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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Improving N-terminal protein annotation of Plasmodium species based on signal peptide prediction of orthologous proteins

Malaria Journal > Ausgabe 1/2012
Armando de Menezes Neto, Denise A Alvarenga, Antônio M Rezende, Sarah S Resende, Ricardo de Souza Ribeiro, Cor JF Fontes, Luzia H Carvalho, Cristiana F Alves de Brito
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-375) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contribution

AMN performed all data analyses. AMN, RSR and AMR work on scripts for data integration. DAM and SSR participated in the experimental validation of new gene models. CJFF coordinated the field work. LHC, CFAB, AMN conceived and designed the study. CFAB coordinated the study. CFAB, AMN and AMR wrote the manuscript. All authors read and approved the final manuscript.



Signal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species.


Signal peptide (SignalP) and orthology (OrthoMCL) were combined in an innovative strategy to identify orthologous groups showing discrepancies in signal peptide prediction among their protein members (Mixed groups). In a comparative analysis, multiple alignments for each of these groups and gene models were visually inspected in search of misannotated proteins and, whenever possible, alternative gene models were proposed. Thresholds for signal peptide prediction parameters were also modified to reduce their impact as a possible source of discrepancy among orthologs. Validation of new gene models was based on RT-PCR (few examples) or on experimental evidence already published (ApiLoc).


The rate of misannotated proteins was significantly higher in Mixed groups than in Positive or Negative groups, corroborating the proposed hypothesis. A total of 478 proteins were reannotated and change of signal peptide prediction from negative to positive was the most common. Reannotations triggered the conversion of almost 50% of all Mixed groups, which were further reduced by optimization of signal peptide prediction parameters.


The methodological novelty proposed here combining orthology and signal peptide prediction proved to be an effective strategy for the identification of proteins showing wrongly N-terminal annotated sequences, and it might have an important impact in the available data for genome-wide searching of potential vaccine and drug targets and proteins involved in host/parasite interactions, as demonstrated for five Plasmodium species.
Additional file 1: Examples of N-terminal alignments of inspected Mixed groups. In the upper panel, three Mixed groups (OG4_10598, OG4_10633 and OG4_47034) placed in the category of No misannotations after visual inspection. Signal peptide predictions positive (+) or negative (-) are shown to the left of gene identifiers, demonstrating the Mixed nature of these groups. A total of 111 groups belong to this category. In the lower panel, two Mixed groups (OG4_54958 and OG4_54960) in which putative misannotated proteins were identified after visual inspection. Proteins in these groups were reannotated and a comparison of alignments before and after reannotations is shown with the respective signal peptide predictions to the left of gene identifiers. A total of 331 groups belong to this category (Reannotated). Reannotated P. vivax genes that were submitted to RT-PCR validation of new gene models are indicated by asterisks. (PDF 3 MB)
Additional file 2: Description of PCR conditions used to validate new gene models. Sequences of primers, amplicon sizes, annealing temperatures and number of cycles used in amplifications of seven new gene models are showed. For each gene model were used three forward primers (control, before and after) and the same reverse primer. (XLS 34 KB)
Additional file 3: List of all reannotated proteins. Reannotated proteins identified by their Gene ID from each species are listed including their orthologous group number, signal peptide prediction before and after annotation, description of putative gene product and new sequence proposed. Species: Pb – Plasmodium berghei, Pf – Plasmodium falciparum, Pk – Plasmodium knowlesi, Pv – Plasmodium vivax, and Py – Plasmodium yoelii. (XLS 401 KB)
Additional file 4: Reannotated proteins with orthologs validated experimentally based on ApiLoc. Reannotated proteins from orthologous groups previously showing mixed signal peptide prediction are shown. This information includes the status of each group after proteins reannotation, the protein reannotated from each group with their species and SP prediction status before and after annotation and the description of the orthologous proteins experimentally validated based on ApiLoc information. Species: Pb – Plasmodium berghei, Pf – Plasmodium falciparum, Pk – Plasmodium knowlesi, Pv – Plasmodium vivax, and Py – Plasmodium yoelii. (XLS 37 KB)
Additional file 5: Classification of orthologous groups after protein reannotations and optimization of signal peptide prediction parameters. Classification of each orthologous group according to signal peptide prediction of their proteins in Positive (all proteins of the group showed predicted signal peptide); Negative (all proteins of the group showed prediction of absence of signal peptide); Mixed (proteins with or without predicted signal peptide in the same group). The classifications were performed before reannotation, after reannotation and after optimization of signal peptide parameters of prediction. Classification of group category after visual inspection showed groups without proteins misannotated (No misannotations); groups with all misannotated proteins corrected (Reannotated); groups with one or more proteins still misannotated (Partially reannotated). (XLS 82 KB)
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