Genome wide analysis of ATP-binding Cassette (ABC) transporter in the eastern honey bee (Apis cerana Fabricius, 1793)

1 Introduction

The highly populated environment is the habitat of honey bees, where they have a strong association and food sharing with their nest-mates (DeGrandi-Hoffman and Chen, 2015). This close association causes widespread pathogens inside the bee colony. Hence, honey bees are at more risk of pathogens. This led researchers to focus on how bees maintain their health and resist the disease. Therefore, the honey bees have followed two strategies for protection against pathogens, such as innate immunity (Danihlík et al., 2015) and social immunity (Goode et al., 2006). In the first strategy, honey bees protect themselves and their colony through innate immunity; for example, the worker bees are the main pollinators, forage on crops and wildflowers. Despite this, foraging caused large exposure of honey bees with harmful pathogens (bacteria /viruses) infection and agricultural pesticides, transmitted from workers to colony bees (Raymann and Moran, 2018). In addition, the honey bees’ innate immunity studies have revealed the younger forager bees have a strong innate immune system than the older foragers (Bull et al., 2012). In the second strategy, the bees’ social immunity studies have revealed the health maintenance function. Worker bees smell the infected and diseased adults and broods, dumped their hive from foreign invaders/dead bodies and clear their bodies (Goode et al., 2006). Six morphologically different A. cerana subspecies are endemic in Asia spread throughout the Asian climatic zone series, and further used for commercial beekeeping and pollination for thousands of years. Due to morphological and behavioral similarities, A. mellifera and A. cerana are close relatives. Despite this, A. cerana contained various distinct features in comparison with A. mellifera. Such as A. cerana workers fanned their hives with pointing heads outward, as opposed to A. mellifera workers bees which ventilated with pointing heads inward; A. cerana foragers are good to accumulate nectars from dispersed flower resources, while in A. mellifera foragers, this character is neglected; A. mellifera used propolis (resinous material) to seal hive apertures and protect against pathogens. Furthermore, A. cerana is an indigenous Asian species that evolved serial striking biological features to conflict with the unfavorable conditions in their habitats. A. cerana foragers under cloudy situation at 7 °C temperature visit many floral species, while A. mellifera goes into a stagnant state at this temperature. In addition, A. cerana also formed a special defense mechanism set to control pathogens, parasites, and predators (Evans and Spivak, 2010).

On comparison further revealed that A. cerana has a hasty, unpredictable, and rapid zigzagging flight that helps to escape from bee-eating and honey birds, while A. mellifera has clumsy and steady flight. A. cerana species is an indigenous host to ectoparasitic mite (Varroa destructor) (Anderson and Trueman, 2000), the most injurious A. mellifera pest, and evolved resistance against pest across a long duration of mutual association significantly. Their worker bees can effectively eradicate mite brood and adults through successive cleaning behavior (Guan_Huang, 2005).

The ABC (ATP-binding cassette) transporters comprise the most significant superfamily protein, present in all organisms (Dean et al., 2001). These transporter proteins share extremely conserved nucleotide-binding domains (NBDs), which consist of signature motifs (the C loop), connecting two walker boxes, Walker A and Walker B (Walker et al., 1982). Additionally, to nucleotide-binding domains (NBDs), the eukaryotic ATP-binding cassette transporter usually contains one/two TMDs (transmembrane domains), comprising six to 11 membrane-spanning alpha-helices and prescribe the substrate specificity. A classic architecture domain of the whole transporter has represented the TMD-NBD-TMD-NBD sequence from N to C terminus; while half transporter contains just one NBD-TMD set. Based on NBDs homology, human transporter proteins ABC have been grouped into seven different sub-families, ABCA-ABCG. The ABCH transporter was firstly identified in Drosophila melanogaster, also present in Zebrafish and arthropods but absent in fungi, plants, and mammals (Tian et al., 2017).

Mainly, the transporter ABC translates membrane-bound proteins that hold many molecules, including sugar, lipids, amino acids, vitamins, peptides, sterols, endogenous metabolites, xenobiotics, hormones, and inorganics across the membrane (Dean et al., 2001). Further, these used energies are released with ATP hydrolysis during NBDs molecular transportation across the plasma membrane. It also performed a function in translation, ribosome assembly, and cell signaling (Sturm et al., 2009). Such as ABCE – ABCF transporter proteins do not work as a transporter but are involved in ribosome assembly, transcription, and translation (Tyzack et al., 2000). Moreover, the ABC transporters in plants are also involved in detoxification, phytoalexin function, and osmolality (Verrier et al., 2008).

