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. 2000 Dec 4;192(11):1563-76.
doi: 10.1084/jem.192.11.1563.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis

R S Naik  1 , O H Branch, A S Woods, M Vijaykumar, D J Perkins, B L Nahlen, A A Lal, R J Cotter, C E Costello, C F Ockenhouse, E A Davidson, D C Gowda
Affiliations

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis

R S Naik et al. J Exp Med. .

Abstract

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

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Figures

Figure 1
Figure 1
HPLC and HPTLC purification of P. falciparum GPIs. (A) The parasite GPIs (10 μg plus 400,000 cpm of [3H]GlcN-labeled GPIs) were chromatographed on a 4.6 × 250–mm C4 reversed phase HPLC column with a linear gradient of 20–60% aqueous 1-propanol (reference 37). (Top) Analysis of parasite GPIs: fractions (1.0 ml) were collected and 3H activity in 5-μl aliquots was measured (•); 0.5-μl aliquots assayed by ELISA for immunoreactivity with Kenyan adult sera (○). (Bottom) Analysis of glycolipids from control erythrocyte membrane debris obtained from 4 ml packed erythrocytes (•), and those of delipidated, pronase-digested erythrocyte ghosts from 2 ml packed erythrocytes (○). 1-μl aliquots were assayed for immunoreactivity with Kenyan sera. Extracts of total erythrocyte lysate were similarly analyzed (not shown). (B) Fluorograms of the [3H]GlcN-labeled GPIs chromatographed on Silica Gel 60 HPTLC plates using CMW (10:10:2.5, vol/vol/vol). Lane 1, total free GPIs before HPLC; lane 2, HPLC-purified matured free GPIs; lane 3, total free GPIs (different preparation from that in lane 1; obtained by culturing parasites in regular medium after replacing medium with radiolabeled precursor to maximally convert intermediates into matured GPIs); and lane 4, HPLC-purified, amino acid–linked GPIs. Each lane contains 200 ng of GPIs plus 20,000 cpm of [3H]GlcN-labeled GPIs. Note that a small amount of free GPIs that remained with parasite pellet even after exhaustive extraction with organic solvents was copurified with amino acid–linked GPIs (lane 4).
Figure 1
Figure 1
HPLC and HPTLC purification of P. falciparum GPIs. (A) The parasite GPIs (10 μg plus 400,000 cpm of [3H]GlcN-labeled GPIs) were chromatographed on a 4.6 × 250–mm C4 reversed phase HPLC column with a linear gradient of 20–60% aqueous 1-propanol (reference 37). (Top) Analysis of parasite GPIs: fractions (1.0 ml) were collected and 3H activity in 5-μl aliquots was measured (•); 0.5-μl aliquots assayed by ELISA for immunoreactivity with Kenyan adult sera (○). (Bottom) Analysis of glycolipids from control erythrocyte membrane debris obtained from 4 ml packed erythrocytes (•), and those of delipidated, pronase-digested erythrocyte ghosts from 2 ml packed erythrocytes (○). 1-μl aliquots were assayed for immunoreactivity with Kenyan sera. Extracts of total erythrocyte lysate were similarly analyzed (not shown). (B) Fluorograms of the [3H]GlcN-labeled GPIs chromatographed on Silica Gel 60 HPTLC plates using CMW (10:10:2.5, vol/vol/vol). Lane 1, total free GPIs before HPLC; lane 2, HPLC-purified matured free GPIs; lane 3, total free GPIs (different preparation from that in lane 1; obtained by culturing parasites in regular medium after replacing medium with radiolabeled precursor to maximally convert intermediates into matured GPIs); and lane 4, HPLC-purified, amino acid–linked GPIs. Each lane contains 200 ng of GPIs plus 20,000 cpm of [3H]GlcN-labeled GPIs. Note that a small amount of free GPIs that remained with parasite pellet even after exhaustive extraction with organic solvents was copurified with amino acid–linked GPIs (lane 4).
Figure 2
Figure 2
