Proteome Analysis of Rhoptry-Enriched Fractions Isolated from Plasmodium Merozoites Tobili Y. Sam-Yellowe,*,† Laurence Florens,‡,# Tongmin Wang,† J. Dale Raine,§ Daniel J. Carucci,| Robert Sinden,§ and John R. Yates, III‡ Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115, Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, California 92037, Immunology and Infection Section, Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, South Kensington, London SW7 2AZ, United Kingdom, and Naval Medical Research Center, Malaria Program (IDD), 503 Robert Grant Avenue, Room 3A40, Silver Spring, Maryland 20910-7500 Received April 5, 2004
The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosinesorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria. Keywords: Plasmodium • blood stages • MudPIT • rhoptry • organelle • proteome
Introduction Malaria remains a serious public health problem with approximately 300 million cases occurring worldwide. One to three million deaths occur annually from malaria, mostly in young children. Many malaria antigens with promise as vaccine targets are either polymorphic or undergo antigenic variation, thereby frustrating vaccine design. Proteins from the rhoptries, micronemes, and dense granulessapical secretory organelles associated with invasion and host cell modificationsare among those being considered as vaccine targets, and some of these are of conserved structure. Merozoite rhoptry proteins partici* To whom correspondence should be addressed: E-mail: ilibot@ hotmail.com. † Department of Biological, Geological and Environmental Sciences, Cleveland State University. ‡ Department of Cell Biology, The Scripps Research Institute. § Immunology and Infection Section, Department of Biological Sciences, Imperial College London. | Naval Medical Research Center, Malaria Program (IDD). # Present address: The Stowers Institute for Medical Research, 1000 50th Street, Kansas City, MO 64110. 10.1021/pr049926m CCC: $27.50
2004 American Chemical Society
pate in parasitophorous vacuole (PV) formation.1,2 Traditional methods have identified only a small number of genes encoding rhoptry proteins. Polyclonal and monoclonal antibodies prepared against whole rhoptries isolated from different Plasmodium species identified several novel proteins. Proteins of low abundance were similarly identified by electrophoresis. These methods, while useful in identifying individual proteins, do not permit analysis of the full protein repertoire of the organelle. The genomes of P. falciparum, P. yoelii, P. berghei, and P. chabaudi are now virtually complete.3,4,5 This, combined with cell fractionation and proteomic analysis, enables the comprehensive identification of organellar proteins. Merozoite rhoptries enriched by subcellular fractionation were subjected to proteomic analysis using multidimensional protein identification technology (MudPIT).6 The analysis of all life stages, with the single exception of the pre-erythrocytic/ hepatic stage, of P. falciparum and/or P. berghei was recently completed using this gel-free proteomic approach5,7 and revealed the presence of stage-specific, strategy-specific and stage-transcending sets of proteins. However, an analysis of Journal of Proteome Research 2004, 3, 995-1001
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research articles individual organelles within Plasmodium has not been reported. In this study, we report a comprehensive analysis of proteins detected in rhoptry-enriched fractions isolated from rodent-infective merozoites.
Material and Methods Parasite Maintenance. P. berghei (strain K-173) (provided by Dr. Mark Wiser, Tulane University), P. chabaudi adami (provided by Dr. Masamich Aikawa, Tokai University, Isehara, Kanagawa, Japan), and P. yoelii strain 17XL (provided by Dr. Carol Long, Allegheny University, Philadelphia PA) were used in this analysis. These parasites were maintained in Carworth Farm White (CFW) mice (outbred) as described.8 Rhoptry Isolation. Rhoptries were isolated from merozoites of P. berghei (strain K-173), P. chabaudi adami and P. yoelii (17XL) using previously described methods9 with modifications.10,8 Multidimensional Protein Identification Technology. P. berghei, P. chabaudi, and P. yoelii rhoptry-enriched fractions (one fraction of each) were digested with endoproteinase Lys-C followed by trypsin, in 0.1 M ammonium bicarbonate, pH 8.5.6 Five replicates of P. yoelii rhoptry fractions were digested with proteinase K (Roche) in 100mM sodium carbonate, pH 11.5.11 Peptide mixtures were analyzed by MudPIT as previously described.7 Interpretation of MS/MS Datasets. A modified version of SEQUEST,12 PEP_PROBE,13 was used to match MS/MS spectra to peptides in a database containing gene model sequences from both P. yoelii and P. berghei genomes,4,5 as well as rodent protein sequences. These sequences can be downloaded from ftp://ftp.sanger.ac.uk/pub/pathogens/P_berghei/Berg.peptides.2.7.2003 (14667 proteins) and ftp://ftp.tigr.org/pub/data/ Eukaryotic_Projects/p_yoelii/annotation_dbs/PYA1.pep (7749 proteins). Cross-correlation scores, DeltCn, and probability and statistical confidence values were used to filter PEP_PROBE outputs using the DTASelect/CONTRAST package14 as reported previously.15
