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• W2120403054 abstract "Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity. Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity. Developing a vaccine to control human malaria is a global health priority. The recent availability of the genome sequence of the protozoan parasite Plasmodium falciparum, the major cause of malaria, allows the use of genomic technologies such as microarray and proteomics to identify novel vaccine and drug targets (1Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. Craig A. Kyes S. Chan M.S. Nene V. Shallom S.J. Suh B. Peterson J. Angiuoli S. Pertea M. Allen J. Selengut J. Haft D. Mather M.W. Vaidya A.B. Martin D.M. Fairlamb A.H. Fraunholz M.J. Roos D.S. Ralph S.A. McFadden G.I. Cummings L.M. Subramanian G.M. Mungall C. Venter J.C. Carucci D.J. Hoffman S.L. Newbold C. Davis R.W. Fraser C.M. Barrell B. Nature. 2002; 419: 498-511Crossref PubMed Scopus (3453) Google Scholar, 2Lasonder E. Ishihama Y. Andersen J.S. Vermunt A.M. Pain A. Sauerwein R.W. Eling W.M. Hall N. Waters A.P. Stunnenberg H.G. Mann M. Nature. 2002; 419: 537-542Crossref PubMed Scopus (562) Google Scholar, 3Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. Nature. 2002; 419: 520-526Crossref PubMed Scopus (1093) Google Scholar, 4Bozdech Z. Llinas M. Pulliam B.L. Wong E.D. Zhu J. DeRisi J.L. PLoS Biol. 2003; 1: E5Crossref PubMed Scopus (1267) Google Scholar, 5Le Roch K.G. Zhou Y. Blair P.L. Grainger M. Moch J.K. Haynes J.D. De La Vega P. Holder A.A. Batalov S. Carucci D.J. Winzeler E.A. Science. 2003; 301: 1503-1508Crossref PubMed Scopus (1026) Google Scholar). Most membrane proteins that coat the surface of the erythrocyte-invasive merozoite form of the parasite are attached to the plasma membrane via a C-terminal glycosylphosphatidylinositol (GPI) 5The abbreviations used are: GPIglycosylphosphatidylinositolDRMdetergent-resistant membranesMSPmerozoite surface proteinSERAserine repeat antigenRAMArhoptry-associated membrane antigenPBSphosphate-buffered salineGFPgreen fluorescent proteinMBPmannose-binding proteinMES4-morpholineethanesulfonic acid. anchor. To date, four GPI-anchored merozoite surface proteins (MSP-1, -2, -4, and -5) have been identified, and two others (MSP-10 and rhoptry-associated membrane antigen (RAMA)) appear to reside at least in part in organelles at the apical end of the parasite (6Holder A.A. Lockyer M.J. Odink K.G. Sandhu J.S. Riveros-Moreno V. Nicholls S.C. Hillman Y. Davey L.S. Tizard M.L. Schwarz R.T. Freeman Robert R Nature. 1985; 317: 270-273Crossref PubMed Scopus (227) Google Scholar, 7Smythe J.A. Coppel R.L. Brown G.V. Ramasamy R. Kemp D.J. Anders R.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5195-5199Crossref PubMed Scopus (224) Google Scholar, 8Cowman A.F. Crabb B.S. Science. 2002; 298: 126-128Crossref PubMed Scopus (30) Google Scholar, 9Black C. Wang L. Wu T. Coppel R. Mol. Biochem. Parasitol. 2003; 127: 59-68Crossref PubMed Scopus (55) Google Scholar, 10Topolska A.E. Lidgett A. Truman D. Fujioka H. Coppel R.L. J. Biol. Chem. 2004; 279: 4648-4656Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Another protein originally localized to the merozoite surface, MSP-8, appears to instead reside in the ring stage (11Drew D.R. Sanders P.R. Crabb B.S. Infect. Immun. 2005; 73: 3912-3922Crossref PubMed Scopus (36) Google Scholar). Most other merozoite surface proteins (e.g. MSP-3/6 family members, MSP-7, and acidic basic repeat antigen (ABRA)) are not directly membrane-associated but are indirectly linked to the surface, probably in most cases via interactions with GPI-anchored proteins (12McColl D.J. Silva A. Foley M. Kun J.F. Favaloro J.M. Thompson J.K. Marshall V.M. Coppel R.L. Kemp D.J. Anders R.F. Mol. Biochem. Parasitol. 