Till now, very little is known about the ABC transporter in the A. cerana genome. So, the current study involved a genome-wide screening of ABC transporter in A. cerana genome. In addition, several bioinformatics analyses were also carried out to explore the basic and advanced features of ABC transporter; including, gene structure, phylogenetic relations, and conserved protein domains. The outcomes of this study will provide the base for further functional analysis of the ABC transporter in A. cerana and can also contribute to a better understanding of their molecular mechanisms.

2 Material and methods

3 Results

3.2

3.2 Multiple sequence alignment and phylogenetic analysis of ABC transporter genes

The multiple sequence alignment demonstrated that many important residues and structural motifs, e.g., L₄XXG₈, GXXGXGK, LSGG, R₄₅, A₄₉, DEPTXXVD, were conserved throughout the ABC transporters (Fig. 1). All ABC transporters' combined unrooted phylogenetic tree was categorized into eight major groups (ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH) with one MRP (Fig. 2). The ABCA group consists of only one gene, i.e., XM_017050518.2. Similarly, ABCC has five genes including XM_017055838.2, XM_017061132.2, XM_028668368.1, XM_017049887.2, and XM_017064673.2 and the ABCD has two genes (XM_017048648.2 and XM_017066360.2). The ABCE has three, and ABCF has six ABC transporters.

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Fig. 1

The multiple sequence alignment.

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Fig. 2

A phylogenetic tree categorized into eight major groups.

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3.3

3.3 Gene structure organization

As presented in Fig. 2, the number, distribution, and length of introns and exons were not immensely different among the entire gene family. The exons ranged from 4 (XM_028668368.1) to 30 (XM_017055838.2). However, some genes such as XM_017061551.2 and XM_028668147.1 have a similar number of exons (13 exons). In addition, XM_028665983.1 and XM_028669661.1 genes have 14 exons but differ in sequence length (Fig. 3).

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Fig. 3

Distribution of exons, introns, and UTR (untranslated regions) in Apis cerana ABC transporters gene sequences.

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3.4

3.4 Conserved motif analysis

The ABC transporter proteins structure was also investigated amino acid sequences. The MEME motif analysis identified several common and unique motifs in A. cerana ABC transporters. Commonly shared motifs usually tended in the same groups indicating similar functions. In the present study results 1^st motif was observed in all genes except XM_028666551.1. motif_2^nd was present in 26 genes out of 28 genes (excluding XM_028668645.1. and M_028668368.1), motif_3^rd was observed in XM_017061132.2, XM_017064673.2, XM_017061950.2, XM_028664559.1, XM_017055838.2, XM_028665690.1, XM_028668368.1 and XM_017053731.2 while motife_4^th was observed in XM_017061132.2, XM_017064673.2, XM_017061950.2, XM_017049887.2, XM_028669553.1, XM_028664559.1, XM_017055838.2, XM_028665690.1 and XM_017053731.2. Motifs_5^th, was observed in seven genes (XM_017061950.2, XM_028669553.1, XM_028664559.1, XM_017055838.2, XM_028665690.1, XM_017053731.2 and XM_017048648.2), motif_6^th was observed in eight genes. Motif_7 and motif_8^th was present in eight genes. In summary, some motifs were family-specific, some group-specific, some clade-specific, and some texa specific. The length of motifs was also varied motif to motif like motif_1^st had 32 amino acids (aa), motif_2^nd had 20 amino acids. We found the motif_3^rd, 5^th and 10^th motifs had 50 amino acids while motifs 4^th and 9t^h had 41aa.Motif 6^th had 47 while 7^th and 8^th motif had 21 amino acid (Fig. 4).

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Fig. 4

De novo MEME motifs’ distributions in the Apis cerana ABC transporters proteins.

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3.5

3.5 Comparative synteny analysis identified ABC transporters

The comparative synteny study analysis among A. cerana, A. dorsata, and A. mellifera established a significant relationship in gene evolution, function, triplication, duplication, and expression. It was observed that the A.cerana XM_028668147.1 gene sequence illustrated synteny with the A. dorsata 0298gene sequence. A. cerana gene sequence XM_028668147.1 demonstrated synteny with A. mellifera gnl|Amel_HAv3.10256 (Fig. 5).