Results and Discussion Rhoptry fractions were isolated from P. berghei, P. chabaudi, and P. yoelii using established protocols. The purity and homogeneity of these sucrose gradient fractions have been assessed previously by transmission electron microscopy.16 In addition, antibodies raised against these rhoptry-enriched fractions were used in immunoelectron microscopy of segmented schizonts and isolated rhoptries from rodent Plasmodium species and confirmed the specificity of the antibodies to the rhoptries.8,17 After compiling results from all three species, MudPIT analysis of these rhoptry-enriched fractions identified with confidence 148 nonredundant proteins (against P. yoelii and/or P. berghei databases) detected by at least 2 peptides or one peptide in two independent runs (Supplemental Table 1). Differential Proteomic Analysis to Identify Proteins Enriched in the Organelle Fraction. Significant contamination with abundant cytoplasmic components, e.g., heat shock and ribosomal proteins, translation elongation factors and metabolic enzymes was evident. To distinguish contaminants from bona fide organelle components, the rhoptry-enriched proteome was compared to a pool of 1139 proteins detected in P. berghei mixed asexual blood stages (Supplemental Table 2). 5 This differential analysis approach has recently proven suc996
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Sam-Yellowe et al.
cessful in revealing two novel parasite proteins on the surface of infected erythrocytes.15 Not surprisingly, more than 90% of the proteins detected were also found in the whole cell analyses, with only 15 proteins uniquely identified from the rhoptry-enriched preparations. To highlight proteins specifically enriched in the rhoptry fractions, the number of spectra and sequence coverage (percentage of the protein sequence covered by detected peptides) were used (Supplemental Table 2): proteins had to be either unique to the rhoptry analysis or detected with higher sequence coverage than in the whole cell lysate analysis (Figure 1). Using these selection criteria, and removing obvious contaminants such as ribosomal proteins and histones, 40 proteins were deemed specifically enriched in the rhoptry preparations (Supplemental Table 2). Although the antibodies described in8,17 clearly are recognizing rhoptries, they do not establish that other organelles or proteins in other membranes cannot be labeled as well. The 40 proteins highlighted by the differential sequence coverage display could originate from organelles that have co-purified with the rhoptries and hence are also enriched. A clear example is the detection of 27 proteasome subunits, 13 of which are “enriched” in the rhoptry fractions (Figure 1). This is likely due to co-fractionation of this large cytoplasmic complex with rhoptries on the sucrose gradient. In addition, rhoptries were isolated from several rodent species, whereas only P. berghei mixed asexual blood stages were analyzed. While there is a high similarity between the genomes of rodent parasites sequenced so far, some genes may not have direct orthologues, as it is the case for the members of the Plasmodium interspersed repeat (pir) gene family. Although sharing sequence similarities, the pir genes are species-specific and no P. yoelii-specific YIR proteins were expected to be detected in P. berghei mixed asexual stages (Supplemental Table 2). The presence of YIR proteins indicated that the P. yoelii rhoptry-enriched fractions were likely contaminated with parasite/host plasma membranes. However, no proteins known to locate to micronemes or dense granules were detected in the rhoptry-enriched fractions, indicating that there was limited, if any, contamination by other apical organelles. Analysis of Amino Acid Sequences for Rhoptry-Targeting Signals. In addition to a classical N-terminal secretory signal peptide, targeting of membrane proteins to the rhoptry organelle has been shown, in Toxoplasma, to occur via the endocytic pathway in an adaptin-dependent manner (reviewed in ref 