Protein changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria parva

Molecular and Biochemical Parasitology, 37 (1989) 159-170

159

Elsevier MOLBIO 01219

Protein changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria parva Chihiro

S u g i m o t o 1'', L u c y M . M u t h a r i a 2, P a t r i c i a A . C o n r a d 1 ' ' ' , T h o m a s T . D o l a n ~, Wendy C. Brown ~'''', Bruno M. Goddeeris 1 and Terry W. Pearson 3

Ilnternational Laboratory for Research on Animal Diseases, Nairobi, Kenya; 'Department of Biochemistry, University of Nairobi, Nairobi, Kenya; and 3Department of Biochemistry and Microbiology, University of Victoria, Victoria. British Columbia, Canada

(Received 13 April 1989; accepted 7 June 1989)

Protein and glycoprotein changes induced in bovine lymphoblasts by infection with Theileria parva were analyzed by high-resolution two-dimensional gel electrophoresis. Uninfected and infected cloned bovine T and B lymphoblasts were biosynthetically labeled with [-~S]methionine and their two-dimensional autoradiographic patterns were compared with each other and with the pattern obtained using purified labeled schizonts. Ten proteins were found in infected cells which were not present in uninfected cells, and seven of these were detected in preparations of purified schizonts. Four glycoproteins were detected on the surface of infected cells labeled with [3H]borohydride while a major glycoprotein present on uninfected cells disappeared or was reduced in infected cells. Other minor changes in protein and glycoprotein patterns were also observed. Key words: Theileria parva; Schizont; T lymphoblast; Two-dimensional gel electrophoresis; Surface protein; Surface glycoprotein

Introduction Theileria p a r v a is a tick-borne protozoan parasite which causes East Coast fever (ECF) in cattle in East, Central and Southern Africa. Sporozoites of T. p a r v a inoculated by the tick vector invade host lymphocytes and develop into intracellular schizonts. The schizonts induce the host cell to undergo blast transformation and continCorrespondence address: T.T. Dolan, ILRAD, P.O. Box

30709, Nairobi, Kenya. Present address: "First Research Division, National Institute of Animal Health, Tsukuba, lbaraki 305, Japan; "'Department of Veterinary Microbiology and Immunology, University of California, Davis, CA, U.S.A.; ""Department of Veterinary Microbiology and Parasitology, Texas A & M University, College Station, TX, U.S.A. Abbreviations: 2D-PAGE, Two-dimensional polyacrylamide

gel electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCGF, T cell growth factor; PBS, phosphate-buffered saline; PBSG, PBS-glucose FBS, fetal bovine serum.

ued proliferation, frequently resulting in fatal lymphoproliferative disease. A major component of the protective immune response to T. parva is that of cytotoxic T lymphocytes, which are genetically restricted by bovine class I major histocompatibility antigens [1-4]. Detailed studies of the cytotoxic responses induced by T. p a r v a have been facilitated by the ability to cultivate schizont-infected cell lines in vitro either by obtaining infected lymphoblastoid cells from infected cattle or by infecting normal bovine lymphocytes in vitro with T. parva sporozoites [5]. Despite extensive studies, the antigens on Theileria-infected cells which stimulate either humoral or cell-mediated immune responses in cattle have not been identified [6,7]. In this study, the proteins and glycoproteins of cloned T. p a r v a - i n f e c t e d bovine lymphoblasts were compared with those of uninfected lymphoblasts from the same cloned cell line. Two-dimensional polyacrylamide gel electrophoresis (2DP A G E ) was used to compare radiolabeled proteins and glycoproteins from the lymphoblasts. In

0166-6851/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

order to determine which proteins were contributed by the parasite schizonts, a new technique [8] was used to purify schizonts from biosynthetically labeled, infected lymphoblasts. The objective of the study was to identify proteins and glycoproteins which result from infection with T. parva and which might be target antigens for the immune response. Materials and Methods