1994; 68: 53-67Crossref PubMed Scopus (101) Google Scholar, 13Pachebat J.A. Ling I.T. Grainger M. Trucco C. Howell S. Fernandez-Reyes D. Gunaratne R. Holder A.A. Mol. Biochem. Parasitol. 2001; 117: 83-89Crossref PubMed Scopus (94) Google Scholar, 14Trucco C. Fernandez-Reyes D. Howell S. Stafford W.H. Scott-Finnigan T.J. Grainger M. Ogun S.A. Taylor W.R. Holder A.A. Mol. Biochem. Parasitol. 2001; 112: 91-101Crossref PubMed Scopus (97) Google Scholar, 15Pearce J.A. Mills K. Triglia T. Cowman A.F. Anders R.F. Mol. Biochem. Parasitol. 2005; 139: 141-151Crossref PubMed Scopus (27) Google Scholar). In contrast to the apical and peripheral classes of blood-stage antigens, the GPI-anchored proteins appear to be essential to blood-stage growth with repeated attempts to genetically disrupt six GPI-anchored merozoite proteins resulting in only one “knock-out,” that of the msp-5 gene (16Cowman A.F. Crabb B.S. Sherman I.W. Molecular Approaches to Malaria. ASM Press, Washington, D. C.2005Google Scholar). 6P. R. Sanders and B. S. Crabb, manuscript in preparation. This, together with considerable data highlighting their potential as targets of protective antibodies, places the merozoite GPI-anchored proteins among the most highly validated blood-stage vaccine targets. glycosylphosphatidylinositol detergent-resistant membranes merozoite surface protein serine repeat antigen rhoptry-associated membrane antigen phosphate-buffered saline green fluorescent protein mannose-binding protein 4-morpholineethanesulfonic acid. GPI-anchored plasma membrane proteins of many eukaryotes, including P. falciparum (17Wang L. Mohandas N. Thomas A. Coppel R.L. Mol. Biochem. Parasitol. 2003; 130: 149-153Crossref PubMed Scopus (27) Google Scholar), appear to be enriched in detergent-resistant membrane (DRM) domains, sometimes referred to as lipid rafts. To further characterize the surface coat of the blood-stage form of P. falciparum parasites, we determined the proteome of DRMs in this life stage and found that these membranes are greatly enriched in GPI-anchored proteins and their interacting partners. Also prominent in DRMs were apical organelle (rhoptry), multimembrane-spanning, and a novel set of proteins that are destined to be exported from the intracellular form parasite into the host erythrocyte. A number of new surface proteins were identified, and three of these were confirmed as merozoite surface antigens that elicit antibody responses in naturally infected individuals. This study provides a more comprehensive picture of the nature of the P. falciparum merozoite surface and generates a range of new possibilities for malaria vaccine development. Preparation of Detergent-resistant Membranes—Sorbitol-synchronized parasite cultures at 8-10% parasitemia were pelleted (1500 rpm Beckman GS-6 centrifuge), and late stage schizonts (∼44 h after invasion) were purified using Miltenyi Biotec Vario MACS CS magnetic separation columns. Parasites were washed twice in culture medium prior to saponin lysis (0.15% saponin, 10 min, on ice). Samples were pelleted at 2800 rpm for 10 min (Beckman GS-6 centrifuge) and washed three times in MES-buffered saline (25 mm MES (Sigma) pH 6.5, 150 mm NaCl). Parasites were resuspended to a volume of 1.5 ml in MES containing a Roche Applied Science Complete protease inhibitor mixture tablet and chilled to 0 °C on an ice water slurry. Parasite samples were cooled to 0 °C, and an equal volume of chilled (0 °C) 1% Triton X-100 (SigmaUltra) in MES at 0 °C was added to give a final concentration of 0.5% Triton X-100. Samples maintained at 0 °C for 30 min (3 ml total) were resuspended every 10 min and then centrifuged at 30,000 rpm (2 °C for 30 min) in a pre-chilled TLA 100.3 rotor in a Beckman Optima MAX-E ultracentrifuge. The supernatant was discarded, and the pellet was resuspended in a total volume of 184 μl of 0.5% Triton