18). This requires tyrosine-based (YXXΦ, where Φ is a large hydrophobic residue)19 and/or di-leucine motifs20 within their C-terminal cytoplasmic tail. Such sorting mechanisms have not been demonstrated in Plasmodium. However, with the single exception of the reticulocyte binding proteins, all known rhoptry proteins do have YXXΦ motifs in their Cterminal domains (Table 1). When considering the proteins detected in the rhoptryenriched fraction, C-terminal tyrosine-sorting motifs were found in six of the nine hypothetical proteins enriched in the rhoptry fractions (Table 2). In addition, seven hypothetical proteins that were detected with numerous peptides in the mixed asexual analysis, and hence did not satisfy our criteria for being enriched in the rhoptry fractions, appeared to also contain such motifs. In addition to the 24 proteins detected with higher sequence coverage in the rhoptry-enriched fraction, 12 proteins displaying the rhoptry-specific targeting signal were deemed worth considering as potential rhoptry proteins, especially those predicted to have a signal peptide as well
research articles
Plasmodium Rhoptry Proteome
Table 1. Structural Properties and Expression Profile of Known Rhoptry Proteins from Plasmodium Merozoites
namea
RhopH1
Pb
Py
PB000405.02.0 PY02932
differential analysise [Rhop-ABS]
structural featuresd
locusb Pf
descriptionc
PFC0110w/ PFC0120w
high molecular weight rhoptry protein 1 RhopH2 PB001562.02.0 PY05296 + PFI1445w high molecular PY05295 weight rhoptry protein 2 RhopH3 PB000377.03.0 PY06325 PFI0265c high molecular weight rhoptry protein 3 Clag9 PB000482.02.0 PY06117 PFI1730w cytoadherence linked asexual protein 9 RAP1 PB000779.00.0 PY00622 PF14_0102 rhoptry-associated protein 1 RAP2 PB301475.00.0 PY03918 PFE0080c rhoptry-associated protein 2 RAP3 PFE0075c rhoptry-associated protein 3 ROPE PB000112.03.0 PY00447 PFB0145c repetitive organellar protein Rhop148 PB000869.00.0 PY03152 PF13_0348 148kDa rhoptry protein RAMA PB103628.00.0 PY00143 AAQ89710 rhoptry associated membrane antigen Stomatin PB300597.00.0 PY03188 PFC0800w Stomatin/ band7-related AG1 PB000586.02.0 PY03828 PFL2140c ADP-ribosylation factor GTPaseactivating protein P36 PB000799.01.0 PY04725 MAL7P1.114 P36-like rhoptry protein PfRH1 PFD0110w Reticulocyte binding protein 1 PfRH2b MAL13P1.176 Reticulocyte binding protein 2b PfRH2a PB000327.03.0 PY00649 PF13_0198 Reticulocyte binding protein 2a PfRH3/Py235 PY04930 PFL2520w 235 kDa rhoptry protein/ Reticulocyte binding protein 3 Py235 PY04630 235 kDa rhoptry protein Py235 PY01185 235 kDa rhoptry protein PB301428.00.0 Reticulocytebinding protein
L
Pf proteomef
SP TM YXXΦ RPT ∆SC ∆spec T M G
-52 x
x
0 YKEI
-7.6 -103 x
x
0 YKEM
-12.8 -192 x
x
1416 1
0 YCPV
1364 1 897 1 1340 1
1 YELI
782 1
-13.2
S RNA levels cluster
x
ES f LS
15
ES f M
15
x
ES f M
15
x
nd x
x
x
x
ES f LS
13
0 YFAF
-50.6 -160 x
x
x
x
ES f M
15
398 1
0 YALF
-52
-79 x
x
x
ES f M
15
400 1
0 YMDM
na
na x
x
ES f M
15
1979 0
0 YSVM yes
nd
nd
x
ES f M
no
1262 0
0 YDHL yes
x
ET f LS
861 1 GPI YQKV
nd
Pf transcriptomeg
yes
x
nd
nd
nd
nd na na na na na
na
nd
ER f LT
8
ES, S
12
374 0
0 YNNI
nd
332 0
0 YLNY
-12
225 0
0 YITF
x
-3 x
-8.4
-1
2931 1
1 no
yes
na
na x
1115 0
1 no
yes
na
3130 1
1 no
yes
2792 1
0 YTQI
yes
2740 1
x
6
ET f LS
no
x
LS f M
15
na x
x
LS f M
15
nd
nd
x
ES f M
15
nd
nd
x
ES f M
15
0 YKNY
nd
nd
2723 1
0 YNII
nd
nd
537 0
0 YEII
nd
nd
x
a Commonly used abbreviation. b P. berghei (Pb), P. yoelii (Py), and P. falciparum (Pf) orthologous genes (when applicable), using the nomenclature established by the International Malaria Genome Project. c Gene annotation. d Length (L), signal peptide (SP, as determined by the SignalP algorithm),37 transmembrane segments (TM, as determined by the TMHMM algorithm),43 C-terminal tyrosine-sorting motif (YXXΦ), and repetitive sequences (RPTs) corresponding to the orthologue with the longest (i.e., most complete) gene (underlined in the locus columns). e Differential proteomic analysis: rhoptry-enriched (Rhop) minus whole cell lysates from mixed asexual blood stages (ABS).5 Differential Sequence Coverage (∆SC) and Differential Spectral Count (∆Spec) are defined in Supplemental Table 2. Numbers are underlined when proteins were detected uniquely in the mixed ABS dataset. f Protein expression data for P. falciparum trophozoite (T), merozoite (M), gametocyte (G), and sporozoite (S) stages.7 g RNA expression levels and temporal pattern clusters for P. falciparum early and late rings (ER/LR), early and late trophozoites (ET/LT), early and late schizonts (ES/LS), merozoites (M), gametocytes (G) and sporozoites (S).41 nd: not detected; na: not applicable.