Cells and parasites. Four cloned bovine T lymphoblastoid cell lines were studied. Three of these lines, 657.G6 [9], 639.B7 [10], and T19.4 [11], were cultured in RPMI-1640 medium (Gibco, Paisley, U.K.) containing 10-20% (v/v) fetal bovine serum (FBS, Hyclone, Logan UT, U.S.A.), 2 mM L-glutamine (Gibco), 50 ~g m l ~ gentamycin (Gibco) and 10-20% (v/v) T cell growth factor (TCGF) as described [12]. A Theileria-specific helper T cell clone, T16.13 was cultured in the presence of TCGF and irradiated stimulator cells [13]. These T cell clones were infected in vitro with T. parva sporozoites as previously described [14] and maintained in the same culture medium as above but without TCGF. Two B cell clones infected with T. parva, D409B1 and D409B4, were also used in this study. Theilerial stocks and sporozoite stabilates (Stb) used in this study were Muguga (Stb 836) for 657.G6, Muguga (Stb 1004) for cell line 639.B7, T16.13, T19.4, D409B1 and D409B4 and Marikebuni (Stb 2245) for 639.B7 and T19.4. A bovine B lymphoblastoid cell line established from a calf with bovine lymphosarcoma, BL20 [15], and a T lymphoblastoid cell line transformed by bovine herpesvirus type 3, MCF629, were also tested. Metabolic labeling of cells with [35S]methionine. The cells were centrifuged at 200 × g for 10 min and resuspended at a concentration of 1 × 106 cells ml I in 10 ml of RPMI-1640 containing only 5% of the normal methionine concentration, 10% (v/v) dialyzed heat-inactivated FBS, 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes; Sigma, St. Louis, MO, U.S.A.), 2 mM L-glutamine, 50 ~g ml- 1 gentamycin and 200 ~Ci [35S]methionine (SJ204; Amersham, U.K.) in a 25-cm2 tissue culture flask (Costar, MA, U.S.A.).

For labeling uninfected T lymphoblasts, TCGF was added to the medium described above. Flasks were gassed with 5% CO2 in air and incubated horizontally for 18 h at 37°C.

Schizont

purification. Cells labeled with [35S]methionine were centrifuged at 2130 x g for 10 min and resuspended in Hepes buffer (10 mM, pH 7.4) containing 2% (w/v) Ficoll 400 (Pharmacia, Uppsala, Sweden). The cells were lysed using 10 I~g ml-t aerolysin toxin [16} and placed on a 45 and 65% (v/v) Percoll (Pharmacia) discontinuous gradient. Schizonts were separated by ultracentrifugation in an SW41 Ti rotor (Beckman Instruments, CA, U.S.A.) at 26000 rev./min for 30 min and collected from the band at the interface between the 45 and 65% Percoll solutions as described [8]. Cell surface glycoprotein labeling by sodium boro[3H]hydride reduction. Cell surface glyco-

proteins were labeled by reduction with sodium boro[3H]hydride as described [17]. Cells (1 x 107) were washed once with ice-cold phosphate-buffered saline (PBS; 10 mM, pH 8.0), resuspended in 1 ml of 10 mM NalO4 in PBS, and incubated for 7 min on ice. To this suspension, 0.2 ml of 0.1 M glycerol in PBS was added and the volume was adjusted to 10 ml by the addition of PBS. After centrifugation at 200 × g for 15 rain at room temperature, the cells were resuspended in 1 ml of PBS (10 mM, pH 7.4) and 2.5 mCi of sodium boro[3H]hydride (TRK45, Amersham) was added. The cells were incubated for 15 min at room temperature, centrifuged at 200 x g for 15 min at 4°C, and washed again with PBS containing 5% (v/v) FBS.

1251-labeling of surface proteins. Cells were centrifuged and washed twice with Dulbecco's PBS containing 0.1% (w/v) glucose (PBSG), and resuspended in PBSG at a concentration of 1 x 10 7 ml-~ 100 I-d of the cell suspension was transferred to a glass tube (12 x 75 mm) previously coated with 10W Ixg of Iodogen (Pierce Chemical Co., IL, U.S.A.) according to the manufacturer's instructions. Two hundred I~Ci of Na125I (IMS 30, Amersham International) was added to the cell suspension and the tube was incubated for 15 min

161 at room temperature. The reaction was stopped by transferring the cell suspension to a microcentrifuge tube. The cells were centrifuged in a microcentrifuge (Eppendorf Ger~itebau, Hamburg, F.R.G.) at 13000 x g for 2 s and then washed three times with PBSG.

Sample solubilization and determination of radioactivity. Prior to 2D-PAGE, radiolabeled samples were solubilized in pH 9.5 solubilization buffer as previously described [16]. Solubilized material was stored at -70°C. To determine the amount of [35S]methionine which was incorporated into cellular or schizont proteins, 2 p.l of the solubilized material lysate was placed on a filter paper strip and allowed to air-dry before boiling for 10 min in fresh 10% (w/v) trichloroacetic acid. The filter paper was washed with distilled water, methanol and acetone and dried under a heat lamp [18]. The radioactivity was determined using a scintillation counter (LS6800, Beckman Instruments).

