ACCELERATED PUBLICATION
Plasmodium falciparum Glycosylphosphatidylinositol-induced TNF-alpha Secretion by Macrophages Is Mediated without Membrane Insertion or Endocytosis*

Matam Vijaykumar, Ramachandra S. Naik, and D. Channe GowdaDagger

From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

Received for publication, January 6, 2001, and in revised form, January, 10, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum are believed to contribute to the pathogenesis of malaria by inducing the secretion of proinflammatory cytokines by macrophages. Previous studies have shown that P. falciparum GPIs elicit toxic immune responses by protein tyrosine kinase (PTK)- and protein kinase C (PKC)-mediated cell signaling pathways, which are activated by the carbohydrate and acyl moieties of the intact GPIs, respectively. In this study, we show that induction of TNF-alpha by P. falciparum GPIs in macrophages is mediated by the recognition of the distal fourth mannose residue. This event is critical but not sufficient for the productive cell signaling; interaction by the acylglycerol moiety of GPIs is also required. These novel interactions are coupled to previously demonstrated PTK and PKC pathways, since the specific inhibitors of these kinases effectively blocked the GPI-induced TNF-alpha production. Surprisingly, sn-2 lyso-GPIs were also able to elicit TNF-alpha secretion. Contrary to the prevailing notion, GPIs are neither inserted to the plasma membranes nor endocytosized. Thus, this study defines the GPI structural requirements and reveals a novel mechanism for the outside-in activation of cell signaling by P. falciparum GPIs in inducing proinflammatory responses.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glycosylphosphatidylinositol (GPI)1 anchors represent a special class of glycolipids consisting of a conserved trimannosylglucosaminyl moiety linked to the inositol residue of PI. GPIs are ubiquitous in eukaryotes (1, 2), and their primary function is to anchor proteins to the cell membranes (1-3). Examples include the Trypanosoma brucei variant surface protein, T. cruzi trans-sialidase, Plasmodium falciparum MSP-1 (merozoite surface protein 1) and MSP-2, Leishmania gp63, Thy-1, acetylcholine esterase, CD14, CD52, CD55, and CD59 (3-7). In parasites, GPIs also occur abundantly as free lipids (not anchored to proteins). GPIs exhibit a wide array of functions (8, 9). GPIs from different species differ in the type of acyl/alkyl substituents and the presence of additional sugar moieties on third and/or first mannose and ethanolamine phosphate residues on the carbohydrate moiety, leading to a broad structural diversity that may relate to their diverse biological activity (3).

GPIs of P. falciparum have been shown to induce secretion of proinflammatory cytokines, including TNF-alpha and up-regulation of cell adhesion molecules (10-12). Because P. falciparum-infected erythrocytes sequester in the microvascular capillaries of vital organs, the above process is expected to synergistically promote adherence of parasite-infected erythrocytes; this in turn potentiate proinflammatory immune responses in deep endothelium because of local high concentration of GPIs and other factors released by accumulated parasites, leading to organ dysfunction and life threatening pathological conditions. Thus, GPIs of P. falciparum have been identified as "pathogenicity factors" (13).

P. falciparum GPIs activate PTK and PKC pathways, which collaboratively regulate the NF-kappa B/rel transcription factors with the downstream expression of proinflammatory immune responses (10-12). The conserved glycan core of GPIs, Manalpha 1-2Manalpha 1-6Manalpha 1-4GlcNalpha 1-6myo-inositol, was reported to be the minimum structure involved in PTK activation, whereas the diacylglycerol moiety has been postulated to be an independent second messenger for the PKC activation (12). Although it has been predicted that a transmembrane receptor with a lectin-like property might be involved in GPI-mediated cell signaling (12), the nature of the receptor and the sugar(s) involved in the receptor recognition has not been elucidated. The suggested involvement of membrane-bound GPI-phospholipase D in hydrolyzing GPI and thus allowing the DAG moiety to enter cells or GPI translocation across cell membrane by the action of a "flippase" to activate PKC remains speculative (12). Although it has been reported that the DAG moiety but not a 1-alkyl-2-acylglycerol residue could activate PKC (12), T. cruzi GPIs containing a 1-alkyl-2-acylglycerol moiety exhibit potent cytokine-inducing ability, and alpha -Gal residues of these GPIs were reported to be crucial for the activity (14, 15). These observations suggest the involvement of multiple recognition mechanisms in signal transduction by GPIs.