X-100 in MES buffer containing protease inhibitors to which an equal volume of 80% (w/v) sucrose (ultra-pure, Invitrogen) in MES buffer was added. The pellet material (368 μl total, 40% sucrose) was transferred to a 2.2-ml open top thin-walled polyallomer tube and overlaid with 1.1 ml of 35% (w/v) sucrose in MES buffer followed by 733 μl of 5% (w/v) sucrose in MES buffer to form a sucrose step gradient (all solutions at 0 °C). The gradient was centrifuged at 55,000 rpm for 18 h (2 °C) with low acceleration and no deceleration in a pre-chilled Beckman TLS-55 swing rotor. Following ultra centrifugation, 15 equal fractions (146 μl) were removed from the top of the gradient, snap-frozen on dry ice, and stored at -80 °C. For proteomic analysis, the 15 sucrose gradient fractions were pooled according to the distribution pattern of MSP-1200 (determined by Western blotting), such that Fractions 1-4 formed Pool 1, Fractions 5-9 formed Pool 2, Fractions 10-13 formed Pool 3, and Fractions 14-15 formed Pool 4 (pellet-associated material). Pooled fractions were diluted to 3 ml with human tonicity-PBS at 0 °C and centrifuged at 30,000 rpm for 1 h (2°C) in a TLA 100.3 rotor in a Beckman Optima MAX-E ultracentrifuge. The supernatant was discarded, and the pellet was washed with an additional 3 ml human tonicity-PBS (0 °C) and recentrifuged as above. Supernatant was discarded, and the pellets were snap-frozen on dry ice for proteomic analysis. Protein Digestion and Multidimensional Protein Identification Technology Analysis—Each membrane/protein pellet was resuspended in 20 μl of 90% formic acid containing 500 mg/ml cyanogen bromide and incubated overnight at room temperature protected from light. 2 volumes (40 μl) of 30% NH4OH were added drop by drop followed by the addition of 3 volumes (180 μl) of saturated NH4HCO3 drop by drop. After verifying that the pH of each solution was ∼8.5, solid urea was added to a final concentration of 8 m. Disulfide bonds were reduced by adding tris(2-carboxyethyl)phosphine to 5 mm and incubated at room temperature for 30 min. Cysteines were alkylated by adding iodoacetamide to 20 mm and incubating at room temperature for 30 min protected from light. Sequencing grade endoproteinase Lys-C (Roche Applied Science) was then added at an estimated ratio of 1:100 (enzyme: substrate, w/w) and incubated at 37 °C for ∼16 h. The solution was then diluted 2-fold by adding an equal volume (345 μl) of 100 mm Tris, pH 8.5, and adding CaCl2 to 2 mm. Sequencing grade modified trypsin (Promega) was then added at an estimated ratio of 1:100 and incubated at 37 °C for ∼16 h. 90% formic acid was then added to a final concentration of 4%. Each sample was essentially loaded on a “three-phase multidimensional protein identification technology column” and developed with six elution steps using an HP1100 high pressure liquid chromatography system (Hewelett Packard) online with an LCQ Deca ion trap mass spectrometer (Thermo) as described previously (18McDonald W.H. Yates III, J.R. Dis. Markers. 2002; 18: 99-105Crossref PubMed Scopus (271) Google Scholar). Data-dependent tandem mass spectrometry spectra were collected as described previously (19Washburn M.P. Wolters D. Yates III, J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4091) Google Scholar). Tandem mass spectrometry spectra were searched against a P. falciparum protein data base (10/03/02 release, with the manually added sequence of RAMA) combined with human, mouse, and rat databases using the search algorithm SEQUEST. Peptide identifications were filtered using default settings (except where noted) using the program DTASelect, and samples were compared using the program Contrast (20Tabb D.L. McDonald W.H. Yates III, J.R. J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1141) Google Scholar). Fluorescent Imaging—GFP fusion proteins for localization studies were encoded in transfection constructs under the regulation of the tetracycline-inducible expression system (21Meissner M. Krejany E. Gilson P.R. de Koning-Ward T.F. Soldati D. Crabb B.