(Table 2). The likelihood of these 36 proteins being bona fide rhoptry proteins is discussed below based on known function, localization, structural features, and/or expression profile. Known Rhoptry Proteins. All three high molecular weight rhoptry proteins (RhopH1, P. yoelii/P. berghei locus name: PY02932/PB000405.02.0; RhopH2, PY05296 + PY05295/ PB001562.02.0; RhopH3, PY06325/PB000377.03.0) were detected with multiple peptides in both rhoptry and mixed asexual analyses (Supplemental Table 2). However, because these proteins are highly abundant in the merozoites, none appeared “enriched” in the rhoptry fraction as defined by the differential analysis. In addition, a high molecular weight protein related
to RhopH3 (PY01798), a protein related to RhopH1 (PY04666), and a large protein annotated as rhoptry protein in the P. yoelii database (PY04959/PB300716.00.0) were detected, the latter two being unique to the organelle fractions. Their previous annotation as rhoptry proteins based on sequence similarities was hence confirmed. Known rhoptry proteins, such as the low molecular weight rhoptry proteins (the rhoptry-associated proteins, RAP), the 235kDa rhoptry proteinsshomologues of the reticulocytebinding proteins of P. falciparum21s, PyAG1, an ADP-ribosylation factor-1 GTPase-activating protein,22 the 229-kDa repetitive organellar protein (ROPE),23 the cytoadherence-linked Journal of Proteome Research • Vol. 3, No. 5, 2004 997
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Sam-Yellowe et al.
Table 2. Structural Properties and Expression Profile of Potential Rhoptry Proteins from Plasmodium Merozoites
Pf
descriptionb
PB001129.00.0 PY02866 PB000975.03.0 PY01045 PB000656.01.0 PY04475
PF08_0115 MAL6P1.70 MAL6P1.215
PB000367.01.0 PY03995
PFE0585c
protein with DnaJ domain coatomer R subunit pyridoxine biosynthetic enzyme, PDX1 myo-inositol-1phosphate synthase lysophospholipase hypoxanthine-guanine phosphoribosyl transferase aspartyl aminopeptidase papain cysteine protease, SERA-3 Papain family cysteine protease Papain family cysteine protease secreted blood stage antigen pAg3 Blood stage membrane protein pAg1 exported protein 2, Exp2 Exported protein 1, Exp1, hepatocyte erythrocyte protein 17 kDa early transcribed membrane protein merozoite surface protein 9 precursor high molecular weight rhoptry protein 3-related rhoptry protein PyRhopH1A-related Pbste (P. berghei subtelomeric gene family e) dentin phosphoryn, putative retinitis pigmentosa GTPase regulator-like protein hypothetical protein, conserved hypothetical protein hypothetical protein
Pb
Py
differential analysisd [Rhop-ABS]
structural featuresc
locusa
PY02009 PF07_0005 PB001303.00.0 PY03478 PF10_0121 exons-3-4-5 PB000725.01.0 PY03205 PY00291
PFI1570c
PB107093.00.0 PY00292
PFB0350c
PB000107.03.0 PY00293
PFB0330c
PB001131.01.0 PY03625
PF08_0003
PB000371.00.0 PY06203
PF08_0003
PB000390.00.0 PY05892 PB000484.01.0 PY04421
PF14_0678 PF11_0224
PY02667 PB000668.01.0 PY02883
PFL1385c
PB000377.03.0 PY01798
PFI0265c
PB300716.00.0 PY04959 PY04666 PB105866.00.0 PY02439
PFI0295c
PB000848.03.0 PY01786
PF08_0137
PY03917 PB000862.00.0 PY07825
