Synthesis of chloroplast galactolipids in apicomplexan parasites

Abstract

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol are major chloroplast lipids of algae and land plants and are synthesized within the plastid envelope. Here we report that in Toxoplasma gondii and Plasmodium falciparum lysates, radiolabeled UDP-galactose is incorporated into monogalactosylcerebrosides, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol due to distinct enzymological activities. Furthermore, DGDG is immunologically detected in apicomplexans.

FIG. 1.

Synthesis of galactolipids in T. gondii and P. falciparum. After incubation with tritiated UDP-galactose, lipids were extracted and separated by TLC (chromatograms [A, B, and C]), followed by phosphorimager detection after a 2-week exposure (B). Glycolipids were compared with standard MGCB from bovine brain and MGDG and DGDG from spinach chloroplast envelope membranes. (A) A mock preparation of parasites, using uninfected Vero cells, incorporated galactose (mock treatment of uninfected Vero cells). When we preincubated the membrane suspension with concanavalin A (50 μg for 30 min at 37°C), a lectin with affinity for the rich mannose decoration of mammalian CGalT (UDP-galactose:ceramide galactosyltransferase), aggregates appeared in the suspension and the pattern of incorporation of tritiated UDP-galactose due to the broken Vero cells disappeared (mock + ConA). T. gondii incorporation of galactose was insensitive to concanavalin A treatment (T. gondii isolated from Vero cells + ConA). (C) Erythrocytes uninfected with P. falciparum did not yield significant amounts of labeled glycolipids (mock treatment of uninfected erythrocytes). Standards were either loaded in separate lanes (A and C) or mixed with the parasite sample to demonstrate comigration of the radiolabeled parasite lipids with the galactolipid standards (B). Shown are syntheses of the following: MGDG and MGCB in T. gondii lysates after a 1-h incubation in the absence of Mg²⁺ (A), digalactolipids in T. gondii lysates after a 1-h incubation in the presence of 5 mM MgCl₂ (B), and MGDG, MGCB, and DGDG in P. falciparum after a 1-h incubation in the absence of Mg²⁺ (C). Counts were integrated for 60 min (A), 30 min (B), and 120 min (C). SL, sulfoquinovosyldiacylglycerol; triGDG, trigalactosyldiacylglycerol; tetraGDG, tetragalactosyldiacylglycerol. (D) Structures of polar portions of MGDG and MGCB. MGDG is made of a glycerol backbone (sn nomenclature of the three carbons) esterified by two fatty acids in the sn-1 and sn-2 positions (FA1 and FA2) and O-galactosylated in the sn-3 position. In MGCB, only the hydrophilic part of the long chain base (LCB) of the ceramide portion is indicated (C1′ to C3′ nomenclature of the first three carbons). The LCB is N-esterified to a fatty acid in C2′, making up the ceramide moiety, and O-galactosylated in the C1′ position.

FIG. 2.

Immunodetection of DGDG in lipid extracts from T. gondii and P. falciparum. (A) Comparison of MGDG, DGDG, and triGDG polar heads. (B) The specificity of the anti-DGDG antibody for the [α-d-galactopyranosyl-(1→6)-O-β-d-galactopyranosyl[ polar head was investigated by dot blot immunostaining (1/100 dilution of anti-DGDG antibody) of 10 μg of lipids purified from spinach chloroplast envelope membranes (first two lanes) or Synechocystis PCC 6803, a cyanobacterium. The anti-DGDG antibody was detected on DGDG spots whereas it did not react with MGDG or triGDG. (C) T. gondii lipids extracted from 2 × 10⁹ free tachyzoites were separated by TLC as shown in Fig. 1. Silica at the level of the DGDG standard was scraped, and lipids were extracted (those with an R_f equal to the R_f of DGDG) and analyzed by dot blot immunostaining. The anti-DGDG antibody reacted with this spot, whereas it did not when the extracted lipids were subjected to mild alkaline hydrolysis and reextracted (+KOH). A similar immunostaining pattern was obtained with a spinach chloroplast DGDG control subjected to identical treatments. The loss of immunostaining after mild KOH hydrolysis showed that the immunostained lipids were deacylated. (D) Dot blot immunostaining with the anti-DGDG antibody of total lipid mixtures from spinach chloroplast envelope membranes (10 μg of lipids), E. coli membranes (10 μg of lipids), T. gondii (total lipid extracts from 2 × 10⁹ tachyzoites), and P. falciparum (total lipid extracts from 10⁹ cells).