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Fig. 5

Synteny analysis among Apis cerana, A. dorsata, and A. mellifera.

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3.6

3.6 Protein-protein interaction network analysis

The PPI network identified the hubs protein of ABC transporters. The ABCC5; XM_017055838.2, ABCG4; XM_017063007.2 and ABCB7; XM_028668368.1 demonstrated strong interaction network among all ABC transporters. In addition XM_017063262.2 had highest number of interaction with other ABC proteins. The XM_017063262.2 interacted with XM_017048648.2, XM_017061132.2, XM_017066491.2, XM_028668368.1, XM_017064673.2, XM_017065726.2 and XM_017055838.2. Similarly ABCC5;XM_017055838.2 showed interaction with ABCB7;XM_028668368.1, ABCG4;XM_017063007.2, XM_017055525.2, and XM_017065726.2 (Fig. 6).

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Fig. 6

The PPI network identified the hubs protein of ABC transporters.

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3.7

3.7 Three-dimensional (3D) protein modeling and molecular docking analysis

Based on the PPI results, we selected three ABC transporters from three ABC groups like ABCG4, ABCC5, and ABCB7. The Swiss model predicted the 3D structures demonstrated that ABCG4 (XM_017063007.2) consists of two chains, A and B, while the other two proteins, ABCC5 (XM_017055838.2) and ABCB7 (XM_028668368.1), had only a single chain. The ribbon and surface structure of ABCG4 presented the dimer formation of the two chains making a complex structure for their functions. We can also conclude that the different ABC transporters groups had different 3D structures, which shows their functional diversity (Fig. 7).

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Fig. 7

Three dimensional structure of ABC transporters.

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Furthermore, these proteins structures were also evaluated for their interaction with ATP molecules. The transmembrane proteins (ABCC5; XM_017055838.2, ABCG4; XM_017063007.2 and ABCB7; XM_028668368.1) docked with the ATP and their binding affinity are given Figure. Of these proteins, the ABCG4 showed the lowest binding affinity with −7.3 kcal/mol and interacted with ATP by residues Asn₁₁₄, Asn₁₁₆ Glu₁₁₇, Phe₁₁₈, Arg₁₁₉, Arg₃₅₆ Gly_359, and Arg₄₅₇. Similarly, ABCC5 had the binding affinity of −7.4 kcal/mol and interacted with ATP by Pro₁₂₇₁, Ser₁₂₇₃, Gln₁₄₉₀, Asp₁₄₉₁, Arg₁₅₅₄, Leu₁₅₅₆, Val₁₅₇₀ residues. The ABCB7 showed the highest binding affinities with −7.5 Kcal/mol and residues Arg₆₉, Thr₇₂, Ser₇₇, Val₇₉, Asp₈₉, Ser₉₀, Asp₉₁, and Glu_92. (Figure). The comparison among the three groups of ABC-transporter protein, e.g., ABCB, ABCC, and ABCG, demonstrated different active sites and binding affinity with ATP molecules. IN summary, the ABCB group has a strong interaction with ATP molecules, followed by ABCC and ABCG (Fig. 8)

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Fig. 8

Molecular docking of Apis cerana ABC transporters.

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4 Discussion

Honey bee A. cerana is one of China native bee species. A. cerana and A. mellifera are close relatives, A. cerana showed different characters compared to A. mellifera. Therefore, A. cerana illustrated strong resistance against Varroa destructor (mite) and getting nectar from flower resources.

