G-protein-coupled receptor genes of Dirofilaria immitis

https://doi.org/10.1016/j.molbiopara.2018.04.001Get rights and content

Highlights

  • In the Dirofilaria immitis genome, 127 GPCR genes were identified.

  • 393 SNP sites were found in 35 GPCR genes investigated for polymorphism.

  • Effects of those SNPs on protein structure were predicted.

  • D. immitis GPCR genes have low genetic variability.

Abstract

The diversity and uniqueness of nematode heterotrimeric G-protein-coupled receptors (GPCRs) provides impetus for identifying ligands that can be used as therapeutics for treating diseases caused by parasitic nematode infections. In human medicine, GPCRs have represented the largest group of ‘drugable’ targets exploited in the market today. In the filarial nematode Dirofilaria immitis, which causes heartworm disease, the macrocyclic lactones (ML) have been used as the sole preventatives for more than 25 years and now there is confirmed ML resistance in this parasite. A novel anthelmintic emodepside, with antifilarial activity, can act on a GPCR. In view of the ML resistance, there is an urgent need to identify new drug targets and GPCRs of D. immitis may be promising receptors. Knowledge of polymorphism within the GPCR superfamily is of interest. A total of 127 GPCR genes have been identified, so far, in the genome of D. immitis. Whole genome sequencing data from four ML susceptible and four ML loss of efficacy populations was used to identify 393 polymorphic loci in 35 D. immitis GPCR genes. Out of 57 SNPs in exonic regions, 36 of them caused a change in an amino acid, out of which 2 changed the predicted secondary structure of the protein. Knowledge about GPCR genes and their polymorphism is valuable information for drug design processes. Further studies need to be carried out to more fully understand the implications of each of the SNPs identified by this study.

Graphical abstract

G-protein-coupled receptors (GPCRs) are potential anthelmintic targets. We found 127 GPCR genes in D. immitis and investigated polymorphism and effects on protein structure.

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Introduction

The G-protein-coupled receptor (GPCR) superfamily constitutes the largest family of transmembrane proteins in eukaryotes. GPCRs are seven transmembrane receptors that signal through heterotrimeric G-proteins. Because of their diversity and essential biological roles, GPCRs have been explored as valuable drug targets in humans. It is estimated that 30–40% of the pharmaceuticals currently in the market target these receptors [1]. Currently, only about 30% of the GPCR family are targeted in the portfolio of drugs developed by pharmaceutical companies and therefore the remaining GPCRs are still targets of interest for the discovery of novel therapeutics [2,3]. The genome of Caenorhabditis elegans has more than 1300 predicted GPCRs [4] that constitute about 7% of all predicted protein-coding genes [5]. While the majority of GPCRs encode nematode-specific chemoreceptors, thought to be essential to compensate for the lack of visual and auditory systems in free-living C. elegans [6], the remaining GPCRs are usually classified according to the GRAFS classification system: Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin families [7].

Neuropeptide and peptide receptors, present in invertebrates, are essential for neuromuscular function in helminths and those receptors with no homolog in vertebrates could serve as potential drug targets [8]. In nematodes, GPCRs that are expressed in the nervous system can be interesting targets for new anthelmintics. GPCRs vary in their biology and ligand specificities and therefore new drugs, targeting GPCRs with nematode-specific activity may be found [8]. In C. elegans, 21 putative neuropeptide GPCRs yielded deleterious phenotypes following knock-out or RNAi analysis [9], and therefore similar GPCRs could be interesting chemotherapeutic targets. Agonists and antagonists for these neuropeptide GPCRs could exhibit therapeutically useful pharmacology. Many neurotransmitters such as the biogenic amines (e.g., serotonin, tyramine, dopamine) act via GPCRs and the function of these receptors may be to modulate fast transmission mediated by ligand-gated ion channels [10]. Some of these GPCRs have distinct pharmacological characteristics and therefore nematode-specific agonists and antagonists could be developed as useful anthelmintics [11]. Classical neurotransmitters including acetylcholine and γ-aminobutryic acid (GABA), also act at GPCRs and regulate nematode locomotion [12]. These GPCRs could be exploited for drug targets to cause paralysis in a nematode. A latrophilin GPCR is already believed to be a minor target for the nematocidal drug emodepside [13,14]. A range of evidence [13,15] links this latrophilin receptor, which binds the black widow spider toxin, latrotoxin, with emodepside action in the pharynx of H. contortus and C. elegans [16,17].

The genome of D. immitis is far from being fully annotated [18] and therefore a complete inventory of GPCR genes is not available. Identification of some of the possible GPCR genes in the genome of D. immitis and an investigation of their single nucleotide polymorphism was of interest as this information may be helpful in investigating GPCR gene products as candidate drug receptors. The present study reports that the heartworm genome contains at least 127 unique GPCR genes. Polymorphism in GPCRs could affect anti-GPCR drug efficacy through altered ligand/modulator binding, receptor activation/inactivation, and/or varied signaling cascades [19] and therefore, knowledge of possible polymorphism in GPCR genes may be relevant for drug design processes. In addition, such knowledge could be used to help ensure that any new drug is active against all of the allelic forms of the target. Genetic changes within GPCR genes may alter their expression levels and/or their functions [20]. Such changes in expression, though not examined in this study, may be a source of variability in receptor properties. Heterogeneity in a drug receptor gene may facilitate resistance development [21] and therefore knowledge of polymorphism in a GPCR gene may be informative to predict possible drug resistance development at the GPCR target. This study was aimed at identifying as many of the putative GPCR genes in D. immitis, by comparative genomics, and to analyze some of those genes for polymorphism.

Section snippets

Identification of GPCR genes in D. immitis

All those GPCR genes that were previously annotated have been included for polymorphism analysis in this study. Information on annotation of those GPCR genes is available as a GFF (Generic Feature Format) file (named as ‘Annotations (GFF3)’) in the dedicated website for the genome of D. immitis (https://parasite.wormbase.org/Dirofilaria_immitis_prjeb1797/Info/Index/). A list of those annotated GPCR genes is given in Supplementary file S1. To identify the remaining, non-annotated GPCR genes,

GPCR genes of all nematodes extracted from databases

A total of 96 nucleotide sequences that could encode different classes of GPCR superfamilies of both free living and parasitic nematodes were extracted from the NCBI and nematode databases. A summary of all the nucleotide sequences that were extracted is available in Supplementary file S2.

Number of GPCR genes identified in the genome of D. immitis

Based on the gene predictions in the nDi.2.2.2 version of the D. immitis genome, by the Blaxter laboratory at the University of Edinburgh, we managed to identify a total of 133 sequences that appear to encode

Discussion

GPCRs are a large family of proteins, but are so far under-exploited drug targets in nematodes. GPCRs represent attractive targets for drug design given the precedence that a major proportion of marketed drugs for human diseases are either direct ligands, or modulators of GPCR evoked signals [33]. Moreover, many of the human GPCRs, are well established as attractive drug targets, based on the two ‘drugability’ criteria; being suitable for heterologous expression and for high throughput

Acknowledgements

This work was supported by a Natural Sciences and Engineering Research Council, Canada grant to RKP (grant No. RGPIN/2777-2012). Research at the Institute of Parasitology was supported by an FRQNT Centre grant to the Quebec Centre for Host-Parasite Interactions. The authors wish to thank the Genome Quebec Innovation Centre for the whole genome sequencing work. We also thank Adebola Razaki for helping us with some Java scripts.

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