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2980-2985Crossref PubMed Scopus (92) Google Scholar). Primer sets used for the amplification of the following genes were: pf12 (5′-GATCACGCGTACCATGTGACTTAACGATGTATACAAATTAG-3′ and 5′-GATCACTAGTTTATAAGATGAATGATAAGAATGATGTGATAATAAAAG-3′); pf38 (5′-GATCACGCGTGTTGATTTTCGAACAGAAAAAGGAA-3′ and 5′-GATCACTAGTTTAAAAGTTTAAAAAGAAAAGATATATA-3′); and pf92 (5′-TACAAAACGCGTACTTTTGAACAAAAGGTACAGA-3′ and 5′-AAGTGAACTAGTCTATATGAAAGAAGAAATAGACAAGGA-3′). PCR products were cut with MluI and SpeI and were cloned in-frame C-terminal to secreted GFP. Anhydrotetracycline was removed from parasite cultures 72 h prior to live imaging (in the presence of 2.5 nm WR99210) to allow expression of the GFP fusion. Prior to microscopy, parasites were incubated in culture medium containing 100 ng/ml 4′,6-diamidino-2-phenylindole. Late blood-stage parasite fluorescence images were captured using a Carl Zeiss Axioskop microscope, with a PCO SensiCam and Axiovision 2 software. For immunofluorescence assays, blood-stage parasites were fixed (22Tonkin C.J. van Dooren G.G. Spurck T.P. Struck N.S. Good R.T. Handman E. Cowman A.F. McFadden G.I. Mol. Biochem. Parasitol. 2004; 137: 13-21Crossref PubMed Scopus (346) Google Scholar) and then dried onto poly-l-lysine-coated slides. Parasites were blocked and permeabilized in 1% bovine serum albumin and 0.2% Triton X-100 in PBS and then probed with mouse anti-Pf41 serum (1:100) and rabbit anti-MSP-119 IgG (23de Koning-Ward T.F. O'Donnell R.A. Drew D.R. Thomson R. Speed T.P. Crabb B.S. J. Exp. Med. 2003; 198: 869-875Crossref PubMed Scopus (75) Google Scholar) (0.02 mg/ml). Polyclonal mouse sera were raised to a recombinant fusion protein containing a 115-amino acid spacer region between the two Cys6 domains of Pf41. This region was amplified with the primer set (5′-TTTTGGATCCCTGGACACGGTGACAATACAAAAGT-3′ and 5′-ACACTCTGCAGTCCAQCGTTTTGAAATAATTTGCT-3′) and was inserted into a modified maltose binding protein expression vector, pMal-c2x (New England Biolabs), that produced a mannose-binding protein (MBP)-Pf41 fusion protein with a C-terminal 6x His tag. The fusion protein was expressed in BL-21 Escherichia coli cells and purified over nickel resin (Qiagen). Bound antibodies were then visualized with Alexa Fluor 568 nm anti-rabbit IgG and Alexa Fluor 488 nm anti-mouse IgG (Molecular Probes) diluted 1:1000. Parasites were mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (Vecta Laboratories). Immunodetection of Detergent-resistant Membrane Proteins—DRM fractions from the sucrose gradient were resuspended in an equal volume of 2 × non-reducing sample buffer and placed at 70 °C for 10 min. Samples were run under non-reducing conditions because a number of the antibodies used in the subsequent western blots (e.g. those recognizing MSP-1, -4, and -5) recognize reduction-sensitive epitopes. Samples (20 μl) were loaded onto a 4-20% gradient Gradipore long-life Tris-HEPES-SDS precast polyacrylamide mini gel, spiking the first fraction with 6 μl of Bio-Rad prestained Precision Plus protein standards. Protein samples were electrophoresed under non-reducing conditions and transferred to Immobilon-P transfer membranes (Millipore) for Western blotting as described (24O'Donnell R.A. de Koning-Ward T.F. Burt R.A. Bockarie M. Reeder J.C. Cowman A.F. Crabb B.S. J. Exp. Med. 2001; 193: 1403-1412Crossref PubMed Scopus (234) Google Scholar). For immunoprecipitation of GFP fusion proteins expressed in P. falciparum, anhydrotetracycline was removed from parasite cultures 72 h prior to solubilization to induce