296s Biochemical SocietvTransactions ( 1 992) 20 IDENTIFICATION AND CHARACTERISATION OF GLYCOSYL-INOSITOLPHOSPHOLIPJDSIN TOXOPLASMA GONDII BORIS STRIEPEN, STANISLAS TOMAVO, JEANFRANCOIS DUBREMETZ* AND RALPH T. SCHWARZ Zentrum fur Hygiene, Philipps-Universit-Marburg, 3550 Marburg, Germany; *INSERM U 42, Villeneuve D'Ascq, France Toxoplasma gondii, a sporozoan parasite, is the causative agent of toxoplasmosis, which is generally asymptomatic but can cause severe complications as connatal infection and secondary oportunistic infection in the course of AIDS. Using human patient sera, previous studies have described a lipophilic low molecular weight ("LMW") antigen, which elicits an IgM response in human primary infection (1). Monoclonal antibodies specific for this antigen have recently been obtained (2). We have used these antibodies to characterise the "LMW" antigen and show here, that it consists of a family of glycosylinositolphospholipids (GIPLs). RH-strain tachyzoites were propagated in cultures of Vero cells. Parasites were released using a Dounce homogenizer and purified by glass wool filtration (3). Parasites were extracted with 1,5 and 0,5 ml CHCI3/MeOH 2:l to remove less polar lipids followed by two extractions with 1 ml CHC13/MeOH/H20 10: 10:3 ( 0 , which extracts the more polar glycolipids such as dolichol derivates and GIPLs (4). CMW-extractable molecules were subjected to SDS-PAGE followed by transfer to nitrocellulose sheets. After saturation with a solution of 5 % dry milk, nitrocellulose sheets were incubated with monoclonal antibodies (T54E10 and T3F12) and sera from humans with an acute toxoplasmosis (kindly provided by Dr. B. Fortier, CHU Lille), followed by antiimmunoglobuline antibodies coupled to alkaline phosphatase. Resolution using NBT/BCIP substrates showed a band with an apparent molecular weight of 4.6 kD. Furthermore, this 4.6 kD antigen was also sensitive to periodate treatment as previously described by Sharma et al. ( 1 ) for the LMW antigen.

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Figure 1: Thin layer radiochromatograms of glycolipids of T. gondii. Analysis of the 'bunanol component' of CMW-extracts after metabolic labeling. A: labeled with [3H]-glucosamine, the table above indicates recognition by mAbs and human patient sera, +: recognized, -: not recognized B: labeled with PHIpalmitic acid C: labeled with PHI-mannose D: labeled with [3H]-ethanolamine In order to characterise this antigen in more detail, infected monolayers were metabolically labeled with tritiated monosaccharides, fatty acids (coupled to BSA), amino acids or ethanolamine at a concentration of 25 pCi/ml in serum-free Dulbecco modified Eagle Medium (DMEM) for 4 h at 37" C

(5). Parasites were purified and extracted as described above and CMW-extracts were dried and partitioned between water and water-saturated n-butanol. The 'butanol component' of CMW-extracts was analysed by TLC using silica 60 HPTLC plates and hexane/CHCI3/MeOH/H2O/HAc 3: 10: 10:2: 1 as solvent system. TLC plates were scanned for radioactivity using a Berthold LB 2842 automatic scanner. After scanning six PHI-glucosamine-labeled peaks were consistently detected. These glycolipids designated peak I to VI with R values of 0,23, 0,3, 0,4, 0,52, 0,59 and 0,68 were also laieled with rHI-mannose, pH]-stearic and pH]-palmitic acid (see Fig. 1). Incorporation of rH]-amino acids was not observed for any of these peaks, whereas three lycolipids (Rpalues of 0,23, 0,3 and 0,4) were labeled with [gHI-ethanolamme. Table 1: Characterisation of glycolipids from Toxoplasma gondii Olymlipidl +vllucs

eI II Ill IV

V VI

labeledvia

'HOlcN %.Man 0.73

030 0.10 052 059 0.68

'H-Pd

)H-Elh

)H-Slu

+ +

+ +

+ +

+

+

C

+ + *

+ + +

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+

ND + ND + ND + N n

+ +

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+ +

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1: TLC-radiochromatograms of butanol component of CMWextracts were shown in Fig. 1; 2 and 3: Cleavage was assesed by scintilation counting and by TLC as described in the text. ND:not determined

Scanned plates were plastified by treating with a saturated solution of polyisobutylmetacrylate in n-hexane for 1 min and were incubated with monoclonal antibodies and patients sera as described above for nitrocellulose. Bands stained by mAbs and sera coincided with radioactive peaks. Human sera (IgM) stained all glycolipids with exception of peak 111, mAb T54ElO recognized predominantly peak I1 and mAb T3F12 peak 111 (in several experiments all three more hydrqphilic peaks I, I1 and 111 were recognized by mAbs). Glycolipids labeled with PHIglucosamine were subjected to enzymatic and chemical treatments (6). The butanol phase of treated glycolipds was analysed by TLC. After PI-PLC, PLD and KOH treatment radioactivity was found almost completly in the water phase. HN02 treatment resulted in a 60% cleavage of all glycolipids. PLA2-treated glycolipids showed a lower chromatographic mobility than untreated molecules. Reactivity of mAb T54E10 to peak I1 was completly abolished by all treatments with exception of PLA2. After PLA treatment the mAb recognized a band with a Rpalue of 0.08 instead of 0.3 in the control. Taken together with the labeling results this data indicates that the low molecular weight antigen of Toxoplasma gondii is a family of glycosyl-inositolphospholipids. We thank D. Becker for expert technical assistance and P. Gerold, C.F. Zmecker and M. Odenthal-Schnittlerfor helpful discussions and C. Roberts for readimg the manuscript. The authors acknowledge support by DFG, PROCOPE from ANRTIDAAD, MSERM, CNRS, Stiftung P.E. Kempkes, Fonds der Deutschen Chemischen Industrie and Hessisches Ministerium f i r Wissenschaft und Kunst. S. Tomavo is a recipient of a fellowship of the Alexander v. Humboldt Stiftung. B. Striepen thanks the Friedrich-Ebert-Stiftuntungfor a scholarship. (1) (2) (3) (4) (5) (6)

Sharma, S.D. et al. (1983) J. Immunol. 131: 977-983 Tomavo, S. et al. (1992) in preparation Grimwood, B.G. et al. (1979) Exp. Parasitol. 48: 282-286 Menon, A.K. et al. (1989) J. Biol. Chem. 263:1970-1977 Tomavo, S. et al. (1989) Mol. Cell. Biol. 9:45764580 Mayor, S. and Menon, A.K. (1990) Methods: A Compendium to Methods in Enzymology 1: 297-305

Identification and characterisation of glycosyl-inositolphospholipids in Toxoplasma gondii.

296s Biochemical SocietvTransactions ( 1 992) 20 IDENTIFICATION AND CHARACTERISATION OF GLYCOSYL-INOSITOLPHOSPHOLIPJDSIN TOXOPLASMA GONDII BORIS STRIE...
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