SYNTHESIS OF GLYCOSYL-PHOSPHATIDYL-INOSITOL LIPIDS BY A CELL-FREE SYSTEM PREPARED FROM ASEXUAL ERYTHROCYTIC STAGES OF THE MALARIA PARASITE PLASMODIUM FALCIPARUM

Abstract

The intracellular protozoan parasite Plasmodium falciparum is the causative agent of malaria in man. The membrane of the merozoites, the invasive stage for the er throcytes, contains, among others, two surface proteins (MSA, anY MSA,), which are presumed to be anchored in the memhrane with a glycosyl-phosphatidyl-inositol (GP1)anchor I l,2,3[. In order to identify and characterize precursorglycolipids f(x GPI-anchors, we have established a cell-free system prepared from P.falciparum trophozoites (33-44 hrs), which are the late erythrocytic stages. Erythrocytes, infected with P.falciparum, were lysed by saponin. The released trophozoites were then lysed by hypotonic shock using a modification of the method described by Masterson et al. 141. Briefly, trophozoites ( 7 . 5~ loy arasites) were resuspended in 6OOpl water containing 0. ImM TECK and Ipglml leupeptin, followed by sonitication (Branson 3200.47kHz) and 30 strokes in a Dounce homogenizer; thereafter an equal volume of IOOmM Na-HEPES (PH 7.4), 50mM KCI. lOmM MgCl , 5mM MnCI,, ,O:ImM TLCK and Ipg/ml leupe tin was added. d i s lysate was divided into aliquots containing 2-5: 108 parasite equivalents. The incubation mixture was completed hy adding ImM ATP, ImM CoA and 2pCi tritiated nucleotide-sugars (UDP-6-[3H]GlcNAc (18.9Ci/mmol, New England Nuclear) or GDP-6-13HIMan (15.1Cilmmol)). Assays were supplemented with I mM GDP-mannose for experiments involving UDP-13HIGlcNAc or with 1mM UDP-GlcNAc for exDeriments HCI, I O O T , 3,3h) of TLC-purified glycolipid V comigrates with a glucosamine standard on High pH Anion Exchange Chromatography (HPAEC, Dionex, Sunnyvale, CA). Three additional more glycolipids, designated glycolipids I, I1 and 111, with R values of Bpd? 0.19, and 0.30, were found in the UDP-[3H]GlcNAc labeled CMextracts. These three gl colipids can also be labeled with GDP-13H]Man. To investigate $e nature of these glycolipids, we have tested their susceptibility to PI-PLC, GPI-PLD, PLA2, HNO , and KOH. The results are summarized in Tahle I. The sensitivity of b e CM-extracted glycolipids to GPI-PLD and nitrous acid treatment share similarities Table 1 Characterisation of Glycolipids synthesized in the cell-free System al CM extract Glycolipldt Rtvalues labeled VIa sensitivity to3 Susceptibility to4 sped.. U-(H)GICNAC G-pHIMan PlPLC PLD PL% H N 4 NaOHI k 0 0 3 + -+ ND II + 0 1 9 + -+ ND 111 + 0 30 + -+ ND I 0.40 + -. ND IV 0 5 5 + ND + + f t 0 5 1 --+ NO V 0 61 + + + + h 012 + + -+ ND VI 1001 7P-Man) 0 81 t B 0 9 1 + -+ ND bl Butanol extpct Glycolipid* Rtvaluas labeled via Sensitivity to G~PHlMan specie. U-IHIGlcNAeC-PHIEtNH4 PlPLc PLD p L b U 0 29 t k -+ t 0 4 1 + + -t + 0 5 1 + d 0.62 + -+ ND -+ ND ! 3 7 + + + + + + NO --+ + + + + + + + + Susceptibility to H N 4 NaOH t t + + + + + +inv~l;ingGDP-[~H)Man and incubated for 45min at 37°C. Glycolipids were extracted twice with Iml chloroform/methanol (CM, 2.1) each, followed hy two extractions with Iml chloroformlmethanol/water (CMW, 1: 1:0.3). The CM-extractable. glycolipids were subjected, to repeated Fol...

Molecular structure of the “low molecular weight antigen” of Toxoplasma gondii: a glucose α1-4 N-acetylgalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic

“…This attached glycolipid is modi®ed by side-chain galactosylation in the Golgi apparatus (Bangs et al, 1988; Englund, 1993). In contrast, in T. gondii and in Plasmodium falciparum the carbohydrate structure of the protein anchors and their corresponding precursor lipid seem to be identical (Gerold et al, 1994;. This ®nding strongly suggests that among the apicomplexan protozoa, peripheral glycosylation of the mannosyl core of GPIs is accomplished prior to transfer to protein, pointing to the activity of glycosyltransferases used for modi®-cation in the ER.…”

Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis

“…The signal was evaluated using a TLC‐scanner (Berthold LB2842). Membranes from P. falciparum were freshly prepared and analyzed as described (Gerold et al ., 1994).…”

Glycosyl-phosphatidylinositols of : Two Common Precursors but a New Protein-anchor

“…Both treatments were performed as described by Gerold et al (1994). The radioactivity released in the aqueous phase after nitrous acid deamination was analysed on Bio-Gel P4 as described below.…”