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 3H-labeled glycoproteins. Aliquots of 3H-labeled cells were solubilized in SDS sample buffer [17]. Electrophoresis was performed on a slab gel containing a 5-15% (w/v) polyacrylamide gradient [19].

Two-dimensional polyacrylamide gel electrophoresis. The radiolabeled samples were thawed and centrifuged in a microcentrifuge to remove insoluble aggregates immediately prior to 2D-PAGE analysis. Samples were applied to isoelectric focusing gels in the ISO-DALT system [20,21]. Ampholines used in the first dimension tube gels were a 2:1 mixture of Ampholytes pH 3.5-10 and pH 4-45 (LKB, Bromma, Sweden). Polyacrylamide gradient gels (7.5-16.5%o, w/v) were used for electrophoresis in the second dimension. To each first dimension tube gel, 2.5 × 106 cpm [35S]methionine-labeled cell proteins or 5 x 104 cpm of [35S]methionine-labeled schizont proteins were applied. For 3H-labeled glycoproteins, samples equivalent to 3 x 106 cells and for 125I-labeled proteins, samples equivalent to 3 × 104 cells were applied.

Detection of radioactivity. Gels were fixed, dried under vacuum at 80°C and exposed to Fuji RX100 film (Fuji Film Co., Tokyo, Japan) for 3--7 days at -70°C with an intensifying screen to detect ~25I-labeled proteins, and for 2--4 weeks at room temperature to detect asS-labeled proteins. Gels with tritiated samples were processed for fluorography as described [22], dried under vacuum at 60°C, and exposed to Fuji RX-100 film for 2-8 weeks at -70°C.

Comparison of two-dimensional gel patterns. Autoradiographs were compared by placing one autoradiograph over another on an X-ray light box. Superimposition of gel spot constellations allowed easy detection of qualitative differences in gel spots and in some cases permitted detection of quantitative differences. This simple procedure is accurate and reliable because the ISODALT multiple 2D gel system allows 10 gels (poured from the same batch of reagents) to be run simultaneously. Interpretation of gel patterns was performed using previously published criteria [23,241 . Results

Comparison of [35S]methionine-labe!ed proteins of uninfected and infected lymphoblasts and purified schizonts. Fig. 1 shows 2D-PAGE autoradiography patterns Of 35S-labeled proteins from cloned 657.G6 uninfected lymphoblasts (Fig. 1A), cloned 657.G6 lymphoblasts infected with T. parva (Muguga) (Fig. 1B) and T. parva (Muguga) schizonts (Fig. 1C) purified from the 657.G6 cell line. Autoradiographs of the 2D gels of infected cells showed a total of 10 proteins that were not present in autoradiographs of gels of uninfected cells (Fig. 1A and B). There were no apparent quantitative differences in the expression of proteins seen in uninfected and infected cells. Of the 10 qualitative differences, 7 were observed on the autoradiograph of gels of the purified schizonts (Fig. 1C). The most discernible and reproducible of these schizont proteins (69, 56, 46 and 41 kDa) are marked on the autoradiographs in Fig. 1A, B and C. The remaining 3 proteins, one of 31 kDa in the neutral region, one of 23 kDa at the acidic end, and a third of 14 kDa at the basic end of the

A

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Fig. 1. Two-dimensional polyacrylamide gel clectrophoresis: autoradiographs of [35S]methioninc-labelcd proteins of uninfected and T. parva-infected cloned T lymphoblasts and purified schizonts. (A) 657.G6 uninfected cloned T lymphoblasts. (B) 657.G6 cells infected with T. parva (Muguga). (C) Schizonts purified from 657.G6 cells infected with T. parva (Muguga). The autoradiograms arc orientated with the acidic end on the left and basic end on the right. The locations of four of the seven major spots found in purified schizonts and infected cells, but not in uninfected cells, are indicated by arrows. Protein spots seen in infected cells, but not in uninfected cells or schizonts, are indicated by boxes. In the first dimension gels, 2 × 1(~' TCA-prccipitablc cpm of uninfected or infected cell lysates or 5 × 10s cpm of schizont lysate ",,,'ere Loaded.

