Dissecting the apicomplexan rhoptry neck proteins

Nicholas I. Proellocks, Ross L. Coppel and Karena L. Waller

Supplementary Table S1. Rhoptry neck protein proteome constructed using published and in silico resources a

Protein / Species / Gene ID/ Accession No. b / e-valuec / General comments about the Proteinprotein
RON1 / Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium berghei
Plasmodium chabaudi
Neospora caninum / TGME49_110010d
PFD0207c
Pv001030
PKH_031040-1
PB000894.00.0
PC300967.00.0
NC_LIV_130800 / The Plasmodium orthologuelogs were identified using PFD0207c in BLAST searches.
N/A / RON1 was first identified in the T. gondii rhoptry proteome and has been localized to the rhoptry neck [29]. The putative Plasmodium spp. RON1 orthologuelogs identified herein possess only weak similarity to TgRON1; thus RON1 might be specific to the Coccidian parasites.
RON2e / Toxoplasma gondii
Toxoplasma gondii
Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi
Theileria parva
Theileria annulata
Babesia bovis
Eimeria tenella
Neospora caninum / TGME49_100100d
TGME49_065080 (Ts3110)
TGME49_094400 (Ts0430)
PF14_0495
Pv117880
PKH_12530-1
PY06813
PB000379.02.0
PC000433.04.0
TP01_0014
TA19390
BBOV_I001630A
AM773998.1
NC_LIV_140230 / N/A
1e -36
1e -66
2e -68
7e -68
4e -62
5e -66
6e -65
6e -76
6e -70
5e -74
2e -60
N/A / RON2 was first identified in the T. gondii rhoptry proteome [29] and has been localized to the rhoptry neck in T. gondii and P. falciparum [9, 29]. RON2 is highly conserved across the Apicomplexa, and is involved in formation of the tight junction [8, 9], possibly through direct interactions with AMA1 [8]. Ts3110 and Ts0430 are thought to be paraloguelogs of TgRON2 [7]
RON3 / Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei-1
Plasmodium berghei-2
Plasmodium chabaudi-1
Plasmodium chabaudi-2
Eimeria tenella
Neospora caninum / TGME49_023920d
PFL2505c
Pv101485
PKH_146960-1
PY01808
PB000909.00.0
PB000304.01.0
PC302125.00.0
PC000025.04.0
AM773997.1
NC_LIV_123080 / 2e -12
1e -07
8e -06
7e -10
2e -09
N/A
N/A
N/A
7e -158
N/A / RON3 was first identified in the T. gondii rhoptry proteome and has been localized to the rhoptry neck [29]. Putative RON3 orthologuelogs with greatest similarity to TgRON3 are found in N. caninum and E. tenella, and thus RON3 might be specific to the Coccidian parasites. All Plasmodium spp. matches are very weak and are unlikely to be represent real orthologuelogs. Intriguingly, the suggested Plasmodium RON3 orthologuelogs in P. bergheiand P. chabaudi (obtained from OrthoMCL DB) areencoded by two genes each located on different chromosomes; each of the P. berghei-1and P. chabaudi-1 orthologuelogs show greatest similarity to the N-termini of the other Plasmodium orthologuelogs, whereas each of the P. berghei-2 and P. chabaudi-2 orthologuelogs show greatest similarity to the C-termini of the other Plasmodium orthologuelogs.
RON4e / Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi
Theileria parva
Theileria annulata
Babesia bovis
Neospora caninum / TGME49_029010d
PF11_0168
Pv091435
PKH_091350-1
PY02157
PB001305.00.0
PC000594.00.0
TP02_0051
TA13245
BBOV_III010920
NC_LIV_100030 / 3e -08
N/A
N/A
N/A
N/A
N/A
6e -07
7e -10
N/A
N/A / RON4 was first identified in the T. gondii rhoptry proteome [29] and has been localied localized to the rhoptry neck in both T. gondii and P. falciparum [6, 11, 29]. RON4 is highly conserved across a large number of Apicomplexa and is involved in tight- junction formation [6, 7, 11, 42]. In T. gondii, RON4 is secreted from the rhoptries and inserted into to the host cell cytosol during invasion [8, 12].
RON5e / Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi-1
Plasmodium chabaudi-2
Theileria parva
Theileria annulata
Babesia bovis
Neospora caninum / TGME49_111470d
MAL8P1.73
Pv089530
PKH_051420-1
PY02282
PB000722.00.0
PC000448.02.0
PC301326.00.0
TP01_1161
TA16660
BBOV_IV011430
NC_LIV_131780 / N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A / RON5 was first identified in the T. gondii rhoptry proteome [29] and has since been localized to the rhoptry neck [8, 12]. RON5 is highly conserved across a large number of Apicomplexa and is involved in tight- junction formation [7, 8, 12]. In T. gondii, RON5 is secreted from the rhoptries and inserted into the host cell membrane during invasion [8]. Interestingly, P. chabaudi encodes two potential RON5 orthologuelogs,whose genes are each located on different chromosome,sand that show higher levels of homology to the central regions of other RON5 orthologuelogs. The difference between the two P. chabaudi orthologuelogs lies in their N- and C-termini. In PcRON5-1, its the N-terminus is homologous to the N-terminus of all other RON5 orthologuelogs; however; its C-terminus is homologous to the central region of other RON5 orthologuelogs. By contrast, the N-terminus of PcRON5-2 is homologous to the central region, whereas its C-terminus is homologous to the C-termini of other orthologuelogs. Together, the two P. chabaudi orthologuelogs combined are similar to the processed RON5N and RON5C of T. gondii.
RON6 / Toxoplasma gondii
Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi
Plasmodium gallinaceum
Cryptosporidium hominis
Cryptosporidium parvum
Theileria parva
Theileria annulata
Babesia bovis
Neospora caninum / TGME49_097960
PFB0680wd
Pv002790
PKH_040500-1
PY05738
PB000472.03.0
PC000850.00.0
Pga10428d08.qlk
Chro.10212
cgd1_1870
TP01_1109
TA16375
BBOV_IV012010
NC_LIV_031940 / 7e -41
3e -130
N/A
N/A
1e -30
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A / RON6 was first identified in P. falciparum and has been localized to the rhoptry neck [41]. Orthologuelogs of RON6 are present in all apicomplexan genomes sequenced to date. These data suggest the function of RON6 is important in all Apicomplexa, even though its function(s) remain unknown. The orthologuelogs of RON6 outside of the Plasmodium genus were identified based solely on the presence of the conserved cysteine-rich domain.
RON8e / Toxoplasma gondii
Neospora caninum / TGME49_106060d
NC_LIV_114740 / N/A / TgRON8 is secreted from the rhoptry neck, is inserted into the host cell cytosol during invasionand is involved in tight tight-junction formation [8]. RON8 appears to be specific to the Coccidian parasites, with a possible orthologuelog also present in Eimeria tenella[8, 12]. No identifiable orthologuelogs were identified in the non-Coccidian parasites, including Plasmodium.
Reticulocyte-Bindingbinding-Like like Proteins proteins (RBLs)
Rh1
Rh2a
Rh2b
Rh3
Rh4
Rh5
PvRBP-1
PvRBP-2
235 kDa Rhoptry Proteins
Pb-reticulocyte binding protein
Reticulocyte binding protein 2 homologuelog a/Hypothetical / Plasmodium falciparum
Plasmodium vivax
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi / PFD0110wd
PF13_0198
MAL13P1.176
PFL2520w
PFD1150c
PFD1145c
Pv098585
Pv090325, Pv094255, Pv121920, Pv101495, Pv101585
Multi-gene family
Multi-gene family
Multi-gene family / 4e -31
N/A
7e -12
9e -11
N/A
6e-69
N/A
N/A
N/A
N/A / Other members of the Reticulocyte reticulocyte Bindingbinding-Like like protein (RBL) family have been suggested to localize to the rhoptry neck based on the localization of their P. falciparum orthologuelogs to the rhoptry neck [34-–37, 66, 76, 81]. Proteins in this family have been shown to bind to RBCs and have an integral role in Plasmodium invasion [38, 76-–78]. The rodent malaria parasites contain a multigene RBL family. In P. yoelii, the Py235 family comprises as many as 50 genes [82]. There are no apparent RBL orthologuelogs in any other Apicomplexa for which there is genome sequence data available.
Pf34 / Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium chabaudi / PFD0955wd
Pv090075
PKH_052400-1
PC000958.02.0 / 2e -36
2e -36
6e -27 / Pf34 was first identified as a potential rhoptry protein based on very weak similarity to a T. gondii rhoptry protein [29], but was later localized to the rhoptry neck in P. falciparum by immunofluorescence [44]. This study also suggested that Pf34 does not have any true orthologuelogs in other Apicomplexa, including T. gondii [44].
PfAARP / Plasmodium falciparum
Plasmodium vivax
Plasmodium knowlesi
Plasmodium yoelii
Plasmodium berghei
Plasmodium chabaudi / PFD1105wd
Pv090210
PKH_052690-1
PY06454
PB402266.00.0
PC401501.00.0 / N/A
3e -06
N/A
N/A
N/A / PfAARP has been localized to the rhoptry neck in P. falciparum [39] and is able to bind RBCs during invasion [39]. There are no apparent AARP orthologuelogs in any other Apicomplexa for which there is genome sequence data are available.