All ABC transporter genes were classified into eight groups: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH. ABCC proteins subfamily has multiple functions, including ion transport, cell surface receptors, and a wide range of substrate translocation from drug to endogenous compounds (Fukuda and Schuetz, 2012; Tian et al., 2017). The human ABCC subfamily members mentioned to MRPs (multidrug resistance-associated proteins) act as sulfonylurea receptors and are involved in potassium channel regulation, transmembrane cystic fibrosis conductance regulation, and chloride channel construction. ABCD family members belong to half-transporter and import acylCoAs and fatty acid into cell organelle (Theodoulou et al., 2006). It is also involved in peroxisome-related development in Caenorhabditis elegans (Petriv et al., 2002). Further, the ABCE transporter family in yeast and human plays a vital function in translation initiation (Zhou et al., 1993). While the other two transporters, ABCE and ABCF, were not described in invertebrates. In penultimate larvae (Tribolium castaneum) significant mortality occurred when knocked down TcABCE-3A (ABCE) gene with RNAi approach (Broehan et al., 2013). In addition, A. cerana containing ABCG transporter protein showed the largest member of ABC subfamily (include 23 members), similar findings were also reported in Diuraphis noxia (Nicholson et al., 2015), Cimex lectularius (bed bug), Anopheles gambiae (Roth et al., 2003), Drosophila (Dean et al., 2001), A. mellifera (Liu et al., 2011), and Pediculus humanus (body louse in humans) (Lee et al., 2010). Sturm et al. (2009) reported that in the D. melanogaster and D. pulex genome, the ABCG transporter expansion result from specific lineage gene duplication (Sturm et al., 2009). The ABCE and ABCF subfamilies consist of unusual ABC transporter proteins, identified through NBDs pair link without TMDs. Thus, their structure was oblique their significance in the biological process rather than transportation. The ABCE1 is considered one of the vital conserved proteins in evolutionary relation and is observed in all organisms except eubacteria. Due to its important role in ribosome synthesis and translation, ABCE1 is vital for entire life stages (Barthelme et al., 2011). The RNase L/ABCE1 of Homo sapiens was primarily identified as an RNase L inhibitor. A. cerana has ten ABCG transporters. The ABCGs have a distinct reverse domain architecture with NBD-TMD (NBD restricted on N-terminus and other is TMD localized at C-terminus). In metazoans, the ABCG transporter considers half-transporters, while after dimerization, it works as functional transporters. Moreover, the ABCGs transporters in fungi and plants are full-transporters and known as PDRs (pleiotropic drug resistance protein) (Kovalchuk and Driessen, 2010). In this study, due to their duplicated structure domain, the PDRs yeast was not integrated with the phylogenetic analyses tree. Similar to other groups, A. cerana has five ABCH transporter, which contains inverse half-transporter that share similar architecture domains identical to ABCG transporter. It was first identified in D. melanogaster, which was only annotated in zebrafish and arthropods (Dean and Annilo, 2005).The ABCH transporter is not studied in mammals, plants, fungi, and C. elegans (Dean et al., 2001).It was studied in zebrafish but not identified in fungi, cod, and catfish (Liu et al., 2013).

The D. melanogaster RNAi screening illustrated that CG9990 D. melanogaster gene silencing was lethal (Mummery-Widmer et al., 2009). The ABCH (Px014955) was abundantly up-regulated in two insecticide-resistant strains of P. xylostella, (You et al., 2013). In T. castaneum, TcABCH-9C knockdown resulted in a hundred percent larval mortality and significantly reduced hatching and fecundity rate. Additionally, the T. castaneum larvae injected with dsRNA of TcABCH-9C showed it is involved in waterproof barrier formation in epicuticle (Broehan et al., 2013). The ABCH subfamily expression in cotton bollworm Manduca sexta and Helicoverpa armigera was vastly induced after treating secondary metabolites (Koenig et al., 2015). The ABC transporter gene structural analysis will be useful in protein functional analysis. Evolutionary background disclosed that exon-intron sequence had figured the evolutionary tree of gene family (Zaynab et al., 2021). Later on, it corresponds to prior scientific approaches retained in some plant genes that may contain no introns or short introns at the period of evolution (Zaynab et al., 2021).

Moreover, the firm gene structure may show a quick-expression response to endogenous and exogenous stimuli. Overall structural analysis suggested that ABC transporter genes showed a similar intron/exon with homogenous functional characters because they originated from duplication events during evolutionary activity. The MEME motif analysis identified several common and unique motifs in A. cerana ABC transporters. Commonly shared motifs usually tended in the same groups indicating similar functions (Cao et al., 2005).

5 Conclusion

The genome-wide study identified twenty-eight ABC transports encoding genes in the A. cerana (Ac) genome. The phylogenetic analysis revealed that AcABCs are also divided into eight groups similar to other organisms. Of these groups, ABCGs were the largest group in A. cerana genome. Different additional computational analyses provided essential data for further functional validation. The sequence associated with structural and functional motifs also showed some common and unique features in ABCs. These identifications might present new insight for advanced functional analysis on the ABC gene group in the A. cerana genome.