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gel, expressed in infected cells were not observed in the autoradiograph of purified schizonts. None of the schizont protein spots could be superimposed on the protein spot patterns of uninfected cells. The 31- and 14-kDa proteins observed in the 657.G6 infected cell line were also identified in autoradiographs of both of the cloned T16.13 and the T19.4 lymphoblasts infected with the Muguga (Fig. 2B) and Marikebuni (Fig. 2C) stocks of T. parva (results with T19.4 cells not shown) and T. parva (Muguga)-infected B cell lines D409B1 and D409B4 (data not shown). These proteins were absent in uninfected T16.13 (Fig. 2A) and T19.4 lymphoblasts (data not shown) and in the bovine lymphosarcoma cell line, BL20, and the virally

Fig. 2. Two-dimensional polyacrylamidegel electrophoresis: autoradiographs of [~SS]methionine-labeled proteins of uninfected and T. parva-infected cloned T lymphoblasts. (A) TI6.13 uninfected cloned T lymphoblasts. (B) Tl6.13 cells infected with T. parva (Muguga). (C) Tl6.13 cells infected with T. parva (Marikebuni). The autoradiograms are orientated with the acidic end on the left and basic end on the right. The locations of four of the seven major spots found in purified schizonts and infected cells, but not in uninfected cells, are indicated by arrows. Protein spots seen in infected cells, but not in uninfected cells or schizonts, are indicated by boxes. In the first dimension gels 2 × l& TCA-precipitablecpm was loaded. transformed cell line MCF629 (data not shown). Again no marked quantitative differences were observed in the expression of proteins between uninfected and infected T16.13 and T19.4 cells. There were no differences observed between the 2D gel patterns of T16.13 lymphoblasts infected with the Muguga stock (Fig. 2B) and the same cells infected with the Marikebuni stock of T. parva (Fig. 2C). Similar results were observed when T19.4 and 657.G6 lymphoblastoid cells infected with the two parasite stocks were compared (data not shown).

Comparison of cell surface glycoproteins. Surface glycoproteins of uninfected cloned 657.G6, 639.B7 and T19.4 lymphoblasts and the same lympho-

164

blasts infected with the Muguga and Marikebuni stocks of T. parva were labeled by sodium boro[~H]hydride reduction and analyzed by SDSP A G E (Fig. 3) and 2D-PAGE (Fig. 4). There were marked differences in surface-labeled glycoproteins between uninfected and infected cells as determined by one-dimensional SDS-PAGE. A major surface glycoprotein of 140 kDa (Fig. 3C) was detected in uninfected 657.G6, 639.B7, and T19.4 lymphoblasts (Fig. 3, lanes 1, 4 and 7) and was not observed in the 657.G6 and T l t ) . 4 cell~ i n f e c t e d w i t h the Marikebuni or Mu-

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guga stocks of T. parva (Fig. 3, lanes 2, 3, 8 and 9). The 140-kDa glycoprotein was much reduced in quantity in 639.B7 cells infected with the same stocks (Fig. 3, lanes 5 and 6). The infected cells all expressed 165- and 215-kDa glycoproteins (Fig. 3a and b) which were not seen in the uninfected cclls. A glycoprotein of 120 kDa (Fig. 3d) was identified on SDS-PAGE profiles of all infected cells except 639.B7 lymphocytes infected with the Muguga stock of T. parva (Fig. 3, lane 6). In the Marikebuni-infected T19.4 cells, there was an additional ~lvcoprotein of 185 kDa (Fig. 3, lane 8).

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Fig. 3. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophorcsis: fluorograph of ~H-labeled surface glycoproreins of uninfected and T. parva-infected cloned T lymphoblasts. (1) 657.G6 uninfected cloned T lymphoblasts. (2) 657.G6 cells infected with T. parva (Marikebuni). (3) 657.G6 cells infected with 7". parva (Muguga). (4) 639.B7 uninfected cloned T lymphoblasts. (5) 639.B7 cells infected with T. parva (Marikebuni). (6) 639.B7 cells infected with T. parva (Muguga). (7) T19.4 uninfected cloned T lymphoblasts. (8) T19.4 cells infected with T. parva (Marikebuni). (9) T19.4 cells infected with T. parva (Muguga). Positions of glycoproteins of 215 (a) 165 (b) and 120 (d) kDa in infected cells and 140 kDa (c) in uninfected cells are indicated on the left. The 140-kDa glycoprotein was detected in a decreased amount in 639.B7 infected cells (lane 5 and 6. arrows). Additional glycoproteins of 185 kDa (lane 8). 185 kDa and 210 kDa (lane 9) in TlC).4-infected cells are indicated by arrows. Samples equivalent re) 1 × I(P cells were loaded in each lane.

