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Short Communication

Short Communication Analyses of the binding between Theileria orientalis major piroplasm surface proteins and bovine red blood cells H. Takemae, T. Sugi, K. Kobayashi, F. Murakoshi, F. C. Recuenco, A. Ishiwa, A. Inomata, T. Horimoto, N. Yokoyama, K. Kato Theileria orientalis is a tick-transmitted, intraerythrocytic protozoan belonging to the phylum Apicomplexa. It is a member of the nontransforming group of Theileria species (Theileria sergenti/buffeli/orientalis) that proliferates in the bovine host as an intraerythrocytic form (Sugimoto and Fujisaki 2002). This Theileria group is now collectively referred to as T. orientalis. It can occasionally cause symptoms that include fever, anaemia and anorexia in infected cattle (Shiono and others 2001). The livestock industry in Japan suffers enormous economic losses due to bovine piroplasmosis caused by this parasite (Ota and others 2009). Major piroplasm surface protein (MPSP) is an immunodominant protein present on the parasite surface during the intraerythrocytic stage (piroplasm) (Kawazu and others 1992). Erythrocyte-stage 30–34 kDa proteins, such as MPSP, are conserved among other bovine Theileria species and Theileria equi (Formerly Babesia equi), a tick-transmitted protozoan parasite of horses (Knowles and others 1997). The MPSP proteins have a putative signal peptide and a putative ­membrane anchor domain at the N-terminus and C-terminus, ­respectively (Kim and others 1998). The MPSP gene is a useful Veterinary Record (2014) H. Takemae, PhD T. Sugi, DVM, PhD F. Murakoshi, F. C. Recuenco, DVM, PhD A. Ishiwa, PhD N. Yokoyama, DVM, PhD K. Kato, DVM, PhD National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan T. Sugi, DVM, PhD F. Murakoshi, F. C. Recuenco, DVM, PhD A. Ishiwa, PhD A. Inomata, T. Horimoto, DVM, PhD K. Kato, DVM, PhD

­molecular marker for the epidemiological study of T. orientalis; however, the molecular components that interact with the MPSP protein remain unknown. The interaction between MPSP and the surface molecules of host red blood cells (RBC) is difficult to investigate experimentally, because no T. orientalis parasites have been adapted to in vitro culture. Here, we examined the binding of MPSP recombinant proteins to RBCs. An RBC binding assay was performed as previously described with modification (Kaneko and others 2000). Briefly, glutathione S-transferase (GST)-fusion recombinant MPSP proteins (GSTMPSP) from five different genotypes (Yokoyama and others 2012) were expressed in Escherichia coli and purified by using GlutathioneSepharose 4B beads (GE Healthcare) under native conditions. The purified GST-MPSP recombinant protein produced a major band on SDS-PAGE with a molecular mass similar to its predicted size of 56 kDa (Fig 1A, lanes 3–7). Five micrograms of GST or GST-MPSP recombinant protein was mixed with 50 μl of packed bovine fresh RBCs and incubated in binding buffer (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM CaCl2; 1 mM MnCl2; 1 mM MgCl2) supplemented with complete EDTA-free protease inhibitor cocktail (Roche) for 2 h at 4°C. After centrifugation, the supernatant containing the unbound protein was pooled. The pelleted bovine RBCs were resuspended in 500 μl of incomplete RPMI-1640 medium containing 25 mM Hepes and 367 μM hypoxanthine and layered onto 500 μl of silicon oil (SIGMA-Aldrich) and then centrifuged at 15,000 g for one minute. The bovine RBCs were recovered from the bottom of the tube, washed with PBS and bound GST-MPSP recombinant proteins were eluted in 50 μl of 1.5 M NaCl. The eluate was purified by using MagneGST Glutathione Particles (Promega). GST-MPSP recombinant proteins, except for type 2, were detected in the elution fraction, whereas almost all the control GST was recovered from the unbound fraction (Fig 1B). The binding of MPSP proteins to bovine RBCs was detected in fresh RBCs, not preserved RBCs. This result suggests the possibility that MPSP binds to bovine fresh RBCs.

doi: 10.1136/vr.102535 Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan K. Kobayashi, DVM, PhD Division of Microbiology and Immunology, Department of Host-Parasite Interaction, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan E-mail for correspondence: [email protected] Provenance Not commissioned; externally peer reviewed Accepted May 25, 2014

