples were subjected to SDS-PAGE. After SDS-PAGE, the protein bands in the gel were electrically transferred to a membrane (immobilon transfer membrane, Millipore). The membrane was blocked with PBS containing 3% skim milk (PBS-SM) and then incubated with anti-NcSRS2 mAb (2C8) (Nishikawa et al., 2000) diluted 1:200 with PBS-SM at 37 ◦ C for 60 min. The membrane was washed three times with PBS for 5 min and then incubated with horseradish peroxidase-conjugated mouse IgG antibody (Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:1000 with PBS-SM at 37 ◦ C for 60 min. The membrane was washed three times with PBS for 5 min and incubasted with enhanced chemo-luminecsence detection reagents (Amersham Pharmacia Biotech) for 1 min and exposed to a film. The amounts of intracellular and extracellular rNcSRS2 were determined using a density meter (Luminous Imager version 2.0, Aisin cosmos, Tokyo, Japan). 2.6. Confocal laser scanning microscopy Sf9 cells infected with baculovirus for 48 h at 5 PFU/cell were fixed with acetone, which optimized detection of protein on the cell surface and the intracellular localization (Xuan
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et al., 1995), and incubated with anti-NcSRS2 mAb diluted 1:100 with PBS containing 3% FBS for 30 min at 37 ◦ C, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Southern Biotechnology, Birmingham, AL) diluted 1:100 with PBS containing 3% FBS for 30 min at 37 ◦ C. Uninfected Sf9 cells were used as a negative control. The cells were examined on a confocal laser scanning microscopy (Leica TCS NT, Heidelberg, Germany). 2.7. Flow cytometric analysis Sf9 cells (1 × 106 ) infected with baculoviruses for 48 h at 5 PFU/cell were reacted with anti-NcSRS2 mAb diluted 1:100 with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide for 30 min at 4 ◦ C, followed by FITC-conjugated goat anti-mouse antibody diluted 1:100 with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide for 30 min at 4 ◦ C. The cells (1 × 104 ) were then analyzed on an EPICS XL flow cytometer (Coulter, Hialeah, FL). 2.8. Phospholipase C treatment The presence of the GPI anchor was assessed by digestion with phosphatidyl-inositolspecific phospholipase C (PI-PLC). Sf9 cells (1 × 106 ) infected with recombinant baculoviruses for 48 h at 5 PFU/cell were washed three times in cold Sf-900II SFM medium (Gibco BRL) without FBS. The cells were incubated with 0.1 U PI-PLC (Sigma) in Sf-900II SFM or in Sf-900II SFM without the enzyme for 2 h at 27 ◦ C. The reaction volume was 50 l. The supernatants were harvested by centrifugation (5 min, 1450 × g, 4 ◦ C) and then mixed with 50 l 2× SDS gel-loading buffer. The 10 l samples were analyzed by SDS-PAGE and immunoblot. 2.9. Peptide sequencing Sf9 cells (1 × 106 ) were infected with Ba/SRS2p41 for 48 h at 5 PFU/cell. The culture medium was harvested by centrifugation (5 min, 1450 × g, 4 ◦ C). The supernatants and the cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane by semi-dry electroblotting. The membrane was stained with Coomassie brilliant blue, and the protein band was excised from the membrane and submitted for HP G1005A protein sequencing system (Takara, Shiga, Japan).
3. Results 3.1. rNcSRS2 expression in the insect-baculovirus expression system We have generated four types of recombinant baculoviruses (full length and three truncated versions of the NcSRS2 gene) (Fig. 1), and these were used to examine the terminal location of rNcSRS2 gene products in the insect-baculovirus expression system.
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Fig. 2. Expression of rNcSRS2 in the insect-baculovirus expression system. Cell lysates (cell), and culture supernatants (sup) of Sf9 cells infected with recombinant baculoviruses for 4 days and analyzed by SDS-PAGE, and immunoblot with anti-NcSRS2 mAb under reducing conditions. The anti-NcSRS2 mAb did not react with the uninfected cells. Molecular weights are given in kDa.
