Protein-protein interactions (PPIs) are the most fundamental biological processes at the molecular level. The experimental methods for testing PPIs are time-consuming and are limited by analogs for many reactions. As a result, a computational model is necessary to predict PPIs and to explore the consequences of signal alterations in biological pathways. Reproductive control of the vector Anopheles gambiae using transgenic techniques poses a serious challenge. To meet this challenge, it would help to define the biological network involving the male accessory gland (MAG) proteins responsible for successful formation of the mating plug . This plug forms in the male and is transferred to the female during mating, hence initiating the PPIs in both sexes. As is the case in Drosophila melanogaster, a close relative of A. gambiae, some MAG proteins responsible for the formation of the mating plug have been shown to alter the post-mating behavior of females.
Methods and results
The STRING database for known PPIs was used to identify orthologs of A. gambiae proteins in Drosophila (Table 1). Twenty-seven proteins are known to form the mating plug in A. gambiae, and 16 others were obtained as strings in the STRING database. Chromosome synteny comparisons for proteins with more than 50% identity between species were carried out using the Artemis Comparison Tool (ACT version 9.0), and this showed 24.39% matches (M), 12.20% mismatches (MM) and 63.41% unmatched (NM). The network built in Cytoscape (version 2.8.0) with the UniProt IDs for these Drosophila orthologs showed 14 complexes, with 4 of them being for Drosophila. The network showed 555 nodes and 2,344 edges. The top 50 identified hubs in the network showed a range of 3 to 30 interactions. The expression values for these proteins in FlyAtlas showed that they are upregulated in the reproductive tissues of both sexes. To understand the processes involved in plug formation, the Reactome database was used, and the hub proteins were identified in 49 of the 2,021 known processes in Drosophila. Twelve proteins were involved in the following processes: metabolism of proteins (8.8e–13), gene expression (2.0e–06), 3’-UTR-mediated translational regulation (7.7e–08), regulation of β-cell development (1.3e–06), diabetes pathways (6.8e–06), signal recognition (preprolactin) (5.0e–07) and membrane trafficking (1.3e–03). Of the top 50 proteins, 92% had orthologs in A. gambiae, with one identified in the mating plug and four others identified as strings to AGAP009584, which is found in the mating plug. Acp29AB was identified in the network and is known to induce post-mating responses in Drosophila, confirming that the network is reproductive and giving an insight into the possible pathways involved. The CG9083 (Q8SX59) protein was ranked first among the hub proteins but has no ortholog in A. gambiae. Interestingly, it has the same protein properties as the Plugin protein (AGAP009368) in A. gambiae, suggesting that Plugin may be the main protein in the PPI reproductive network in A. gambiae. The Whelan and Goldman (WAG) maximum likelihood tree evaluations of the plug proteins in A. gambiae and their orthologs in Drosophila showed that these proteins are involved in similar biological processes in both species, but the A. gambiae protein evaluation provided a better explanation for the expected process as it clustered in both pre-mated and post-mated PPIs.
Table 1. Orthologs of Anopheles gambiae proteins in Drosophila identified using the STRING database
This table shows the 27 proteins known to be in the mating plug of A. gambiae, derived predominantly from the male. The 16 strings predicted as orthologs in Drosophila, using the STRING database, have varying scores. Scores above 60 can be trusted following their alignments. Plugin, which has the lowest score, has no good ortholog in Drosophila. Most of the proteins are encoded on chromosome arms 2L and 3R in both species. The chromosome synteny comparisons using ACT showed 24.39% matches (M), 12.20% mismatches (MM) and 63.41% unmatched (NM). The presence of gaps between the alignments resulted in the observed MM and NM. The nucleotide sequences at the chromosomal locations where the proteins NOVEL ACP1 and NOVEL ZCP7 are encoded were used to identify similar proteins and their orthologs.
The identification of A. gambiae proteins in this network creates more targets for functional analysis and reproductive control of the malaria vector.