FIG 1: Red blood cell (RBC) binding of major piroplasm surface protein (MPSP) recombinant proteins. (A) Bacterially expressed glutathione S-transferase (GST) and GST-MPSP recombinant proteins from five different genotypes were purified by using Glutathione-Sepharose 4B beads. Each recombinant protein (5 μg) was separated by 12.5 per cent SDS-PAGE and subjected to Coomassie Brilliant Blue staining (GST, lane 2; GST-MPSP (Type 1), lane 3; GST-MPSP (Type 2), lane 4; GST-MPSP (Type 3), lane 5; GST-MPSP (Type 4), lane 6; GST-MPSP (Type 5), lane 7). (B) 5 μg each of GST, GST-MPSP (Type 1–5) recombinant protein was incubated with packed bovine RBCs. After the unbound protein was separated by centrifugation of the bovine RBCs layered onto silicon oil, the bound protein was eluted with 1.5 M NaCl and purified by using glutathione beads. The input (2 per cent per total volume), unbound (2 per cent per total volume), and eluted proteins were analysed by Western blotting with an anti-GST antibody

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Short Communication GST

GST-MPSP (Type 1)

G 1 2 3 4 5 6 7 8 9 10 G

G 1 2 3 4 5 6 7 8 9 10 G

G 11 12 13 14 15 16 17 18 19 20 G

G 11 12 13 14 15 16 17 18 19 20 G

G 21 22 23 24 25 26 27 28 29 30 G

G 21 22 23 24 25 26 27 28 29 30 G

G 11 12 13 14 15 16 17 18 19 20 G GST-MPSP (Type 2)

GST-MPSP (Type 3)

GST-MPSP (Type 4)

GST-MPSP (Type 5)

N-glycan (1-8) 1:M9 2:NA2 3:A2 4:NA2F 5:MGA2B 6:NA3 7:A3 8:NA4 O -glycan (9-10) 9:STn 10:T GAG (11-16) 11: Heparin 12: 2-O-Desulfated Heparin 13: 6-O-Desulfated Heparin 14: N-Desulfated Heparin 15: N-Desulfated reN-Acylated Heparin 16: Chondroitin sulfate Lewis antigen (17-20) 17:SLea 18:SLex 19:SLea 20:SLex Lac (21-27) 21:Lac 22: α2-3 Sialyl lactose 23: α2-6 Sialyl lactose 24: α2-3 Sialyl LacNAc 25: α2-6 Sialyl LacNAc 26: Lacto-N-tetraose 27: Lacto-N-neo-tetraose ABO blood group (28-30) 28: A trisaccharide 29: B trisaccharide 30: H(O) disaccharide H. Takemae et al.

FIG 2: Glycoarray analysis. glutathione S-transferase (GST) or GST-major piroplasm surface protein (MPSP) (Type 1 to 5; 10 μg each) recombinant protein was incubated with the glycoarray plate. The plate was then incubated with an anti-GST antibody. Alexa Fluor 546 goat anti-mouse IgG served as the secondary antibody. Fluorescence intensity of the plate-bound protein was measured with a fluorescence scanner. The oligosaccharides spotted on the glycoarray plate are listed. G shows the grid marker

The interaction of MPSP with various oligosaccharides was investigated by using an in vitro glycoarray system (Fig 2). The glycoarray plate BS-X1707 (Sumitomo Bakelite, Tokyo, Japan) was incubated with 10 μg of GST or GST-MPSP recombinant protein according to the manufacturer’s instructions. After being washed, the plate was incubated with 10 μg/ml of anti-GST antibody produced in mouse (Medical & Biological Laboratories, Nagoya, Japan), following by the secondary antibody reaction using Alexa Fluor 546 goat anti-mouse IgG (Life Technologies). The plate was then scanned with ScanArray Lite (PerlinElmer, Waltham, Massachusetts, USA). The fluorescent signal of GST-MPSP was detected at the spot corresponding to heparin and desulfated heparin, whereas the control GST did not react with these oligosaccharides (Fig 2, 11–15). MPSP did not bind to N-glycans, O-glycans, Lewis antigens, lactose derivatives or ABO blood group antigens (Fig 2). Fc-fusion MPSP proteins from type 1 and 2 prepared using a baculovirus expression system bound to heparin and heparin derivatives, whereas Fc protein bound only to heparin (data not shown). GST-MPSP from five different genotypes produced almost identical binding patterns, suggesting that MPSP binds to heparin/heparin derivatives and that the genotype of the MPSP does not affect its binding to GAGs. In this study, we showed that T. orientalis MPSP binds to bovine RBCs and that heparin/heparin derivatives are MPSP binding components. The MPSP recombinant proteins from type 1 and 3 bear two potential glycosaminoglycan (GAG)-binding motifs, characterised by XBBXBX and XBBBXXBX (where B is lysine or arginine and X is any other amino acid) (Cardin and Weintraub 1989) and these from type 2, 4 and 5 bear one GAG-binding motif. This supports the result of our glycoarray assay in which all MPSP recombinant proteins from five different genotypes bound to heparin and modified heparin derivatives (Fig 2), although it is unclear why the MPSP recombinant protein from type 2 did not bind to bovine RBCs in the RBC binding Veterinary Record | August 9, 2014