By immunoblotting using the anti-NcSRS2 mAb, two major bands were detected in the cell lysate of each group of recombinant baculovirus-infected Sf9 cells: 48 and 42 kDa in Ba/SRS2p44; 46 and 40 kDa in Ba/SRS2p41; 43 and 37 kDa in Ba/SRS2p37; 31 and 25 kDa in Ba/SRS2p28 (Fig. 2). The anti-NcSRS2 mAb did not react with the uninfected cells (data not shown). Some proteolytic degradation of the rNcSRS2 may account for the presence of some minor bands. The two major bands detected in all cell lysates differed by 6 kDa in molecular weight. Culture supernatants of Ba/SRS2p41 and Ba/SRS2p37-infected cells showed the 40 and 37 kDa bands, respectively. Analysis of the N-terminal of the 40 kDa protein from the cell lysate and supernatant of Ba/SRS2p41-infected Sf9 cells yielded the amino acid sequence APFKSEN, indicating that the 53-amino acid peptide from the N-terminal was cleaved from the rNcSRS2. We infer that the higher-molecular weight proteins (48 kDa in Ba/SRS2p44, 46 kDa in Ba/SRS2p41, 43 kDa in Ba/SRS2p37, 31 kDa in BaSRS2p28) were precursors of rNcSRS2 that contain an N-terminal signal peptide, and that lower-molecular-weight proteins (42 kDa in Ba/SRS2p44, 40 kDa in Ba/SRS2p41, 37 kDa in Ba/SRS2p37, 25 kDa in Ba/SRS2p28) were mature proteins. The molecular weight (42 kDa) of the mature form of the full length rNcSRS2 (Fig. 2, lane 2) was larger than that of the authentic NcSRS2 (40 kDa) (Fig. 2, lane 1), with the latter having been detected as well in the cell lysate and supernatant of Ba/SRS2p41-infected Sf9 cells. 3.2. Kinetic analysis of rNcSRS2 expression Secreted rNcSRS2 was detectable in the supernatants of Ba/SRS2p41 and Ba/SRS2p37infected Sf9 cells (Fig. 3). While the non-expression of rNcSRS2 in the supernatant of
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Fig. 3. Kinetics of rNcSRS2 synthesis. Cell lysates and culture supernatants of Sf9 cells infected with recombinant baculoviruses for 24, 48 and 72 h analyzed by SDS-PAGE, and immunoblot with anti-NcSRS2 mAb under reducing conditions. Molecular weights are given in kDa.
Ba/SRS2p44-infected Sf9 cells was clearly attributable to the hydrophobic C-terminal tail, in the rNcSRS2 (Ba/SRS2p28), despite the truncation of the hydrophobic C-terminal tail, its expression was not detected in the supernatant. The detection of the precursor rNcSRS2 in the supernatant of Ba/SRS2p41-infected Sf9 cells may be due to an artifact of this insect–baculovirus system, such as the cytopathic effects. 3.3. Cellular localization of the rNcSRS2 The expression of the full length rNcSRS2 was observed mainly on the cell surface of Sf9 cells, while that of the truncated rNcSRS2 was limited to the internal compartment of infected cells (Fig. 4). The anti-NcSRS2 mAb did not react with the uninfected cells (data not shown). By flow cytometric analysis, expression levels of the truncated rNcSRS2, were shown, to be reduced on the insect cell surface of Sf9 cells compared to those of the full length rNcSRS2 (Fig. 5). The intact rNcSRS2 product was transported completely to the cell surface, while the truncated rNcSRS2 with deletions of 25 amino acid residues (Ba/SRS2p41) or 62 C-terminal residues (Ba/SRS2p37) were synthesized, but nonetheless remained in the internal compartment of infected cells. Moreover, the truncated recombinant (Ba/SRS2p28) lacking 147 C-terminal residues including the C-terminal hydrophobic
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Fig. 4. NcSRS2 localization in Sf9 cells infected with recombinant baculoviruses for 48 h. (A) Ba/SRS2p44infected cells. (B) Ba/SRS2p28-infected cells. Similar localization of rNcSRS2 was observed in Ba/SRS2p41 or BaSRS2p37-infected cells. The anti-NcSRS2 mAb did not react with the uninfected cells; bar = 10 m.