PF08_0074
PB000110.02.0 PY00763 PB000502.02.0 PY03424 exon2 (985_6) PB000730.02.0 PY02301 PB000642.01.0 PY07482 PB108442.00.0 PY01759 PB102173.00.0 PY01145 PB102172.00.0 PY01146 + PY03763 PB000970.00.0 PY06925 PB106275.00.0 PY05401 PB108030.00.0 PY02638 PB108293.00.0 PY00800 PB402197.00.0 PB108056.00.0 PY02057 PB000878.02.0
PFI1270w PFI0580c
PF14_0344 hypothetical protein MAL13P1.237 hypothetical protein MAL13P1.308 hypothetical protein hypothetical protein hypothetical protein PF11_0302
PF10_0063
hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein, conserved
L
SP TM YXXΦ RPT ∆SC
∆spec
Pf transcriptomef
Pf proteomee T M G
900 0 1537 0 301 0
0 YNGL 0 YENI 0 LL
3.6 0.4 13
-1 -1 4
x x
x x
x x
604 0
0 YSSF
-7.4
-43
x
x
x
424 0 231 0
0 YNTI 0 YNEI
7.9 30.2
-39 378
x
x
570 0 1204 0
0 YNQV 0 YFCY
5.1 3
2 -2
1132 0
0 YFDL yes -10.1
1223 1
0 YTKL yes
675 1
S
RNA levels
cluster
x
S LT G, ER f LT
no no 5
x
G, LT fLS
6
x
ERfLR, M LRfES
4 6
x x x x G, LR f ES na na na na na
8 na
-55
x
ESfLS
13
-4.5
-81
x
LTfLS
13
0
19.7
8
x
ER, M
4
675 1
0
18.8
25
x
ER, M
4
287 1 169 1
0 YTHI 1
-21.6 43.8
ETfES, M LR f LT, M
7 7
175 1
1 YGIY
28.6
11
na na na na na
na
743 1
0
5.3
-2
x
x
ES f LS
13
897 1
0 YKEM
-35.7 -309
x
x
ES f M
15
x x ET f ES na na na na na na na na na na
no na na
x
7
-11.5 x 8 x
1807 0 454 0 313 1
2 YEKL 4 YTLY 0 yes
1 9 9.3
2 4 0
1219 1
0 YSQY yes
10.9
12
725 0
1 YFNL yes
5.2
2
248 0
0 YRGF
-24
-41
222 1 413 1
0 YNKL 3 YRPF
-47.5 -6.1
-2.9 -20 -24 -162 -19.2 -371 -2.8 -38 6.8 5
x
x x
x x
x
ER f LT, M
na na na na na
na
x
x
x
ERfES, M
no
-71.5 x -10
x x
x
ERfET, M ER, S
no 1
x x x na na
x x x na na
x x x na na
x na na na
x ERfLT, M na na na na na na na na na
x
993 374 2598 179 285
1 0 0 0 0
0 0 0 1 1
452 378 173 334
1 1 1 1
0 0 YLDI yes 2 YIHI 0 YTLI yes
2.1 1.4 12.1 6.3
-28 -24 2 4
x na na na
1 YASI 0
3 12.2
7 -82
na na na na na x x x x ER f ES
1554 1 107 0
YKKY YNGL YKIY YSRI
x
yes
x
ERfLR, M, S ERfES, M x ESfLS, M, S na na na na
4 14 na na no na na na na no
a P. berghei (Pb), P. yoelii (Py), and P. falciparum (Pf) orthologous genes (when applicable), using the nomenclature established by the International Malaria Genome Project. b Gene annotation. c Length (L), signal peptide (SP, as determined by the SignalP algorithm),37 transmembrane segments (TM, as determined by the TMHMM algorithm),43 C-terminal tyrosine-sorting motif (YXXΦ), and repetitive sequences (RPTs) corresponding to the orthologue with the longest (i.e. most complete) gene (underlined in the locus columns). d Differential proteomic analysis: rhoptry-enriched (Rhop) minus whole cell lysates from mixed asexual blood stages (ABS).5 Differential Sequence Coverage (∆SC) and Differential Spectral Count (∆Spec) are defined in Supplemental Table 2. e Protein expression data for P. falciparum trophozoite (T), merozoite (M), gametocyte (G), and sporozoite (S) stages.7 f RNA expression levels and temporal pattern clusters for P. falciparum early and late rings (ER/LR), early and late trophozoites (ET/LT), early and late schizonts (ES/LS), merozoites (M), gametocytes (G) and sporozoites (S).41 na: not applicable.
