3.2 Results
3.2.1 Compilation of a list of proteins from which the diagnostic targets were
68
69
Table 3.2 Twenty most abundant proteins according to proteome data from Foth et al., (2011)
* Note the transcription ranking according to the LeRoch et al. (2003) data was included here and not the average expression values shown in Table 3.1.
▲ These columns include the Foth et al. (2011) data
Finally Tables 3.1 and 3.2 were combined, resulting in the 35 most abundant proteins throughout the parasite intra-erythrocytic cycle as shown in Table 3.3.
The list of 35 proteins was narrowed down to seven proteins of interest for use as diagnostic targets and those of interest were highlighted in green, where excluded proteins were highlighted in red. The criteria used are explained briefly to follow.
#
PlasmoDB /
NCBI ID Protein name Transcription
ranking * Ring
▲ Trophozoite
▲ Schizont
▲ 1 PF14_0598 Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) 3 10 10 10
2 PF08_0054 Heat shock protein 70-1 4 10 10 10
3 PFI1090w S-adenosylmethionine
synthetase 16 10 10 10
4 PFI0875w Heat shock protein 70-2 (BiP) 24 10 10 10
5 PF08_0019 Receptor for activated C kinase (PfRACK) = Guanine
nucleotide-BP
28 10 10 10
6 PF10_0155 Enolase 65 10 10 10
7 PF07_0029 Heat shock protein 86 121 10 10 10
8 PFI1105w Phosphoglycerate kinase 205 10 10 10
9 MAL13P1.214 Phosphoethanolamine N-
methyltransferase, putative 812 10 10 10
10 PFL1070c HSP90 (Endoplasmin homolog
precursor, putative) 1078 10 10 10
11 PFI1475w Merozoite surface protein 1
(MSP1) 454 10 9 10
12 PFL2215w Actin I 138 10 9 9
13 PFF1300w Pyruvate kinase, putative 788 9 9 10
14 PF14_0655 RNA helicase 45 (H45, eIF4A) 1041 9 10 9
15 MAL13P1.56 M1-family aminopeptidase 2916 9 10 9
16 PFF0435w Ornithine aminotransferase 14 9 9 9
17 PF14_0425 Fructose-bisphosphate aldolase 20 9 9 9
18 PF13_0141 L-lactate dehydrogenase
(LDH) 35 9 9 9
19 MAL8P1.17 Protein disulfide isomerase
(PDI-8) 47 9 9 9
20 PF11_0208 Phosphoglycerate mutase 64 9 8 8
70
Table 3.3 Compilation of the 35 most abundant proteins, highlighting potential diagnostic targets in green and proteins that were excluded in red
* denotes proteins that are either membrane bound or associated with the insoluble cytoskeleton.
Pan-Plasmodium and species-specific peptides were chosen based on ClustalΩ alignments and BLASTp searches.
Available P. falciparum crystal structures were searched for on the RCSB PDB database.