domain, known to be a crucial in signaling anchor protein attachment (Tomavo et al., 1992), was synthesized, albeit remained inside infected cells. 3.4. PI-PLC treatment Sf9 cells infected with recombinant baculoviruses were treated with PI-PLC to find out whether the rNcSRS2 were anchored by GPI. The rNcSRS2 expressed by Ba/SRS2p44, Ba/SRS2p41 and Ba/SRS2p37 were detected in the culture supernatants, and were of the mature protein form (Fig. 6). In the presence of PI-PLC, the intact rNcSRS2 expressed by Ba/SRS2p44 secreted 27.6% of the molecule, while 9.8, and 8.5% of the molecules were secreted in the truncated rNcSRS2 expressed by Ba/SRS2p41 and Ba/SRS2p37, respectively. Protein synthesis during incubation period may account for the detection of precursor and mature rNcSRS2 expressed by Ba/SRS2p41-infected cells in the presence and absence of PI-PLC. There was no detectable rNcSRS2 release in PI-PLC-treated Ba/SRS2p28-infected cells.
Fig. 5. Flow cytometry for Sf9 cells infected with recombinant baculoviruses for 48 h. The rNcSRS2 expression on Sf9 cells infected with wild type baculovirus (light line) and recombinant baculoviruses (heavy line) is shown.
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Fig. 6. Treatment of rNcSRS2 with PI-PLC. Sf9 cells infected with recombinant baculoviruses for 48 h were treated with PI-PLC (+) or incubated in medium alone (−). After treatment, the cells were centrifuged, and the resulting supernatants were analyzed by SDS-PAGE and immunoblotting using the anti-NcSRS2 mAb under reducing conditions. Molecular weights are given in kDa.
4. Discussion Previous studies have revealed an open reading frame of 1203 nucleotides encoding a 401-amino acid protein in the authentic NcSRS2 gene, and based on the N-terminal peptide sequence of purified NcSRS2, it contains a 53-amino acid signal peptide (Howe et al., 1998). We have obtained data demonstrating the expression of an intact rNcSRS2 protein on the cell surface of infected Sf9 cells, suggesting a similar transport mechanism between the parasite and insect cells. The distinction between the precursor and mature form of rNcSRS2 gene products expressed in the infected cells based on our findings on the analysis of the N-terminal peptide sequence of the rNcSRS2, revealed similar cleavage sites of the signal peptide in both the parasite and insect cells. The molecular weight of the mature form of the truncated rNcSRS2 with deletions of 25 amino acid residues was the same as that of the authentic NcSRS2. Present data are consistent with earlier studies that showed a relationship between the removal of the 53-amino acid signal peptide and the 25-amino acid hydrophobic C-terminal tail with the mature NcSRS2 protein (Howe et al., 1998). The cleavage of the intact rNcSRS2 from the cell surface after treatment with PI-PLC is a clear indication that the intact recombinant was a GPI-anchored molecule, and implies that GPI-attachment is extremely essential in the successful transport of NcSRS2 to the
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cell surface. With PI-PLC treatment, truncated rNcSRS2 expressed by Ba/SRS2p41 and Ba/SRS2p37 were present in the supernatants in lower amounts compared to the secretion of intact rNcSRS2. In a related study on murine cell surface protein Ly-6E, the addition of only 50% phosphaditylinositol-glycan (PI-G) moiety to the mutant proteins produced proteolytic cleavage resulted to the replacement of the C-terminal residues with a cytoplasmic tail of the transmembrane form of the lymphocyte function-associated antigen (Caras, 1991). Proteolytic cleavage occurring at other amino acid sites and GPI-attachment at new site(s) as earlier proposed (Su and Bothwell, 1989) could possibly be happening in rNcSRS2. However, there is a possibility of an experimental artifact such that PI-PLC treatment may lead to the release of non-GPI anchored proteins. This is one facet that is worth investigating in future studies. In the light of a comparatively more exhaustive study on the GPI-anchored surface proteins such as SAG1 and SAG3 (Tomavo et al., 1992; Manger et al., 1998; Dzierszinski et al., 2000) in T. gondii, a closely related species of N. caninum, an in-depth analysis of the function(s) of NcSRS2 would require additional work on its biosynthesis and other details of its transport to the cell surface which have not been examined in the present study. In conclusion, we have demonstrated that the N. caninum NcSRS2 is a transmembrane protein that contains a GPI-anchor molecule expressed in insect cells, and that the hydrophobic C-terminal domain in the NcSRS2 is an essential component for GPI-membrane attachment and transport to the cell surface. In T. gondii surface antigen SAG1, Azzouz et al. (2000) reported the inappropriateness of the insect baculovirus system for the expression of GPI-anchored proteins. The expression of GPI-anchored protein in insect cells may be due to the difference in the transport property of individual proteins. We have likewise shown the usefulness of the insect-recombinant baculovirus expression system in studies on N. caninumNcSRS2.