a Abbreviations: The the letters represented in each acronym are givenunderlinedin italic. RON, Rhoptry Neck neck protein; Rh, Reticulocytereticulocyte-binding like homologuelog; PvRBP, Plasmodium vivax Reticulocytereticulocyte-Binding binding Proteinprotein; RBC;,Red red Blood blood Cellcell; PfAARP,;Plasmodium falciparumApical apical Asparagineasparagine-Rich rich Proteinprotein. N/A, not available (see below).

b Genes listed in the third column are those identified as the most similar in the genomes when searched with query gene sequences([indicated by an asterisk [*]) (*)] from T. gondii and P. falciparum (listed in the second column).

c Similarity was assessed using BLAST and other tools available at a number of online genome databases, including PlasmoDB (plasmodb.org), ToxoDB (toxodb.org), EuPathDB (eupathdb.org), OrthoMCL DB ( and NCBI BLAST ( The e-values that were obtained are listed in column four. N/A indicates where an e-value was not obtained as because the orthologuelog was retrieved via additional bioinformatic searches, such as BLAST, using conserved regions (in the case of RON6) to search alternate alternative databases, such as OrthoMCL DB. Further research is required to confirm that these putative orthologuelogs asencodeingtrue location-corresponding and functionally orthologous proteins. In each case, the Neospora orthologuelog was obtained from the Toxodb (toxodb.org) database.

d Sequences used to probe the genome databases to identify orthologuelogs.

e Proteins that have been shown to be involved in the tight junction in at least Toxoplasma gondii.

Supplementary references to Table S1

81Hayton, K. et al. (2008) Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe 4, 40–51

82Gruner, A.C. et al. (2004) The Py235 proteins: glimpses into the versatility of a malaria multigene family. Microbes Infect. 6, 864–873

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Proellocks et al. Supplementary Table S1. Page 1