# Gene ID Protein name Known
function Conserved amongst human malaria
species Human
orthologue Pan-Plasmodial
peptides Species-specific
peptides Crystal structure available
1 PF11_0040 Early transcribed membrane protein 11.2 yes only P.f. no no no
2 PFB0120w Early transcribed membrane protein 2, putative yes only P.f. no no no
3 PF14_0598 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) yes all 5 yes yes yes yes
4 PF08_0054 Heat shock 70 kDa protein yes P.f., P.v., P.k. yes yes no
5 PF11_0224 Circumsporozoite-related antigen yes only P.f. no no no
6 PF11_0508 Hypothetical protein no
7 PF10_0019 Early transcribed membrane protein yes only P.f. no no no
8 PF10_0159 Glycophorin-binding protein 130 precursor yes only P.f. no no no
9 PF10_0372 Hypothetical protein no
10 PF14_0486 Elongation factor 2 yes P.f., P.v., P.k. yes yes no
11 PF11_0043 60S acidic ribosomal protein p1, putative yes P.f., P.v., P.k. no yes no
12 PFA0420w Hypothetical protein no
13 PFL2515c Hypothetical protein no
14 MAL6P1.91 Ornithine aminotransferase yes P.f., P.v., P.k. yes yes no
15 PF13_0346 Ubiquitin-60S ribosomal protein L40 yes P.f., P.v. yes no no
16 PFI1090w S-adenosylmethionine synthetase, putative yes P.f., P.v., P.k. yes yes no
17 PFE0070w Interspersed repeat antigen, putative yes only P.f. no no no
18 PF11_0039 Early transcribed membrane protein 11.1 yes only P.f. no no no
19 PF14_0678 Exported protein 2 yes only P.f. no no no
20 PF14_0425 Fructose-bisphosphate aldolase yes P.f., P.k. yes no yes
21 PFI0875w Heat shock protein 70-2 (BiP) yes only P.f. yes no no
22 PF08_0019 Receptor for activated C kinase (PfRACK) = Guanine nucleotide-BP yes P.f., P.v., P.k. yes yes no
23 PF10_0155 Enolase yes P.f., P.v., P.k. yes yes no
24 PF07_0029 Heat shock protein 86 yes P.f., P.v., P.k. yes yes yes Only the N-terminus
25 PFI1105w Phosphoglycerate kinase yes P.f., P.v., P.k. yes yes no
26 MAL13P1.214 Phosphoethanolamine N-methyltransferase, putative (PMT) yes P.f., P.v., P.k. no yes yes yes
27 PFL1070c Heat shock protein 90 (Endoplasmin homolog precursor, putative) yes P.f., P.v., P.k. yes yes yes Only the N-terminus
28 PFI1475w Merozoite surface protein 1 (MSP1) yes* all 5 no yes yes
29 PFL2215w Actin I yes* P.f., P.v., P.k. no yes yes
30 PFF1300w Pyruvate kinase, putative yes P.f., P.v., P.k. yes yes yes yes
31 PF14_0655 RNA helicase 45 (H45, eIF4A) yes P.f., P.v., P.k. yes yes yes no
32 MAL13P1.56 M1-family aminopeptidase yes P.f., P.v., P.k. no yes yes yes
33 PF13_0141 L-lactate dehydrogenase (LDH) yes all 5 yes yes yes yes
34 MAL8P1.17 Protein disulfide isomerase (PDI-8) yes P.f., P.v., P.k. yes yes yes yes
35 PF11_0208 Phosphoglycerate mutase yes P.f., P.v., P.k. yes yes yes yes
71 All proteins with unknown functions were excluded since only proteins involved in metabolic processes were of interest. Any membrane-bound or cytoskeletal proteins were also excluded from further analysis as indicated in Table 3.3 with an asterisk. This was decided based on the inherent difficulties of expression of membrane bound proteins and the additional requirements to solubilise these if used in an RDT or ELISA test format. Only proteins of known function, which were conserved in P. falciparum, P. vivax and P. knowlesi species, were further pursued, as this would potentially allow tests to specifically detect all three infections. Ideally a protein unique to the parasite and not present in the human genome would be targeted, but this was not used as an exclusion criterion. The primary amino acid sequences of the proteins with human orthologs were evaluated and those with close to 100%
identity were excluded since antibodies against the P. falciparum proteins would detect the human proteins and not be of use for diagnosis. Finally proteins without crystal structures were excluded since a criterion for the selection of peptide epitopes was to verify their presence on the surface of the proteins, where they are more likely to be detected by antibodies.
GAPDH was chosen as the first diagnostic target since it was the highest expressed of the seven remaining proteins in Table 3.3. PMT was chosen as the second target as it was the highest expressed protein of those without human orthologs. LDH was included as the model control protein in this study as a current diagnostic protein. The available amino acid sequences of each protein were aligned and potential species-specific and common malaria peptide epitopes were identified as described in section 3.2.2.