Acknowledgements We thank Dr. J.P. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for supplying N. caninum, NC-1 isolate. We appreciate the helpful inputs of Dr. Florencia G. Claveria of the Biology Department, De La Salle University, Manila, Philippines during her sabbatical stay at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine. The first author is supported by a Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References Azzouz, N., Kedees, M.H., Gerold, P., Becker, S., Dubremetz, J.F., Klenk, H.D., Eckert, V., Schwarz, R.T., 2000. An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection. Glycobiology 10, 177–183. Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 1441–1444.
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Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells Yoshifumi Nishikawa a , Khajornsak Tragoolpua a,b , Levi Makala a , Xuenan Xuan a , Hideyuki Nagasawa a,∗ a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan b Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand Received 18 January 2002; received in revised form 24 July 2002; accepted 14 August 2002
Abstract We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; Surface protein; GPI; NcSRS2; Baculovirus; Insect cells
∗ Corresponding author. Tel.: +81-155-49-5644; fax: +81-155-49-5643. E-mail address: [email protected] (H. Nagasawa).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 2 5 6 - X
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1. Introduction Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii (Dubey et al., 1988). It is frequently diagnosed as the cause of bovine abortion of epidemic proportion worldwide (Dubey and Lindsay, 1996). Vertical transmission contributes significantly to the spread of N. caninum, with most congenital infections resulting in the birth of healthy calves (Bjorkman et al., 1996; Pare et al., 1996; Schares et al., 1998). Surface proteins of obligate intracellular parasites collectively serve a number of essential functions because they represent the initial point of contact and interaction with the host cell and components of the host immune system. The cloned N. caninum surface protein (NcSRS2) (Hemphill et al., 1997; Howe et al., 1998) could be found in dense granules and rhoptries and on the surface of tachyzoites (Hemphill, 1996; Hemphill and Gottstein, 1996; Hemphill et al., 1997; Howe et al., 1998). It functions in host cell adhesion and invasion (Hemphill et al., 1997; Nishikawa et al., 2000), and has been reported as an efficacious antigen component in enzyme-linked immunosorbent assay (Schares et al., 2000a; Nishikawa et al., 2001c) and is a potential target/candidate antigen in the development of vaccine against N. caninum infection (Nishikawa et al., 2001a,b). Approximately six surface protein molecules have been identified in tachyzoites of N. caninum (Schares et al., 1999), including the NcSRS2 that is anchored to the parasite cell surface by a glycosyl-phosphatidylinositol (GPI) molecule (Howe et al., 1998; Schares et al., 2000b). In earlier studies, we have detected expression of the full length NcSRS2 gene on the surface of N. caninum tachyzoites (Nishikawa et al., 2000), and in insect cells infected with recombinant baculovirus (Nishikawa et al., 2001c), indicating that NcSRS2 is exclusively in the cell surface. In the light of increasing evidence pointing to the importance of GPI anchored proteins in enhancing the function(s) of the major surface proteins of protozoan parasites, in this study, we examined the terminal location of the full length and three truncated versions of rNcSRS2, using the insect-baculovirus expression system that has both capabilities of performing posttranslational modifications and high yields of recombinant proteins.