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Fig. 4. Two-dimensional polyacrylamide gel electrophoresis: fluorographs of 3H-labeled surface glycoproteins of uninfected and T. parva-infected cloned T lymphoblasts. (A) 657.G6 uninfected cloned T lymphoblasts. (B) 657.G6 cells infected with T. parva (Marikebuni). (C) 657.G6 cells infected with T. parva (Muguga). (D) 639.B7 uninfected cloned T lymphoblasts. (E) 639.B7 cells infected with T. parva (Marikebuni). (F) 639.137 cells infected with T. parva (Muguga). (G) T19.4 uninfected cloned T lymphoblasts. (H) T19.4 ceils infected with T. parva (Marikebuni). (I) T19.4 cells infected with T. parva (Muguga). Glycoproteins of 215 (a), 165 (b) and 120 kDa (d) in infected cells, and 140 kDa (c) in uninfected cells, are indicated in the fluorograms. The positions in the fluorograms where these proteins were expected but not found are indicated by open arrows. The spot train of a 33-kDa glycoprotein is indicated by boxes and marked (e). Other spots in the fluorograms of the infected cells which did not match spots in the fluorograms of uninfected cells are indicated by boxes. Samples equivalent to 3 × 106 cells were loaded on the first dimensional gels.

lbl~ This glycoprotein and another of 280 kDa were seen in the same cells infected with T. parva (Muguga) (Fig. 3, lane 9). The three glycoproreins of 215,165 and 120 kDa were also identified in 7. pan'a-infected B lymphoblasts, D409BI and D409B4, but not in a lymphosarcoma cell line, BL20, or in a virally transformed cell line, MCF629. On 2D gels, the 165-kDA (b) and 215-kDa (a) glycoproteins (Fig. 4B, C, E, F, H, and I) in the infected cells, and the 140-kDa glycoprotein (c) in uninfected cells (Fig. 4A, D and G) were identified as a series of spots extending from the neutral region to the basic end in the upper part of the gels. The glycoprotein of 120 kDa (d) observed in all the infected cells appeared on 2DP A G E as a train of spots running at the acidic end of the gcls (Fig. 4B, C, E, F, H and I). An additional glycoprotein of 33 kDa (c), which was not defined in one dimensional S D S - P A G E , was found in all infected cells as a doublet in the neutral region (Fig. 4B, C, E, F, II and 1). "['he expression of this glycoprotein was most abundant in T19.4 cells infected with the Marikebuni stock of T. parva (Fig. 4H). These tritiated surface glycoproteins of 215, 165, 120 and 33 kl)a were absent from uninfected cells (Fig. 4A, D and G). A trace amount of the glycoprotein of 140 kDa which was found in the uninfected cells was detected in 639.B7 lymphoblasts infected with the Muguga stock (Fig. 4F,c). Other 31t-labeled glycoprotein spots that were detected in some of the infected lymphoblasts, but not in the corresponding uninfected cells, are also indicated by boxes in Fig. 4. Comparison o f cell surface proteins. Cell surface proteins of uninfected 657.G6, 639.B7 and T19.14 lymphoblasts and the same cells infected with the Marikebuni and Muguga stocks were labeled with

1251 and analyzed by 2 D - P A G E (Fig. 5). In the 2D gel autoradiographs of all infected cells, spot trains were present at the acidic end of the gels and were not seen in the autoradiographs of gels of uninfected cells (except T19.4). The apparent molecular weight of this iodinated protein was 120 kDa and the spot of this protein could be superimposed on the spot of the 121) kDa glycoprotein shown in Fig. 4 (spot d). Other t2Sl-labeled protein spots which were present in some of the infected lymphoblasts but absent in uninfected cells are also indicated by boxes in Fig. 4. Thcre were no major diffcrenccs between 2 D - P A G E profiles of the cells infected with the Marikcbuni stocks and the same cell infected with the Muguga stock of T. parva. Discussion

Several proteins which were present in Theileria-infected, cloned bovine T lymphoblasts were not identified in the uninfected cloned T lymphoblasts or the purified schizonts. Uninfected TCGF-dependent or antigen-specific T cell clones (rather than unstimulated lymphocytes) were used for comparison with infected cells to reduce the possibility that the protein differences detected were the result of blastogenesis alone and were not infection-specific. The protein changes detected could not be attributed to T C G F , since infected cells cultured with or without T C G F showed identical 2D gel patterns (data not shown). Analysis of the 2D gel autoradiograph patterns of [35S]methionine-labeled proteins showed that the majority of labeled proteins were present in similar quantities in infected and uninfected cells. There were, however, 10 proteins found in infected cells which were not detected in uninfected ceils of the same cloned cell lines. The