Mass spectrometry analysis of P. falciparum GPIs. HPLC- and HPTLC-purified GPIs were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry in negative or positive ion mode. (A) Negative ion mass spectrum of the purified free GPIs (Fig. 1 B, lane 2). (B) Negative ion mass spectrum of phospholipase A2–treated free GPIs showing (M-H)− ions of unconverted GPIs and GPIs lacking substituent at sn-2 position. (C) Positive ion mass spectrum of inositol-acylated glycan moiety released by HF treatment of GPIs. (D) Negative ion mass spectrum of inositol-acylated glycan moiety released by HF treatment of GPIs. (E) Negative ion mode mass spectrum of the amino acid–linked GPI fraction I (Fig. 1 B, lane 4). The mass spectrum of fraction II (Fig. 1 B, lane 4) contained (M-H)− ions at m/z 2147.3 at significantly higher proportions compared with fraction I, in addition to an ion at m/z 2175.3 (not shown). (F) Negative ion mass spectrum of phospholipase A2–treated amino acid–linked GPIs showing (M-H)− ions of unconverted GPIs and GPIs lacking substituent at sn-2 position. (A–F) The major GPI(s) that represent(s) the molecular ions are indicated. E, ethanolamine; P, phosphate; M4, Man4; Gn, GlcN; I, inositol; AG, acylglycerol; DAG, diacylglycerol; S, serine. (G) The cleavage sites for the attachment of GPIs in P. falciparum MSPs.
Figure 2
Figure 2
Mass spectrometry analysis of P. falciparum GPIs. HPLC- and HPTLC-purified GPIs were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry in negative or positive ion mode. (A) Negative ion mass spectrum of the purified free GPIs (Fig. 1 B, lane 2). (B) Negative ion mass spectrum of phospholipase A2–treated free GPIs showing (M-H)− ions of unconverted GPIs and GPIs lacking substituent at sn-2 position. (C) Positive ion mass spectrum of inositol-acylated glycan moiety released by HF treatment of GPIs. (D) Negative ion mass spectrum of inositol-acylated glycan moiety released by HF treatment of GPIs. (E) Negative ion mode mass spectrum of the amino acid–linked GPI fraction I (Fig. 1 B, lane 4). The mass spectrum of fraction II (Fig. 1 B, lane 4) contained (M-H)− ions at m/z 2147.3 at significantly higher proportions compared with fraction I, in addition to an ion at m/z 2175.3 (not shown). (F) Negative ion mass spectrum of phospholipase A2–treated amino acid–linked GPIs showing (M-H)− ions of unconverted GPIs and GPIs lacking substituent at sn-2 position. (A–F) The major GPI(s) that represent(s) the molecular ions are indicated. E, ethanolamine; P, phosphate; M4, Man4; Gn, GlcN; I, inositol; AG, acylglycerol; DAG, diacylglycerol; S, serine. (G) The cleavage sites for the attachment of GPIs in P. falciparum MSPs.
Figure 3
Figure 3
P. falciparum GPIs contain predominantly C18:1 fatty acid at sn-2. The parasites were metabolically labeled with various 3H–fatty acids. Free GPIs were isolated, treated with bee venom phospholipase A2 (PLA2), and analyzed by HPTLC. −, untreated GPIs; +, phospholipase A2–treated GPIs.
Figure 5
Figure 5
ELISA for naturally elicited anti-GPI antibodies in human sera. The HPLC-purified, free GPIs were coated onto 96-well microtiter plates either at the indicated amounts per well (A) or at 2 ng per well (B). The wells were blocked with TBS-casein, and then incubated with human sera, 1:100 or serially diluted with TBS-casein, 0.05% Tween 20. After washing the plates, the bound antibodies were detected with HRP-conjugated goat anti–human IgG (H and L chains) using ABTS substrate. •, Kenyan adult sera 1; ▴, Kenyan adult sera 2; ▪, Kenyan adult sera 3; ○, USA adult control serum.
Figure 4
Figure 4
The proposed structures of P. falciparum GPIs. The fatty acids and their molar proportions are indicated.
Figure 6
Figure 6
Inhibition of anti-GPI antibody binding to GPIs by phospholipids. The HPLC-purified, free GPIs were coated on to microtiter plates (2 ng), blocked with TBS-casein, and overlaid with representative Kenyan sera (1:200 diluted) incubated with the indicated phospholipids and purified GPIs. The bound antibodies were measured by HRP-conjugated goat anti–human IgG (H and L chains) using ABTS substrate. Shown are the data from a representative of 10 different sera analyzed. Black bar, without inhibitor (Control); hatched bars, 2.5 ng; white bars, 5 ng; gray bars, 10 ng; stippled bars, 20 ng.
Figure 7
Figure 7