2. Materials and methods 2.1. Parasites Neospora caninum tachyzoites (Nc-1 strain) were maintained in human foreskin fibroblast cells (Hs68) cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS). For the purification of tachyzoites, the parasites and host cell debris were washed in cold phosphate-buffered saline (PBS) and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0 m-pore filter (Millipore, Bedford, MA). 2.2. Cells and virus The Autographa californica nuclear polyhedrosis virus and its recombinant viruses were grown in Spodoptera frugiperda (Sf9) cells in a TC-100 insect medium (Gibco BRL, Grand
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Island, NY) supplemented with 10% FBS and 0.26% bacto tryptose broth (Difco, Detroit, MI). 2.3. Cloning of full length and truncated NcSRS2 genes To clone full length and truncated NcSRS2 genes into baculovirus, two primer sets summarized in Fig. 1A were used. The template used in the polymerase chain reaction (PCR) was N. caninum tachyzoite cDNA produced by ZAP-cDNA Synthesis kit (Toyobo, Osaka, Japan). The resulting PCR fragments were blunted by Klenow fragment, and were then ligated with the baculovirus transfer vector pBacPAK8 (Clontech, Palo Alto, CA), which had been previously digested with SmaI. 2.4. Construction of the recombinant baculoviruses, which express NcSRS2 Sf9 cells were co-transfected with a transfer vector and BaculoGold® Baculovirus DNA (PharMingen, San Diego, CA) using LipofectinTM reagent (Gibco BRL). After 4 days of incubation at 27 ◦ C, the culture supernatant containing recombinant viruses expressing NcSRS2 gene was harvested and subjected to plaque purification. After three cycles of purification, recombinant viruses-expressing NcSRS2 were obtained. 2.5. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot Sf9 cells (1 × 106 ) infected with recombinant baculovirus at 5 plaque forming units per cell (PFU/cell) were cultured with 1 ml medium. At the end of culture, the culture medium was harvested by centrifugation (5 min, 1450 × g, 4 ◦ C), and the cells were suspended in 1 ml PBS. Purified N. caninum tachyzoites (2 × 107 ) suspended in 100 l PBS, and samples of Sf9 cells (cells or culture supernatants) were mixed with 100 l, and 1 ml, 2× SDS gel-loading buffer (100 mM Tris–HCl (pH 6.8), 100 mM 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol), respectively, under reducing conditions. Uninfected Sf9 cells (1 × 106 ) were used as a negative control. The samples were heated at 95 ◦ C for 5 min and 10 l samples were subjected to SDS-PAGE. After SDS-PAGE, the protein bands in the gel were electrically transferred to a membrane (immobilon transfer membrane, Millipore). The membrane was blocked with PBS containing 3% skim milk (PBS-SM) and then incubated with anti-NcSRS2 mAb (2C8) (Nishikawa et al., 2000) diluted 1:200 with PBS-SM at 37 ◦ C for 60 min. The membrane was washed three times with PBS for 5 min and then incubated with horseradish peroxidase-conjugated mouse IgG antibody (Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:1000 with PBS-SM at 37 ◦ C for 60 min. The membrane was washed three times with PBS for 5 min and incubasted with enhanced chemo-luminecsence detection reagents (Amersham Pharmacia Biotech) for 1 min and exposed to a film. The amounts of intracellular and extracellular rNcSRS2 were determined using a density meter (Luminous Imager version 2.0, Aisin cosmos, Tokyo, Japan). 2.6. Confocal laser scanning microscopy Sf9 cells infected with baculovirus for 48 h at 5 PFU/cell were fixed with acetone, which optimized detection of protein on the cell surface and the intracellular localization (Xuan