Recently we established, by mass spectrometry, the structures of GPIs purified from intraerythrocytic P. falciparum (16). Here, we studied the specific structural elements and nature of interaction involved and the mechanism of cell signaling in GPI-induced TNF-alpha secretion. The data show that a novel interaction mediates cell signaling and that the terminal fourth mannose and 1-acylglycerol residues are critical for GPI activity. Cell signaling is mediated by a previously unrecognized mode of outside-in signal transduction without inserting into cell membranes or internalization of GPIs.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- RPMI 1640, Dulbecco's modified Eagle's culture medium, fetal bovine serum, and cell culture reagents were purchased from Life Technologies, Inc. Human blood and serum were from Interstate Blood Bank (Memphis, TN). Bee venom phospholipase A2 (1800 units/mg), jack bean alpha -mannosidase (30 units/mg), soybean 1-palmitoyl-2-linoleoylphosphatidylinositol, bovine liver 1-stearoyl-2-arachidonylphosphatidylinositol, synthetic dipalmitoylglycerol and dioleoylglycerophosphate, trifluoroacetic acid, alpha -methyl mannoside, alpha -methyl glucoside, alpha -methyl galactoside, alpha -(1-2)-linked mannobiose, soluble yeast mannan, and saponin were from Sigma. [6-3H]Glucosamine (23 Ci/mmol) and [3H]myristic acid (55 Ci/mmol) were from Amersham Pharmacia Biotech. Genistein and GF 109203X were gifts from Dr. Toshio Kitazawa.

P. falciparum Cell Culture-- Culturing of intraerythrocytic P. falciparum (FCR-3 strain), synchronization, and mycoplasma testing were performed as described (17-19).

Metabolic Labeling of GPIs-- Metabolic labeling of parasites with [3H]GlcN (50 µCi/ml) and [3H]myristic acid (50 µCi/ml) was performed as described (17).

Isolation of GPIs from P. falciparum-- Purification of parasites and isolation of GPIs were performed as described (16). Briefly, cultures with 20-30% parasitemia were harvested at mid-schizont stage, enriched to 70-80% by gelatin floatation (20), and lysed with 0.015% saponin. The erythrocyte debris were removed, and the parasites were washed, lyophilized, and stored at -80 °C (16). The parasite GPIs were extracted with chloroform/methanol/water (10:10:3, v/v/v) and partitioned between water and water-saturated 1-butanol as described (16).

Nitrous Acid Treatment-- The HPLC-purified GPIs (20 µg) were treated with HNO2 in the presence of sodium taurodeoxycholate (21, 22). The released PI moieties were extracted with water-saturated 1-butanol and washed with water. The aqueous phase was desalted on Bio-Gel P-4.

Cleavage by HF-- HPLC-purified GPIs (10 µg) were treated with 50% aqueous HF (21, 22), and the reaction mixture was diluted with 20 volumes of ice cold water, lyophilized, and partitioned between water and water-saturated 1-butanol. The glycan and diacylglycerol moieties were purified by HPLC (16).

Deacylation of GPIs-- The HPLC-purified GPIs (2 µg) were deacylated with methanol, 30% ammonia (1:1, v/v) (16), and the glycan moiety was solubilized in water and dried in a Speed Vac.

Treatment with Mannosidase and Phospholipase A2-- HPLC-purified GPIs (10 µg plus 500,000 cpm of [3H]GlcN-labeled GPIs) were treated separately with jack bean alpha -mannosidase and bee venom phospholipase A2 (16, 23). The products were purified by HPLC and analyzed by HPTLC.

Macrophage Cell Culture-- The J774A.1 murine macrophage cell line (ATCC) was cultured as monolayers in DMEM with 10% FBS. For subculturing and cytokine induction, cells were detached by scraping.

Induction of TNF-alpha in Macrophages by P. falciparum GPIs-- Freshly detached macrophages suspended in DMEM, 10% FBS were seeded in 96-well microtiter plates (2.5 × 106 cells/well) and incubated at 37 °C. After 2 h, the nonadhered cells were removed, and cells were incubated with 200 µl of DMEM containing 10% FBS and varying amounts of GPIs, and other compounds added from stock solution in 80% ethanol, water, or Me2SO. Cells treated with organic solvents (0.1% final concentration) only were used as controls. For inhibition studies, the compounds were added 1 h prior to the addition of GPIs. The amount of TNF-alpha secreted was measured using a sandwich ELISA kit (R & D Systems).