asexual protein Clag9,24 PfRhopH148,25 and the membraneassociated rhoptry protein (RAMA)26 were not among the proteins identified in the rhoptry samples, although some were detected in the whole cell analysis (Table 1). This may suggest they are of relatively low abundance or it may reflect the maturity of the rhoptries at the time of isolation. The rhoptries 998
Journal of Proteome Research • Vol. 3, No. 5, 2004
were isolated from segmented schizonts following one to two rounds of Percoll synchronization of the parasites in mice before collection of the rodent parasites for fractionation. Mature schizonts, not fully segmented, could also be found in the enriched schizont preparations collected for homogenization and subsequent fractionation. The occurrence of different
Plasmodium Rhoptry Proteome
Figure 1. Differential Sequence Coverage Analysis. The percentage of protein sequence covered by peptides identified in the rhoptry-enriched fractions was compared with the one measured in whole cell lysates of P. berghei mixed asexual blood stages. Positive differential sequence coverage indicates proteins enriched in the rhoptry fractions. The proteins were grouped by broad functional classes. Stars indicate proteins predicted to have a signal peptide and/or at least one transmembrane domain. The detailed list of proteins is provided as Supplemental Table 2.
schizont stages may have accounted for the presence of immature rhoptries in the preparations, for variation in the levels of specific rhoptry proteins and for some of the differences seen in the distribution of the rhoptry proteins identified. Proteases. Ten distinct proteases, many of which have been reported as being involved in hemoglobin digestion within the food vacuole, were identified in the rhoptry fractions (Supplemental Table 2). These include plasmepsin (PY06899/PB000298.03.0), berghepain-2 (PY00783/PB000980.00.0), M1-family aminopeptidase (PY01557/PB000843.02.0) and the falcilysinrelated proteins, yoelilysin/bergilysin (PY07032/PB000738.02.0). The detection of these proteases is likely due to contamination of the rhoptry preparation by food vacuoles, although falcilysin has recently been shown to also localize to other vesicular structures within the parasite.27 Three cysteine proteases, encoded by consecutive genes on P. yoelii contig MALPY00082 (PY00293/PB000107.03.0; PY00292/PB107093.00.0; PY00291), were among the identified proteins, SERA-3 being enriched in the rhoptry fractions. These proteases belong to the papain family, have been shown to localize to the merozoite surface and/or the parasitophorous vacuole,28 and all contain the C-terminal tyrosine-sorting motif. A leucine aminopeptidase (PY01898/PB000863.03.0), a putative M24 metallopeptidase (PY00855/PB000628.00.0), and an aspartyl aminopeptidase (PY03205/PB000725.01.0) were also detected; the later has the C-terminal YXXΦ motif and is enriched in the rhoptry fractions (Table 2). Such proteases could be involved in processing merozoite proteins targeted to the rhoptry organelles. Metabolic Enzymes. Whereas the large majority of enzymes detected in the rhoptry fractions were cytoplasmic contaminants (i.e., abundant in the whole cell analysis), three enzymes involved in coenzyme biosynthesis or in lipid metabolism appeared clearly enriched in the rhoptry fractions (Figure 1). The pyridoxine biosynthetic enzyme, (PDX1; PY04475/PB000656.01.0), is involved in an alternative pathway of pyridoxine 5-phosphate (PNP) biosynthesis. Vitamin B6, in its active form pyridoxal phosphate, is an essential coenzyme of many en-
research articles zymes with diverse functions. Because PNP synthase is not present in humans, who need to ingest vitamin B6 in their diet, the enzyme can be regarded as a potential target for the development of novel drugs. The purine salvage enzyme, hypoxanthine-guanine xanthine phosphoribosyl transferase (HGXPRT; PY03478/PB001303.00.0), catalyzes the phosphoribosylation of hypoxanthine, guanine, and xanthine. The malaria parasite is incapable of de novo purine biosynthesis, which makes HGXPRT a drug target.29 In addition, HGXPRT has been described as a major target antigen for cell-mediated immunity in malaria.30 A BLAST search31 on PY02009, annotated as “immediate early protein homolog” in the P. yoelii genome, returned 10 P. falciparum proteins, 6 of which are described as putative lysophospholipases. Lysophospholipase converts lysophosphatidylcholine to lysophosphatidic acid (LPA), a potent bioactive phospholipid mediator. LPA is a known inducer of platelet aggregation, endothelial hyperpermeability, and proinflammatory responses. The plasmodial lysophospholipase represents a potential target for anti-malarial chemotherapy, and lysophospholipase inhibitors have been shown to arrest the growth and abolish the reinvasion capacity of P. falciparum in culture.32 Although not enriched in the rhoptry fraction, myoinositol 1-phosphate synthase (PY03995/PB000367.01.0) is another enzyme of interest because it catalyses the primary reaction in the synthesis of inositol. Inositol is a precursor of membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites. In addition to mitochondria, malaria parasites contain several organelles that have been associated with specific metabolic pathways: the food vacuole is where hemoglobin degradation occurs and the apicoplast is involved in fatty-acid and isoprenoid metabolism.3 None of the four enzymes