HPTLC immunochromatogram of P. falciparum GPIs. The HPTLC-purified, free GPIs (100 ng each) were chromatographed on HPTLC plates, blocked with 1% BSA, and incubated for 2 h in 1:100-diluted sera. The bound antibodies were detected with 125I-labeled goat anti–human IgG (5 μCi/ml). Lanes 1 and 2, free and amino acid–linked GPIs, respectively, treated with Kenyan adult sera; lanes 3 and 4, HNO2-released lipid from free and amino acid–linked GPIs, respectively, treated with Kenyan adult sera; lanes 5 and 6, free and amino acid–linked GPIs, respectively, treated with control USA adult sera; lanes 7 and 8, PIs from bovine liver and soybean, respectively; lane 9, PG; lane 10, CL, treated with Kenyan adult sera. Shown are representatives of 10 Kenyan and USA adult sera analyzed. Parasite lipids other than GPIs and their intermediates, extracted with chloroform/methanol (2:1, vol/vol), and glycolipids from control erythrocytes were completely nonreactive to Kenyan sera (not shown).
Figure 8
Figure 8
Age-dependent anti-GPI antibody response in people living in malaria endemic area. Sera from a cohort of children and adults were analyzed by ELISA using HPLC-purified, free GPIs (see Fig. 5). An antibody level (OD) greater than the mean of USA adult control sera plus 2 SD was considered positive. (A) Percentage of individuals in the following anti-GPI antibody responder categories: negative at both 1–3-mo-spaced time points sampled, negative (white bars); positive at one time point, intermittent (hatched bars); and positive at both time points, positive (black bars). n ≥ 40 in each age group between ∼0.5 and 3.5 yr, and n = 100 and 50 in the 7–8-yr and 20–25-yr age groups, respectively. A χ2 test found that the antibody responder category was different among age groups (P < 0.001). (B) Mean hemoglobin (g/dl, white bars), temperature (−29.5°C, hatched bars), and anti-GPI antibody level [log10(OD+1)] (•) for the indicated age groups. Analysis of variance found that the anti-GPI antibody level, hemoglobin, and temperature were different among age groups (P < 0.0001).
Figure 9
Figure 9
Serum anti-GPI antibody response and resistance to malaria in children 0.5–3.5 yr of age. A general linear model (GLM) was used to investigate the correlation between anti-GPI antibody responder category and temperature or hemoglobin level, while controlling for age and parasite density. (A) Mean temperature (°C) for 0.5–3.5-yr-old negative (white bars), intermittent (hatched bars), and positive (black bars) anti-GPI antibody responder categories. (B) Mean hemoglobin (g/dl) for 0.5–3.5-yr-old negative (white bars), intermittent (hatched bars), and positive (black bars) anti-GPI antibody responder categories (see legend to Fig. 8). The anti-GPI antibody responder category and parasite density were independently associated with hemoglobin (P < 0.0147 and P = 0.0000, respectively). The associations were independent of the nonsignificant association between age and hemoglobin (P = 0.0581). Antibody responder category and parasite density were independently associated with temperature (P = 0.0012 and 0.0001, respectively). Age was not found significantly correlated with temperature (P > 0.0581).
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References

    1. Oaks S.C., Jr., Mitchell V.S., Pearson G.W., Carpenter C.C.J. MalariaObstacles and Opportunities 1991. National Academy Press; Washington, DC: pp. 309
    1. Holder A.A. Malaria vaccines. Proc. Natl. Acad. Sci. USA. 1999;96:1167–1169. - PMC - PubMed
    1. Doolan D.L., Hoffman S.L. Multi-gene vaccination against malariaa multistage, multi-immune response approach. Parasitol. Today. 1997;13:171–178. - PubMed
    1. Ockenhouse C.F., Sun P.F., Lanar D.E., Wellde B.T., Hall B.T., Kester K., Stoute J.A., Magill A., Krzych U., Farley L. Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria. J. Infect. Dis. 1998;177:1664–1673. - PubMed
    1. Shi Y.P., Hasnain S.E., Sacci J.B., Holloway B.P., Fujioka H., Kumar N., Wohlhueter R., Hoffman S.L., Collins W.E., Lal A.A. Immunogenicity and in vitro protective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine. Proc. Natl. Acad. Sci. USA. 1999;96:1615–1620. - PMC - PubMed

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