Uptake of GPIs by Macrophages-- Macrophages in microtiter plates (2.5 × 106cells/well) were incubated with [3H]GlcN- or [3H]myristic acid-labeled GPIs (100,000 cpm each) in DMEM, 10% FBS for 6, 24, or 48 h. The culture supernatants were collected, and aliquots were counted for radioactivity by liquid scintillation counting. The cells were detached by trypsinization, centrifuged, and washed with DMEM. The combined washings and cells solubilized in 1% Triton X-100, 1% SDS were measured separately for radioactivity.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation and Purification of Specific Cleavage Products of P. falciparum GPIs-- Many parasitic protozoa, including P. falciparum, synthesize GPIs severalfold in excess of the amount required to anchor proteins to cell surfaces. These GPIs are likely to play major roles in membrane properties and the modulation of host immune responses. P. falciparum GPIs have been shown to elicit inflammatory cytokines including TNF-alpha (10-13, 16).

To determine the structural feature(s) of parasite GPIs necessary for the induction of TNF-alpha and to identify the receptor on the host cells, GPIs were subjected to specific enzymatic and chemical degradations, and the products were purified and tested. The HPLC-purified GPIs (EtN-P-Man4-GlcN-acyl-IP-DAG, designated as Man4-GPI; retention time, 92 min) were treated with jack bean alpha -mannosidase and bee venom phospholipase A2 to obtain GPIs lacking the distal fourth mannose residue (EtN-P-Man3-GlcN-acyl-IP-DAG, Man3-GPI; HPLC retention time 92 min) and sn-2 fatty acid (EtN-P-Man4-GlcN-acyl-IP-MAG, sn-2 lyso-GPI; HPLC retention time 70 min), respectively (Fig. 1). Both enzymes converted >95% GPIs into their respective products (Fig. 1). The acylated-inositol carbohydrate moiety with and without the phosphate group on inositol (Man4-GlcN-acyl-IP and Man4-GlcN-acyl-Ino) and the carbohydrate moiety (OCH-CH2-P-Man4-AHM) that lacked the PI residue of GPIs were prepared by HF cleavage and nitrous acid deamination, respectively (Fig. 1). HPTLC indicated that these were >95% pure (Fig. 2 and not shown). The compound that lacked all three acyl substituents (EtN-P-Man4-GlcN-IP-G) was prepared by hydrolysis with ammonia in aqueous methanol. The identity of the above products was established by determining mannose content, by identifying the glycan core by specific degradative cleavage, and by analysis of products by HPTLC and mass spectrometry (16, 17, 24).



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Fig. 1.   Schemes showing the enzymatic and chemical cleavage sites for the preparation of GPI components. Treatment of GPIs with alpha -mannosidase gives EtN-P-Man3-GlcN-acyl-IP-DAG. Treatment with phospholipase A2 gives EtN-P-Man4-GlcN-acyl-IP-MAG; HF gives Man4-GlcN-acyl-IP, Man4-GlcN-acyl-Ino, and DAG; and nitrous acid forms OHC-CH2-P-Man4-AHM and acyl-IP-DAG.



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Fig. 2.   HPTLC analysis of P. falciparum GPI cleavage products. Analysis of the HPLC-purified cleavage products of GPIs was carried out using Silica Gel 60 HPTLC plates (Whatman) using chloroform/methanol/water (10:10:2.5, v/v/v). After development, the plates were air-dried, sprayed with En3Hancer (PerkinElmer Life Sciences), and exposed to x-ray film at -80 °C. Lane 1, HPLC-purified GPIs; lane 2, mannosidase-treated GPIs; lane 3, PLA2-treated GPIs; lane 4, HF-released carbohydrate moiety extracted with water-saturated 1-butanol (Man4-GlcNalpha 1-acylIno);lane 5, HF-released carbohydrate moiety recovered in aqueous phase (Man4-GlcNalpha 1-acylIno-P).