expression of the transgene. Schizonts were solubilized in 1% Triton X-100, PBS, pH 7.6, at room temperature for 30 min in the presence of Roche Applied Science Complete protease inhibitor mixture. Samples were preabsorbed with control (from individuals not exposed to malaria) IgG-coated Sepharose (100 μl of a 50% suspension, 2 h, 4 °C) and then incubated with Pool M human IgG-coated Sepharose (from individuals not exposed to malaria) (Melbourne) beads (100 μl of a 50% suspension) and incubated at 4 °C for 4 h. Pool M (as with Pool P; see below) was prepared from pooled sera obtained from highly exposed (P. falciparum immune) Papua New Guinean adults (24O'Donnell R.A. de Koning-Ward T.F. Burt R.A. Bockarie M. Reeder J.C. Cowman A.F. Crabb B.S. J. Exp. Med. 2001; 193: 1403-1412Crossref PubMed Scopus (234) Google Scholar). The unbound fraction (∼1 ml) was retained, and beads were washed 3 × 10 min in immunoprecipitation buffer (PBS, pH 7.6, containing 1% Triton X-100 and protease inhibitors). Bound material was eluted with the addition of an equal bed volume of non-reducing SDS sample buffer (on ice, 5 min) followed by the subsequent addition of an equal bed volume of non-reducing sample buffer at 70 °C for 10 min. P. falciparum Culture and Transfection—P. falciparum 3D7 strain parasites were cultured and synchronized using standard procedures (25Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2840) Google Scholar, 26Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6185) Google Scholar). Ring-stage parasites (∼1% parasitemia) were transfected with 100 μg of purified plasmid DNA (Plasmid Maxi kit, Qiagen) as described previously (27Crabb B.S. Cowman A.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7289-7294Crossref PubMed Scopus (237) Google Scholar), using modified electroporation conditions (28Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (413) Google Scholar). The DRM Proteome of P. falciparum Schizonts Is Dominated by Surface, Rhoptry, Multimembrane-spanning, and Exported Proteins—Sucrose gradient flotation was used to purify DRMs from schizonts, an intraerythrocytic stage that consists of maturing merozoites enclosed in a parasitophorous vacuole. The effectiveness of this approach was examined by western blotting (Fig. 1). Each of the GPI-anchored proteins examined was recovered in the buoyant fractions. These proteins were generally well separated from the single-pass type 1 integral membrane proteins, which remained in the bottom fractions. It was evident that peripheral proteins located in the parasitophorous vacuole or rhoptry organelles also tended to associate with DRMs (Fig. 1). These proteins each possess a signal sequence but do not have an obvious membrane anchor element, suggesting that they interact with a membrane-associated protein. Unlike all other proteins examined by western blotting, SERA5 and SERA6 were only observed in DRM extracts from schizont-infected erythrocytes not treated with saponin (a process that removes much of the surrounding erythrocyte proteins). This was expected as it is well known that the peripheral SERA proteins do not remain with the parasite pellet upon saponin lysis (29Fox B.A. Xing-Li P. Suzue K. Horii T. Bzik D.J. Exp. Parasitol. 1997; 85: 121-134Crossref PubMed Scopus (38) Google Scholar, 30Miller S.K. Good R.T. Drew D.R. Delorenzi M. Sanders P.R. Hodder A.N. Speed T.P. Cowman A.F. de Koning-Ward T.F. Crabb B.S. J. Biol. Chem. 2002; 277: 47524-47532Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Thus, the SERAs are potentially membrane-associated by virtue of adherence to protein(s) that are themselves solubilized by saponin. Alternatively, membrane-bound SERA proteases may be activated and degraded by autolysis following exposure to the extracellular environment; recombinant forms of both SERA5 (31Hodder A.N. Drew D.R. Epa V.C. Delorenzi M. Bourgon R. Miller S.K. Moritz R.L. Frecklington D.F. Simpson R.J. Speed T.P. Pike R.N. Crabb B.S. J. Biol. Chem. 2003; 278: 48169-48177Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and SERA6 7A. Hodder and B. S. Crabb, unpublished data. are capable of autolysis. To analyze the proteome of schizont-stage DRMs, extracts from both untreated and saponin-treated parasites were combined into four pools (Fig. 1), and the proteome of washed membranes present in each pool was determined. Pools 1-3 represent buoyant membrane fractions that the western blotting experiments in Fig. 1 suggested would comprise genuine DRM-associated proteins. Pool 4 represents material that remains at the bottom of the sucrose gradient and is predicted to comprise mostly non-DRM parasite proteins that remain with a pellet of whole parasites and/or parasite organelles that are not effectively solubilized by the cold Triton X-100 extraction. However, we did expect some genuine DRM proteins to be present in Pool 4. 94 and 70 parasite proteins were detected in the saponin and non-saponin DRM proteomes, respectively (Fig. 2 and supplemental Tables S1 and S2). There was strong overlap between proteomes with 60% of the non-saponin DRM proteins also found in the saponin-treated DRM proteome; a total of 122 DRM proteins were detected. Only high confidence proteins were included in these lists, criteria that included the detection of at least two different peptides for each protein. Proteins were ordered according to the degree of buoyancy in their respective sucrose gradients (Fig. 2 and supplemental Tables S1 and S2). Overall, both DRM proteomes (incorporating all four pools) were substantially enriched for proteins predicted to be membrane-associated by means of an encoded signal sequence and/or a transmembrane domain or known acylation site, from 24% in the whole schizont proteome (32Le Roch K.G. Johnson J.R. Florens L. Zhou Y. Santrosyan A. Grainger M. Yan S.F. Williamson K.C. Holder A.A. Carucci D.J. Yates III, J.R. Winzeler E.A. Genome Res. 2004; 14: 2308-2318Crossref PubMed Scopus (357) Google Scholar) to 65-73% in the DRM proteomes. As expected, the enrichment for membrane proteins was greater (75-76%) in Pools 1-3 with the bottom fractions (Pool 4) containing a higher number of apparently contaminating proteins presumably associated with poorly solubilized parasite pellet material. Among the DRM proteins without a signal sequence or transmembrane domain were a number of proteins known to be associated with membrane proteins (e.g. the mitochondrial Tom40 homologue). Thus, the vast majority of proteins in the buoyant fractions, especially from the saponin-lysed material, appeared to be genuine DRM proteins. It was anticipated that the DRM proteome from saponin-lysed parasites would include fewer contaminants because this preparation is devoid of contaminating material from the cytosol of uninfected and infected erythrocytes. Four classes of protein were particularly prominent in buoyant fractions (Pools 1-3): GPI-associated proteins (defined below), rhoptry proteins, multimembrane-spanning proteins, and proteins predicted to be exported from the parasite into the host erythrocyte cytosol by virtue of a PEXEL motif (33Marti M. Good R.T.M.R. Knuepfer E. Cowman A.F. Science. 2004; 306: 1930-1933Crossref PubMed Scopus (713) Google Scholar, 34Hiller N.L. Bhattacharjee S. van Ooij C. Liolios K. Harrison T. Lopez-Estrano C. Haldar K. Science. 2004; 306: 1934-1937Crossref PubMed Scopus (659) Google Scholar). With respect to this latter group, the presence of a number of ring-infected erythrocyte surface antigen (RESA) homologues suggests that these virulence-associated exported proteins may largely constitute a subset of PEXEL motif proteins that are initially stored in merozoite dense granule organelles prior to their export after erythrocyte invasion (35Rug M. Wickham M.E. Foley M. Cowman A.F. Tilley L. Infect. Immun. 