Fig. 5. Two-dimensionalgel autoradiographs of t>l-labeled surface proteins of uninfected and T. parva-mfccted chined T lymphohlasts. (A) 657.G6 uninfected cloned T lymphoblasts. (B} b57.Ge~cells infected with 7". parva (Marikebuni). (C) b57.G6 cells infected with T. parva (Muguga). (D) 639.B7 uninfected cloned T I}mphoblasts. (E) 639.B7 cells infected with T. pan'a (Marlkebuni). (F) 639.B7 cells infected with 7. parva (Muguga). (G) 3"19.4 uninfected cloned T b.mphoblasts. (II)119.4 cells infected with T. parva (Marikebuni). (I) 1"19.4 cells infected with T. parva (Muguga). Proteins of t20 kDa found in all infected cells and "119+4 uninfected cells, hut not in other uninfected cells, are indicated b,, solid arrows. The corresponding positions in the autoradiograms of the uninlected cells are indicated by open arrows. Other spots in the autoradiograms of infected cells ~hich did not match spots in the autoradiograms of uninfected cells are indicated by boxes. Samples e q u i v a l e n t t o 5 x Ill ~ cells v,cre loaded on the tirst dimension gels.

168 presence of sevcn of these proteins in preparations of purified schizonts indicated that they were proteins on or within T. parva schizonts. The other three proteins (31, 23 and 14 kDa) were found in 657.G6 cells infected with T. parva (Muguga). Two of these, the 31 and 14 kDa proteins, were found in all T. parva-infected T and B cells, and appear to be infection-specific. These two proteins are not associated with lymphoblasts transformed by causes other than T. parva. They are either products of the parasites transported into the host cell cytoplasm, or products of the host cell induced as a result of infection. The intracellular localization of these two proteins was not determined. Molecular changes were also detected on the surface of the Theileria-infected bovine lymphoblasts. After surface-labeling with boro[3H]hydr ide, glycoproteins of 215, 165, 120 and 33 kDa were detected on the surface of infected T lymphoblasts, while a major surface glycoprotein of 140 kDa, present in the uninfected cells, disappeared or was quantitively reduced following infection. The glycoproteins of 215, 165 and 120 kDa were also detected in T. parva-infected B lymphoblasts but not in BL20 or MCF629. A protein of 120 kDa was also identified by surface t25I-labeling of infected cells. This surface protein appeared to correspond to the 3H-labeled glycoprotein of 120 kDa. This molecule was detected in ~251-1abeled uninfected T19.4 lymphoblasts and is probably not infection-specific. We were unable to demonstrate the presence of the 120-kDa protein on the surface of T19.4 uninfected cells by boro[3H]hydride reduction, perhaps because it is not glycosylated to the same extent as in the infected cells. The other glycoproteins of 215, 165 and 33 kDa did not label with the 125I perhaps because of a lack of tyrosine residues or because of masking by extensive glycosylation.

Cell surface carbohydrates arc of special importance in cell-cell interaction, adhesiveness, cellular growth regulation and cell differentiation [25]. Changes in cell surface glycoprotein expression are often associated with neoplastic transformation and lymphocyte blast transformation [26-28]. Since Theileria-infected cell lines exhibit a seemingly unlimited growth potential in vivo [5,29], it is not unrealistic to expect that the surface changes observed on Theileria-infected cells are in some way related to the transformation and proliferation events. The surface changes induced by T. parva infection also attract immunological interest, because cytotoxic T cells must recognize changes in the parasitized cell surface [30] while cytotoxic T cells are able to detect parasite strain differences [11,30,31]. The present study provides evidence for parasite-induced changes in host cell proteins during T. parva infection. These changes, especially at the cell surface, may be particularly important in the induction of host immune responses to this parasite. In addition, some of the proteins may play a role in the autologous proliferation of infected lymphoblasts.

Acknowledgements We thank Dr. D. Grab ( I L R A D ) for supplying aerolysin, Drs. I. Morrison (ILRAD) and H. Reid (Animal Disease Research Association, Moredun Institute, Edinburgh, U.K.), for cell lines, Mr. R. Njamunggeh and Ms. K. Logan for technical assistance and Doris Churi and Jennifer Duggan for typing the manuscript. C. Sugimoto is a visiting scientist from the Tropical Agriculture Research Centre, Tsukuba, Japan. B.M. Goddeeris was supported by the General Administration for Development Cooperation of Belgium. This is I L R A D publication number 745.

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