Induction of TNF-alpha Secretion in Macrophages by P. falciparum GPIs-- P. falciparum GPIs are known to elicit proinflammatory cytokine responses by the activation of PTK and PKC pathways by the carbohydrate and lipid moiety, respectively; these signals cooperatively activate NF-kB/rel to elicit cytokine responses (12). Although it has been predicted previously that a lectin-type receptor is involved in the PTK activation by GPIs, the nature of the sugar residue and the receptor were not identified, and the mechanism of GPI interaction with macrophages was not established. In this study, we tested purified GPIs and various cleavage products of GPIs for the induction of TNF-alpha using murine macrophages. As reported previously (12, 16), Man4-GPI was able to elicit TNF-alpha secretion by macrophages (Fig. 3A). However, in contrast to the previous report (12), Man3-GPI was completely inactive, suggesting that the recognition of the terminal mannose residue by the macrophage is crucial for the biologic activity of GPIs (Fig. 3A). In agreement with this observation, alpha -methyl mannoside, alpha -(1-2)-linked mannobiose, and soluble yeast mannan inhibited the ability of P. falciparum GPIs to induce TNF-alpha secretion by >95%, whereas alpha -methyl glucoside and alpha -methyl galactoside were not inhibitory (Fig. 3B). To determine whether the carbohydrate moiety of the parasite GPIs alone is sufficient for the production of TNF-alpha , macrophages were treated with HF-released carbohydrate moieties, Man4-GlcN-acyl-IP and Man4-GlcN-acyl-Ino, and with deacylated GPI, EtN-P-Man4-GlcN-IP-G. All three products failed to elicit TNF-alpha secretion (Fig. 3A). In addition, they effectively inhibited the activity of GPIs (Fig. 3B). The parasite GPI precursor lipids extracted with chloroform/methanol (2:1, v/v), bovine liver and soybean PIs, dipalmitoylglycerol, and dioleoylglycerophosphate were also unable to elicit TNF-alpha secretion (Fig. 3B and not shown). Together, these results suggested the critical involvement of the distal fourth mannose and the requirement of intact GPIs for the activity (12). Furthermore, in agreement with the previously reported involvement of PTK and PKC activation by P. falciparum GPIs in eliciting TNF-alpha secretion, genistein and GF 109203X, the inhibitors of the respective kinases (10-12), blocked >90% of GPI activity (Fig. 3B).



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Fig. 3.   Measurement of TNF-alpha secreted by macrophages in response to P. falciparum GPIs. Murine macrophages were treated with indicated amounts of GPIs or cleavage products for 48 h, and then culture supernatants were assayed for TNF-alpha as described under "Experimental Procedure." For inhibition studies (panel B), macrophages were incubated with the desired compound for 1 h before the addition of GPIs. The plotted values are averages of two independent experiments; in each case TNF-alpha was assayed in duplicates. A, at each concentration, the bars from left to right, respectively, represent intact GPIs, sn-2 lyso-GPIs, Man3-GPI, HF-released carbohydrate moieties, and HNO2-released PI moiety. B, macrophages were incubated with various inhibitors 1 h prior to treatment with 300 ng of intact GPIs/well. Control, no inhibitor; Man3-GPI, 300 ng of mannosidase-treated GPIs; HNO2-PI, 300 ng of the nitrous acid-released PI moiety; Me Man, 350 ng of methyl mannoside; Mannobiose, 600 ng; HF-Glycan, 300 ng of HF-released carbohydrate moiety; Me Glc, 350 ng of methyl glucoside; Me Gal, 350 ng of methyl galactoside; Genistein, 5 µM; GF 109203X, 500 nM; Bovine PI, 300 ng of bovine liver PI.

Because the lipid moiety of GPIs is also critically required for GPI activity (see above), we examined whether the HNO2-released PI moiety, the parasite lipids extracted with chloroform/methanol (2:1), bovine liver and soybean PIs, and diacylglycerols could block the activity of GPIs. Interestingly, none of these compounds could inhibit the GPI activity (Fig. 3B). Furthermore, prior incubation of macrophages with Man3-GPI had no effect on the ability of Man4-GPI to elicit TNF-alpha production (Fig. 3B). These data suggest that initial recognition of the distal fourth mannose residue is critical for the interaction of the lipid moiety to activate the PKC pathway for productive cell signaling. Thus, the lipid moiety-induced activation of PKC (12) must be tightly regulated and coupled to PTK activation by the recognition of the distal fourth mannose.