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et al., 1995), and incubated with anti-NcSRS2 mAb diluted 1:100 with PBS containing 3% FBS for 30 min at 37 ◦ C, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Southern Biotechnology, Birmingham, AL) diluted 1:100 with PBS containing 3% FBS for 30 min at 37 ◦ C. Uninfected Sf9 cells were used as a negative control. The cells were examined on a confocal laser scanning microscopy (Leica TCS NT, Heidelberg, Germany). 2.7. Flow cytometric analysis Sf9 cells (1 × 106 ) infected with baculoviruses for 48 h at 5 PFU/cell were reacted with anti-NcSRS2 mAb diluted 1:100 with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide for 30 min at 4 ◦ C, followed by FITC-conjugated goat anti-mouse antibody diluted 1:100 with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide for 30 min at 4 ◦ C. The cells (1 × 104 ) were then analyzed on an EPICS XL flow cytometer (Coulter, Hialeah, FL). 2.8. Phospholipase C treatment The presence of the GPI anchor was assessed by digestion with phosphatidyl-inositolspecific phospholipase C (PI-PLC). Sf9 cells (1 × 106 ) infected with recombinant baculoviruses for 48 h at 5 PFU/cell were washed three times in cold Sf-900II SFM medium (Gibco BRL) without FBS. The cells were incubated with 0.1 U PI-PLC (Sigma) in Sf-900II SFM or in Sf-900II SFM without the enzyme for 2 h at 27 ◦ C. The reaction volume was 50 l. The supernatants were harvested by centrifugation (5 min, 1450 × g, 4 ◦ C) and then mixed with 50 l 2× SDS gel-loading buffer. The 10 l samples were analyzed by SDS-PAGE and immunoblot. 2.9. Peptide sequencing Sf9 cells (1 × 106 ) were infected with Ba/SRS2p41 for 48 h at 5 PFU/cell. The culture medium was harvested by centrifugation (5 min, 1450 × g, 4 ◦ C). The supernatants and the cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane by semi-dry electroblotting. The membrane was stained with Coomassie brilliant blue, and the protein band was excised from the membrane and submitted for HP G1005A protein sequencing system (Takara, Shiga, Japan).
3. Results 3.1. rNcSRS2 expression in the insect-baculovirus expression system We have generated four types of recombinant baculoviruses (full length and three truncated versions of the NcSRS2 gene) (Fig. 1), and these were used to examine the terminal location of rNcSRS2 gene products in the insect-baculovirus expression system.
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Fig. 2. Expression of rNcSRS2 in the insect-baculovirus expression system. Cell lysates (cell), and culture supernatants (sup) of Sf9 cells infected with recombinant baculoviruses for 4 days and analyzed by SDS-PAGE, and immunoblot with anti-NcSRS2 mAb under reducing conditions. The anti-NcSRS2 mAb did not react with the uninfected cells. Molecular weights are given in kDa.
By immunoblotting using the anti-NcSRS2 mAb, two major bands were detected in the cell lysate of each group of recombinant baculovirus-infected Sf9 cells: 48 and 42 kDa in Ba/SRS2p44; 46 and 40 kDa in Ba/SRS2p41; 43 and 37 kDa in Ba/SRS2p37; 31 and 25 kDa in Ba/SRS2p28 (Fig. 2). The anti-NcSRS2 mAb did not react with the uninfected cells (data not shown). Some proteolytic degradation of the rNcSRS2 may account for the presence of some minor bands. The two major bands detected in all cell lysates differed by 6 kDa in molecular weight. Culture supernatants of Ba/SRS2p41 and Ba/SRS2p37-infected cells showed the 40 and 37 kDa bands, respectively. Analysis of the N-terminal of the 40 kDa protein from the cell lysate and supernatant of Ba/SRS2p41-infected Sf9 cells yielded the amino acid sequence APFKSEN, indicating that the 53-amino acid peptide from the N-terminal was cleaved from the rNcSRS2. We infer that the higher-molecular weight proteins (48 kDa in Ba/SRS2p44, 46 kDa in Ba/SRS2p41, 43 kDa in Ba/SRS2p37, 31 kDa in BaSRS2p28) were precursors of rNcSRS2 that contain an N-terminal signal peptide, and that lower-molecular-weight proteins (42 kDa in Ba/SRS2p44, 40 kDa in Ba/SRS2p41, 37 kDa in Ba/SRS2p37, 25 kDa in Ba/SRS2p28) were mature proteins. The molecular weight (42 kDa) of the mature form of the full length rNcSRS2 (Fig. 2, lane 2) was larger than that of the authentic NcSRS2 (40 kDa) (Fig. 2, lane 1), with the latter having been detected as well in the cell lysate and supernatant of Ba/SRS2p41-infected Sf9 cells. 3.2. Kinetic analysis of rNcSRS2 expression Secreted rNcSRS2 was detectable in the supernatants of Ba/SRS2p41 and Ba/SRS2p37infected Sf9 cells (Fig. 3). While the non-expression of rNcSRS2 in the supernatant of
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Fig. 3. Kinetics of rNcSRS2 synthesis. Cell lysates and culture supernatants of Sf9 cells infected with recombinant baculoviruses for 24, 48 and 72 h analyzed by SDS-PAGE, and immunoblot with anti-NcSRS2 mAb under reducing conditions. Molecular weights are given in kDa.