assay (Fig 1B). Heparin is only present in mast cells, whereas heparan sulfate is present on all cell surfaces including RBCs. Heparin and heparan sulfate can block the invasion of erythrocytes by Plasmodium falciparum merozoites and cytoadherence of parasitised erythrocytes to endothelial cells (Xiao and others 1996). In Babesia bovis, specific sulfated glycoconjugates, such as dextran sulfate, heparin, heparan sulfate, fucoidan and chondroitin sulfate B strongly inhibit parasite growth (Bork and others 2007). Heparin also inhibits the invasion of host RBCs by T. orientalis merozoites in a dose-dependent manner (Hagiwara and others 1997). Thus, sulfated polysaccharides, such as heparin and heparan sulfate inhibit parasite entry into RBCs. Our study suggests that MPSP could bind to heparan sulfate on bovine RBCs. Further studies of other heparin-binding molecules will ­provide a better understanding of T. orientalis attachment and invasion to RBCs.


This study was supported by grant-in-aids for Young Scientists, Exploratory Research, Scientific Research on Innovative Areas (3308) from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN), the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry and the Program to Disseminate Tenure Tracking System from the Japan Science and Technology Agency (JST).


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Short Communication HAGIWARA, K., TAKAHASHI, M., ICHIKAWA, T., TSUJI, M., IKUTA, K. & ISHIHARA, C. (1997) Inhibitory effect of heparin on red blood cell invasion by Theileria sergenti merozoites. International Journal of Parasitology 27, 535–539 KANEKO, O., FIDOCK, D. A., SCHWARTZ, O. M. & MILLER, L. H. (2000) Disruption of the C-terminal region of EBA-175 in the Dd2/Nm clone of Plasmodium falciparum does not affect erythrocyte invasion. Molecular and Biochemical Parasitology 110, 135–146 KAWAZU, S., SUGIMOTO, C., KAMIO, T. & FUJISAKI, K. (1992) Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction. Molecular and Biochemical Parasitology 56, 169–175 KIM, S. J., TSUJI, M., KUBOTA, S., WEI, Q., LEE, J. M., ISHIHARA, C. & ONUMA, M. (1998) Sequence analysis of the major piroplasm surface protein gene of benign bovine Theileria parasites in east Asia. International Journal of Parasitology 28, 1219–1227 KNOWLES, D. P., KAPPMEYER, L. S. & PERRYMAN, L. E. (1997) Genetic and biochemical analysis of erythrocyte-stage surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria species. Molecular and Biochemical Parasitology 90, 69–79 OTA, N., MIZUNO, D., KUBOKI, N., IGARASHI, I., NAKAMURA, Y., YAMASHINA, H., HANZAIKE, T., FUJII, K., ONOE, S., HATA, H., KONDO, S., MATSUI, S., KOGA, M., MATSUMOTO, K., INOKUMA, H. & YOKOYAMA, N.

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Analyses of the binding between Theileria orientalis major piroplasm surface proteins and bovine red blood cells H. Takemae, T. Sugi, K. Kobayashi, F. Murakoshi, F. C. Recuenco, A. Ishiwa, A. Inomata, T. Horimoto, N. Yokoyama and K. Kato Veterinary Record 2014 175: 149 originally published online June 18, 2014

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Analyses of the binding between Theileria orientalis major piroplasm surface proteins and bovine red blood cells.

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