In a previous study, sn-2 lyso-GPIs have been reported to be inactive (12). However, in this study, structurally characterized sn-2 lyso-GPIs obtained by phospholipase A2-treated Man4-GPI were able to induce TNF-alpha secretion in a dose-dependent manner to levels similar to those of Man4-GPI (Fig. 3A). Thus, our data demonstrate that the sn-2 fatty acyl residue is not required and 1-monoacylglycerol moiety of P. falciparum GPIs is sufficient for TNF-alpha induction.

Localization of GPIs in Macrophage Cultures-- To determine whether recognition of P. falciparum GPIs by macrophages involves a mannose-specific receptor binding and/or uptake of GPIs, macrophages were incubated with radiolabeled GPIs, HF-released carbohydrate moiety, or deacylated GPIs. The distribution of these compounds between culture medium and cells was analyzed. About 98% of the radioactivity in intact GPIs was in the culture supernatants, and about 1-2% was associated with the cells. This could be due to nonspecific spontaneous insertion of GPIs into cell membranes, a known phenomenon (25, 26). The HF-released carbohydrate moieties and deacylated GPIs remained exclusively in the culture medium. These results suggest that although the distal fourth mannose residue is critical for the activity, GPI is not bound or internalized by macrophages. In contrast, the macrophage mannose receptor binds and internalizes compounds containing terminal mannose residues (27, 28). These data taken together suggest that TNF-alpha production by P. falciparum GPIs is by unique interactions involving initial recognition of the distal fourth mannose residue by a mannose-specific component presumably localized in plasma membrane in association with PTK.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, highly purified GPIs from intraerythrocytic stage P. falciparum and structurally characterized components of GPIs were assessed for TNF-alpha induction in macrophages to define the active elements and the nature of receptor recognition. The data presented here show that both the carbohydrate and lipid moieties of GPIs are required for the activity. This is in agreement with previous findings that the induction of proinflammatory cytokine responses by P. falciparum and T. cruzi trypomastigote GPIs involve a dual requirement for the glycan and diacylglycerol moieties; the GPI glycan moieties alone could not elicit TNF-alpha secretion (12, 15). Our study also identifies the critical requirement of the distal fourth mannose residue for GPI activity. Furthermore, contrary to the previous report (12), the results described here show that sn-2 lyso-GPIs obtained by phospholipase A2 treatment of the parasite GPIs are able to induce TNF-alpha production to a level comparable with that induced by intact GPIs.

The distal fourth mannose is a necessary component of P. falciparum GPIs for signaling TNF-alpha production. The following data support this conclusion. 1) The removal of the distal fourth mannose residue completely abrogates the ability of the parasite GPIs to induce TNF-alpha secretion in macrophages. 2) alpha -Methyl mannoside, alpha -(1-2)-linked mannobiose, mannan, the carbohydrate moiety released from GPIs by HF, and deacylated GPIs all could effectively inhibit the ability of P. falciparum GPIs to elicit TNF-alpha secretion. 3) alpha -Methyl glucoside and alpha -methyl galactoside were completely noninhibitory. Together, these data establish that induction of TNF-alpha secretion by P. falciparum GPIs occurs by the recognition of the distal fourth mannose. This finding is in contrast to a previous report that P. falciparum Man3-GPI was also bioactive (12). The reason for the discrepancy is not clear. However, it is possible that the reported activity of Man3-GPI could be due to the presence of a significant amount of Man4-GPI association in Man3-GPI fraction.

The requirement of the distal fourth mannose residue for the P. falciparum GPIs to induce TNF-alpha secretion agrees with the previously reported cytokine-inducing activity of T. cruzi trypomastigote mucin GPIs, which also contain unsubstituted fourth mannose (14). The GPIs of T. cruzi trypomastigote mucin have been shown to be severalfold more active than the GPIs of P. falciparum (15). A comparison of the structures shows that, unlike P. falciparum GPIs, the GPIs of T. cruzi trypomastigote mucin contain distal fourth alpha -mannose as well as alpha -galactose residues linked to the first mannose. Therefore, it appears that the presence of alpha -galactose residues further potentiates interaction of T. cruzi GPIs for effective cell signaling.