Ba/SRS2p44-infected Sf9 cells was clearly attributable to the hydrophobic C-terminal tail, in the rNcSRS2 (Ba/SRS2p28), despite the truncation of the hydrophobic C-terminal tail, its expression was not detected in the supernatant. The detection of the precursor rNcSRS2 in the supernatant of Ba/SRS2p41-infected Sf9 cells may be due to an artifact of this insect–baculovirus system, such as the cytopathic effects. 3.3. Cellular localization of the rNcSRS2 The expression of the full length rNcSRS2 was observed mainly on the cell surface of Sf9 cells, while that of the truncated rNcSRS2 was limited to the internal compartment of infected cells (Fig. 4). The anti-NcSRS2 mAb did not react with the uninfected cells (data not shown). By flow cytometric analysis, expression levels of the truncated rNcSRS2, were shown, to be reduced on the insect cell surface of Sf9 cells compared to those of the full length rNcSRS2 (Fig. 5). The intact rNcSRS2 product was transported completely to the cell surface, while the truncated rNcSRS2 with deletions of 25 amino acid residues (Ba/SRS2p41) or 62 C-terminal residues (Ba/SRS2p37) were synthesized, but nonetheless remained in the internal compartment of infected cells. Moreover, the truncated recombinant (Ba/SRS2p28) lacking 147 C-terminal residues including the C-terminal hydrophobic
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Fig. 4. NcSRS2 localization in Sf9 cells infected with recombinant baculoviruses for 48 h. (A) Ba/SRS2p44infected cells. (B) Ba/SRS2p28-infected cells. Similar localization of rNcSRS2 was observed in Ba/SRS2p41 or BaSRS2p37-infected cells. The anti-NcSRS2 mAb did not react with the uninfected cells; bar = 10 m.
domain, known to be a crucial in signaling anchor protein attachment (Tomavo et al., 1992), was synthesized, albeit remained inside infected cells. 3.4. PI-PLC treatment Sf9 cells infected with recombinant baculoviruses were treated with PI-PLC to find out whether the rNcSRS2 were anchored by GPI. The rNcSRS2 expressed by Ba/SRS2p44, Ba/SRS2p41 and Ba/SRS2p37 were detected in the culture supernatants, and were of the mature protein form (Fig. 6). In the presence of PI-PLC, the intact rNcSRS2 expressed by Ba/SRS2p44 secreted 27.6% of the molecule, while 9.8, and 8.5% of the molecules were secreted in the truncated rNcSRS2 expressed by Ba/SRS2p41 and Ba/SRS2p37, respectively. Protein synthesis during incubation period may account for the detection of precursor and mature rNcSRS2 expressed by Ba/SRS2p41-infected cells in the presence and absence of PI-PLC. There was no detectable rNcSRS2 release in PI-PLC-treated Ba/SRS2p28-infected cells.
Fig. 5. Flow cytometry for Sf9 cells infected with recombinant baculoviruses for 48 h. The rNcSRS2 expression on Sf9 cells infected with wild type baculovirus (light line) and recombinant baculoviruses (heavy line) is shown.