highlighted here have a definite subcellular localization, in particular, none are predicted to have an apicoplast transit peptide (http://www.plasmodb.org/). However, all four contain Cterminal tyrosine or di-leucine rhoptry-sorting motifs (Table 2). Besides proteins, rhoptries contain large amount of lipids, which may be incorporated into the parasitophorous vacuole membrane during secretion of rhoptries.33 The localization of enzymes involved in lipid metabolism to apical organelles would hence make biological sense if they were directly involved in the invasion process or the establishment of the parasitophorous vacuole. Secreted Proteins. Among the proteins detected in the rhoptry analysis, eleven are known to be secreted outside the parasite plasma membrane. These proteins fall into two main classes of antigens that have been associated with either the surface of infected red blood cells (IES), or the parasitophorous vacuole (PV) surrounding the intracellular parasite. All but three appeared enriched in the rhoptry fractions (Supplemental Table 2). The secreted blood stage antigen pAg-3 (PY03625/PB001131.01.0) and the membrane blood stage antigen pAg-1 (PY06203/PB000371.00.0) were among the most abundant proteins in the rhoptry fractions (Supplemental Table 2). Although PyAg-1 and PyAg-3 share limited sequence similarities, both contain conserved tryptophan-rich domains and are orthologues of P. falciparum tryptophan/threonine rich antigen (PF08_0003). The PyAg antigens were both detected in culture supernatants, and have also been shown to be associated with the membrane of P. yoelii-infected erythrocytes.34,35 In agreeJournal of Proteome Research • Vol. 3, No. 5, 2004 999
research articles ment with being secreted proteins, P. falciparum tryptophan/ threonine rich antigen and PyAg-1 (PY06203) share a similar 2-exon gene structure, where a first short exon of around 180 bases encodes an N-terminal secretory sequence (Supplemental Figure 1A). However, the published first exon for PyAg-3 (PY03625) is longer (270 bases) and does not encode a signal peptide. An alternate choice for first exon encoding a potential signal peptide was found upstream of PY03625 (http://www.plasmodb.org/) in the P. yoelii genome (Supplemental Figure 1A). Three out of four proteins known to localize to the parasitophorous vacuole membrane (PVM) appeared enriched in the rhoptry fractions (Supplemental Table 2). It has recently been shown that members of the high molecular weight rhoptry complex (RhopH2 and CLAG9) are secreted from the rhoptries and transferred to the ring-infected red blood cell where they appear to be associated with the parasitophorous vacuolar membrane.36,24 Whether the exported protein 1 (PY04421/ PB000484.01.0), early transcribed membrane protein (PY02667), and merozoite surface protein 9 (PY02883/PB000668.01.0) are transferred to the parasitophorous vacuole membrane by release of the rhoptry contents upon invasion remains to be determined, but their presence in the rhoptry fractions indicates a fate similar to the RhopH complex. Proteins of Unknown Function. In the overall analysis of the merozoite rhoptry-enriched proteome from P. berghei, P. chabaudi, and P. yoelii, 18 hypothetical or unknown proteins were identified. These were proteins for which no function could be deduced by homology to known proteins in the public databases, suggesting that they are Plasmodium-specific molecules. Seven of these proteins were predicted to contain a signal peptide (SP) by the SignalP algorithm37 and five were predicted to contain at least one transmembrane (TM) domain using the TMHMM algorithm38 (Supplemental Table 2). All but three of the SP/TM-domain containing hypothetical proteins were enriched in the rhoptry fractions (Figure 1). Targeting to the rhoptry organelles has been shown to require the presence of an N-terminal secretory signal sequence. However, because both P. yoelii and P. berghei genomes were sequenced using a shotgun approach, numerous genes are partial, and their boundaries are tentative. We have recently shown that, even in the P. falciparum genome, the absence of an N-terminal signal peptide could be the result of 5′ exons missed by the gene prediction algorithms.39 In the present analysis, we manually re-annotated the boundaries of three hypothetical genes. PY05296 was found to encode the N-terminus of RhopH2. On the basis of homology with PB102172.00.0, PY01146 and PY03763, located at the end and the beginning of P. yoelii, contigs MALPY00303 and MALPY01112, respectively, are likely to encode only one protein (Supplemental Figure 1B). Finally, on the basis of alignments with PB000502.02.0 and PFI0580c, the second exon of gene PY03424 is likely to encode an independent protein. This is also supported by the fact that the three peptides detected in our MS/MS analysis (Supplemental Table 1) mapped within the C-terminus of PY03424, i.e., the region encoded by exon 2. Similar to the P. berghei and P. falciparum orthologous genes, a short open reading frame 5′ of PY03424 exon 2 was detected in the whole P. yoelii genome (http://www.plasmodb.org/), encoding a potential signal peptide (Supplemental Figure 1C). One of the proteins we classified as unknown is annotated as a putative dentin phosphoryn (PY01786/PB000848.03.0) in the P. yoelii genome, and was abundantly detected in rhoptry1000
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Sam-Yellowe et al.