Leishmania mexicana glycoinositolphospholipids containing a 1-alkyl-2-acylglycerol moiety and the conserved glycan core with alpha -(1-3)mannose linked to the first mannose residue but lacking ethanolamine phosphate on the third mannose could not induce TNF-alpha production; however, these compounds could activate PTK (12). By comparing these results with those from the GPIs of P. falciparum and T. brucei variant surface protein, it has been suggested that GPIs containing diacylglycerol but not those with 1-alkyl-2-acylglycerol can elicit cytokine production (12). However, because GPIs from T. cruzi trypomastigote mucin with a 1-alky-2-acylglycerol residue can efficiently induce TNF-alpha production (15), the presence of 1,2-diacylglycerol as opposed to 1-alkyl-2-acylglycerol moiety is not a crucial requirement for GPI activity.

Not all GPIs containing terminal fourth mannose residue and 1,2-diacylglycerol or 1-alkyl-2-acylglycerol moiety can elicit TNF-alpha secretion in macrophages. As shown previously by Almeida et al. (15), the GPI fraction of T. cruzi epimastigote mucin that contains unsubstituted fourth mannose, as in the case of active GPI fraction of trypomastigote mucin and P. falciparum GPIs, is completely inactive. Whereas P. falciparum GPIs and the active GPI fraction of T. cruzi trypomastigote both contain unsaturated acyl substituent at the sn-2 position (15, 16), the inactive GPI fraction of the T. cruzi trypomastigote mucin contains a saturated acyl substituent at sn-2; the inactive GPIs of epimastigote mucin have a ceramide moiety. Therefore, as in the case of the active trypomastigote mucin GPIs (15), the unsaturated acyl group at the sn-2 position seems to be required for the activity of intact P. falciparum GPIs; however, P. falciparum GPIs lacking sn-2 acyl substituent are also fully active (see below).

It has been reported previously that sn-2 lyso-GPIs, obtained by the treatment of P. falciparum GPIs with phospholipase A2, were unable to induce cytokine secretion (12). However, in our study, the HPLC-purified structurally characterized sn-2 lyso-GPIs obtained from different GPI preparations were able to reproducibly elicit TNF-alpha at levels similar to those elicited by intact GPIs. The reason for this discrepancy is not known. However, our data suggest that P. falciparum GPIs lacking sn-2 acyl residue provide the necessary structural requirements for cell surface interactions.

The mechanism of GPI-cell interaction in signaling is poorly understood. Previous studies have predicted a receptor-mediated GPI binding and insertion or internalization (12, 15). Interestingly, in the present study, the P. falciparum GPIs were neither bound to plasma membranes nor internalized by macrophages. However, our data clearly indicate the critical participation of the terminal mannose residue. Although the signal transduction by the well known macrophage mannose receptor to elicit cytokine responses involves phagocytic or endocytic mechanism (29-32), our data suggest that neither of these processes was involved in the case of P. falciparum GPIs. Furthermore, because neither the PI moiety released by HNO2 nor Man3-GPI is able to inhibit the activity of Man4-GPI, the interaction of the acylglycerol moiety for PKC activation requires prior recognition of the terminal mannose residue of GPIs. Moreover, because GPIs were not inserted to cell membrane or internalized, the interaction of the GPI acyl moiety in cell signaling represents a novel mechanism that is likely to be tightly coupled to PTK activation. Further understanding of the details of these interactions might offer specific targets for malaria therapy.


    FOOTNOTES

* This study was supported by Grant AI41139 from NIAID, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 3900 Reservoir Rd., N. W., Washington, D. C. 20007. Tel.: 202-687-3840; Fax: 202-687-7186; E-mail: gowda@bc.georgetown.edu.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C100007200


    ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; PTK, protein tyrosine kinase; PKC, protein kinase C; FBS, fetal bovine serum; Man, mannose; PI, phosphatidylinositol; IP, inositol phosphate; EtN, ethanolamine; Ino, inositol; AHM, anhydromannose; DAG, diacylglycerol; MAG, 1-monoacylglycerol; G, glycerol; HF, hydrofluoric acid; HPTLC, high performance thin-layer chromatography; genistein, 4',5,7-trihydroxyisoflavone; GF 109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; TNF-alpha , tumor necrosis factor alpha ; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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