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Fig. 6. Treatment of rNcSRS2 with PI-PLC. Sf9 cells infected with recombinant baculoviruses for 48 h were treated with PI-PLC (+) or incubated in medium alone (−). After treatment, the cells were centrifuged, and the resulting supernatants were analyzed by SDS-PAGE and immunoblotting using the anti-NcSRS2 mAb under reducing conditions. Molecular weights are given in kDa.
4. Discussion Previous studies have revealed an open reading frame of 1203 nucleotides encoding a 401-amino acid protein in the authentic NcSRS2 gene, and based on the N-terminal peptide sequence of purified NcSRS2, it contains a 53-amino acid signal peptide (Howe et al., 1998). We have obtained data demonstrating the expression of an intact rNcSRS2 protein on the cell surface of infected Sf9 cells, suggesting a similar transport mechanism between the parasite and insect cells. The distinction between the precursor and mature form of rNcSRS2 gene products expressed in the infected cells based on our findings on the analysis of the N-terminal peptide sequence of the rNcSRS2, revealed similar cleavage sites of the signal peptide in both the parasite and insect cells. The molecular weight of the mature form of the truncated rNcSRS2 with deletions of 25 amino acid residues was the same as that of the authentic NcSRS2. Present data are consistent with earlier studies that showed a relationship between the removal of the 53-amino acid signal peptide and the 25-amino acid hydrophobic C-terminal tail with the mature NcSRS2 protein (Howe et al., 1998). The cleavage of the intact rNcSRS2 from the cell surface after treatment with PI-PLC is a clear indication that the intact recombinant was a GPI-anchored molecule, and implies that GPI-attachment is extremely essential in the successful transport of NcSRS2 to the
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cell surface. With PI-PLC treatment, truncated rNcSRS2 expressed by Ba/SRS2p41 and Ba/SRS2p37 were present in the supernatants in lower amounts compared to the secretion of intact rNcSRS2. In a related study on murine cell surface protein Ly-6E, the addition of only 50% phosphaditylinositol-glycan (PI-G) moiety to the mutant proteins produced proteolytic cleavage resulted to the replacement of the C-terminal residues with a cytoplasmic tail of the transmembrane form of the lymphocyte function-associated antigen (Caras, 1991). Proteolytic cleavage occurring at other amino acid sites and GPI-attachment at new site(s) as earlier proposed (Su and Bothwell, 1989) could possibly be happening in rNcSRS2. However, there is a possibility of an experimental artifact such that PI-PLC treatment may lead to the release of non-GPI anchored proteins. This is one facet that is worth investigating in future studies. In the light of a comparatively more exhaustive study on the GPI-anchored surface proteins such as SAG1 and SAG3 (Tomavo et al., 1992; Manger et al., 1998; Dzierszinski et al., 2000) in T. gondii, a closely related species of N. caninum, an in-depth analysis of the function(s) of NcSRS2 would require additional work on its biosynthesis and other details of its transport to the cell surface which have not been examined in the present study. In conclusion, we have demonstrated that the N. caninum NcSRS2 is a transmembrane protein that contains a GPI-anchor molecule expressed in insect cells, and that the hydrophobic C-terminal domain in the NcSRS2 is an essential component for GPI-membrane attachment and transport to the cell surface. In T. gondii surface antigen SAG1, Azzouz et al. (2000) reported the inappropriateness of the insect baculovirus system for the expression of GPI-anchored proteins. The expression of GPI-anchored protein in insect cells may be due to the difference in the transport property of individual proteins. We have likewise shown the usefulness of the insect-recombinant baculovirus expression system in studies on N. caninumNcSRS2.
Acknowledgements We thank Dr. J.P. Dubey (United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and Epidemiology Laboratory) for supplying N. caninum, NC-1 isolate. We appreciate the helpful inputs of Dr. Florencia G. Claveria of the Biology Department, De La Salle University, Manila, Philippines during her sabbatical stay at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine. The first author is supported by a Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. References Azzouz, N., Kedees, M.H., Gerold, P., Becker, S., Dubremetz, J.F., Klenk, H.D., Eckert, V., Schwarz, R.T., 2000. An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection. Glycobiology 10, 177–183. Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 1441–1444.
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