enriched fractions (Supplemental Table 2). The P. falciparum orthologue (PF08_0137) is clearly predicted to have a signal peptide (http://www.plasmodb.org/). Phosphoryns are the major noncollagenous proteins in dentin,40 and are believed to play a crucial role in mineral nucleation and hydroxyapatite growth during dentin mineralization. The presence of dentin phosphoryn homologues in rhoptry-enriched fractions is intriguing. Dentin contains a number of extracellular matrix proteins, some of which may have adhesive properties. P. yoelii, P. berghei, and P. falciparum dentin phosphoryns show clear repetitive motifs that are species-specific (Supplemental Figure 1D). Interestingly, of the 10 hypothetical/unknown proteins enriched in the rhoptry fractions, 6 show repetitive elements within their sequence (Table 2). Since one function of repetitive motifs is to increase avidity of interaction, such structural features may have a role in merozoite attachment and invasion into the erythrocyte.
Concluding Remarks Subcellular fractionation methods coupled with multidimensional protein identification technology (MudPIT) provides a powerful approach to investigate the rhoptry proteome. However because preparing “pure” organelles is virtually impossible, the identification of abundant cytoplasm contaminants is inevitable. On the basis of the differential proteomic analysis and/or the presence of sorting signals, a set of 36 proteins (Table 2) is defined as potentially locating to the rodent Plasmodium rhoptries. Of these, 25 have direct orthologues in the human parasite genome. Large scale gene41,42 and protein7 expression studies of the P. falciparum life cycle have been carried out and show that all but one of the P. falciparum orthologues are expressed during the intra-erythrocytic cycle. Further characterization of the proteins identified in the present analysis will yield important insights concerning the biogenesis of the rhoptries and the role of the organelle in the biology of Plasmodium spp.
Acknowledgment. This study was supported by funds from Cleveland State University’s Promoting Research Initiatives with Major External Sponsors (PRIMES) award, Established Full-Time Faculty Research and Developmental Award (EFFRD) and an NIH grant AI36470 to TSY. JRY acknowledges the support of the Office of the Naval Research (Cooperative Agreement N00014-01-2-0003), the US Army Medical Research and Material Command and the National Institutes of Health (RR11823). J.D.R. is funded by a Wellcome Trust Studentship. The opinions expressed are those of the authors and do not reflect the official policy of the Department of the Navy, Department of Defense, or the U.S. government. We thank LaShonda Everett for excellent technical assistance. The whole genome shotgun sequence data for Plasmodium berghei was produced by the Pathogen Sequencing Unit at the Wellcome Trust Sanger Institute and can be obtained from the following internet site: http://www.sanger.ac.uk/Projects/P_berghei. Supporting Information Available: Supplemental Table 1: Parasite proteins identified in rhoptry-enriched fractions from rodent-infective Plasmodium species. Supplemental Table 2: Comparison of the proteins identified in rhoptryenriched fractions with whole cell lysate proteomic analysis of Plasmodium asexual blood stages. Supplemental Figure 1: Hydrophobic cluster analysis of potential Plasmodium rhoptry
research articles
Plasmodium Rhoptry Proteome
proteins. This material is available free of charge via the Internet at http://pubs.acs.org.
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