2004; 72: 6095-6105Crossref PubMed Scopus (59) Google Scholar). Analogous to proteomic analyses of mammalian lipid rafts (36Foster L.J. De Hoog C.L. Mann M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5813-5818Crossref PubMed Scopus (729) Google Scholar), the fractionation procedure appears to have enriched for lipid-anchored membrane proteins, with a strong peptide coverage of GPI-associated proteins in the most buoyant fractions (Fig. 2). Good peptide coverage was obtained for known, seemingly abundant GPI-anchored proteins such as MSP-2 and MSP-4, peptides of which were surprisingly poorly represented, or absent altogether (Fig. 2, absent) in previously described whole schizont P. falciparum proteomes (2Lasonder E. Ishihama Y. Andersen J.S. Vermunt A.M. Pain A. Sauerwein R.W. Eling W.M. Hall N. Waters A.P. Stunnenberg H.G. Mann M. Nature. 2002; 419: 537-542Crossref PubMed Scopus (562) Google Scholar, 3Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. Nature. 2002; 419: 520-526Crossref PubMed Scopus (1093) Google Scholar, 32Le Roch K.G. Johnson J.R. Florens L. Zhou Y. Santrosyan A. Grainger M. Yan S.F. Williamson K.C. Holder A.A. Carucci D.J. Yates III, J.R. Winzeler E.A. Genome Res. 2004; 14: 2308-2318Crossref PubMed Scopus (357) Google Scholar). Thus, our procedures used to isolate, extract, and digest DRMs have facilitated the identification of a subset of relatively abundant proteins that were seemingly not effectively solubilized in the whole cell extracts examined in previous studies. Thirteen GPI-associated proteins were detected, which can be categorized as follows: (i) known GPI-anchored proteins, (ii) predicted GPI-anchored proteins (i.e. possessing an N-terminal signal sequence and a characteristic C-terminal hydrophobic domain), (iii) proteins that bind to GPI-anchored proteins (MSP-7 (13Pachebat J.A. Ling I.T. Grainger M. Trucco C. Howell S. Fernandez-Reyes D. Gunaratne R. Holder A.A. Mol. Biochem. Parasitol. 2001; 117: 83-89Crossref PubMed Scopus (94) Google Scholar)), and (iv) proteins that are predicted bind to GPI-anchored proteins (i.e. protein homologues of groups 2 or 3 and the SERAs). Most GPI-associated proteins detected in the non-saponin extract were also present in the saponin DRM proteome; only SERA5 and SERA6 were unique to the non-saponin list consistent with the western blot data described above. Genes encoding all of the 13 GPI-associated proteins are transcribed in blood stages and are mostly co-regulated with maximal levels of expression late in the blood-stage cycle (Fig. 2 and supplemental Fig. S1) (4Bozdech Z. Llinas M. Pulliam B.L. Wong E.D. Zhu J. DeRisi J.L. PLoS Biol. 2003; 1: E5Crossref PubMed Scopus (1267) Google Scholar, 5Le Roch K.G. Zhou Y. Blair P.L. Grainger M. Moch J.K. Haynes J.D. De La Vega P. Holder A.A. Batalov S. Carucci D.J. Winzeler E.A. Science. 2003; 301: 1503-1508Crossref PubMed Scopus (1026) Google Scholar). Four of the six known GPI-anchored merozoite proteins (MSP-1, -2, -4, and RAMA) were in the DRM proteomes, and each was prominent in buoyant fractions. The absence of MSP-10 from the list was expected as this protein does not appear to incorporate into DRMs (17Wang L. Mohandas N. Thomas A. Coppel R.L. Mol. Biochem. Parasitol. 2003; 130: 149-153Crossref PubMed Scopus (27) Google Scholar). It is less clear why MSP-5 was not detected, although it is perhaps only a minor component of the merozoite surface. Consistent with this, gen" @default.
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• W2120403054 title "Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum" @default.
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