bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Insight into mosquito GnRH-related neuropeptide receptor 2 specificity revealed through analysis of naturally occurring and 3 synthetic analogs of this neuropeptide family 4 Azizia Wahedi1, Gerd Gäde2* and Jean-Paul Paluzzi1* 5 1 Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J1P3, 6 Canada 7 2 Department of Biological Sciences, University of Cape Town, Private Bag, Rondebosch 8 7701, South Africa 9 10 *corresponding authors 11 Prof. Jean-Paul Paluzzi, Department of Biology, York University, 4700 Keele Street, 12 Toronto, Ontario, M3J1P3, Canada, Email: [email protected] 13 Prof. Gerd Gäde, Department of Biological Sciences, University of Cape Town, Private Bag, 14 Rondebosch 7701, South Africa, Email: [email protected] 15 16 17 18 19 20 21 Keywords: 22 GnRH-related neuropeptides, G protein-coupled receptor, structure activity relationships, 23 adipokinetic hormone (AKH), corzazonin (CRZ), AKH/CRZ-related peptide (ACP) 24 1 bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 Adipokinetic hormone (AKH), corzazonin (CRZ) and the AKH/CRZ-related peptide (ACP) 27 are peptides considered homologous to the vertebrate gonadotropin-releasing hormone 28 (GnRH). All three Aedes aegypti GnRH-related neuropeptide receptors have been 29 characterized and functionally deorphanized, which individually exhibit high specificity for 30 their native ligands, which prompted us to investigate the contribution of ligand structures in 31 conferring receptor specificity. In the current study, we designed a series of analogs based on 32 the native ACP sequence in A. aegypti and screened them against the ACP receptor using a 33 heterologous system to identify critical residues required for receptor activation. Specifically, 34 analogs lacking the carboxy-terminal amidation, replacing aromatic residues, as well as 35 truncated analogs were either completely inactive or had very low activities even at high 36 concentration. The next most critical residues were the polar threonine in position 3 and the 37 blocked amino-terminal pyroglutamate, with activity of the latter partially recovered using an 38 alternatively blocked analog. ACP analogs with alanine substitutions at position 2 (valine), 5 39 (serine), 6 (arginine) and 7 (aspartic acid) positions were less detrimental as were 40 replacements of charged residues. Interestingly, replacing asparagine with an alanine at 41 position 9, creating a C-terminal WAA-amide, resulted in a 5-fold more active analog which 42 may be useful as a lead superagonist compound. Similarly, we utilized this high-throughput 43 approach against an A. aegypti AKH receptor (AKHR-IA) testing a number of mostly 44 naturally-occurring AKH analogs from other insects to determine how substitutions of 45 specific amino acids in the AKH ligand influences receptor activation. AKH analogs having 46 single substitutions compared to the endogenous A. aegypti AKH revealed position 7 (serine) 47 was well tolerated whereas changes to position 6 (proline) had pronounced effects, with 48 receptor activity compromised nearly ten-fold. Substitution of position 3 (threonine) or 49 analogs with combinations of substitutions were quite detrimental with a significant decrease 2 bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 in AKHR-IA activation. Interestingly, analogs with an asparagine residue at position seven 51 displayed improved receptor activation compared to the native mosquito AKH. Collectively, 52 these results advance our understanding of how two GnRH-related systems in A. aegypti 53 sharing the most recent evolutionary origin sustain independence of function and signalling 54 despite their relatively high degree of ligand and receptor homology. 55 56 3 bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 Introduction 58 In invertebrates there exist three neuropeptide systems of which the mature peptides as well 59 as their cognate G protein-coupled receptors (GPCRs) are structurally similar, and 60 collectively, they are related to the vertebrate gonadotropin releasing hormone (GnRH) 61 system and suggested to form a large peptide superfamily (Gäde et al., 2011; Hansen et al., 62 2010; Li et al., 2016; Roch et al., 2011). These neuropeptide systems are called the 63 adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family, the 64 corazonin (CRZ) family and the structurally intermediate adipokinetic hormone/corazonin- 65 related peptide (ACP) family. AKHs are primarily or exclusively produced in neurosecretory 66 cells of the corpus cardiacum, and a major function is mobilization of energy reserves stored 67 in the fat body, thus providing an increase of the concentration of diacylglycerols, trehalose 68 or proline for locomotory active phases in the haemolymph. In accordance with this function, 69 AKH has been shown to activate the enzymes glycogen phosphorylase and triacylglycerol 70 lipase and transcripts of the AKHR are most prominently found in fat body tissue (see 71 reviews by (Gäde, 2004; Gäde and Auerswald, 2003; Marco and Gäde, 2019a). As with most 72 endocrine regulatory peptides, additional functions such as inhibition of anabolic processes 73 (protein and lipid syntheses), involvement in oxidative stress reactions and egg production 74 inter alia are known making AKH a truly pleiotropic hormone (see reviews by (Kodrík, 2008; 75 Kodrík et al., 2015). This is also true for CRZ which is mainly synthesized in neuroendocrine 76 cells of the pars lateralis of the protocerebrum and released via the corpora cardiaca (Predel et 77 al., 2007). Although originally described as a potent cardiostimulatory peptide (Veenstra, 78 1989), it does not generally fulfil this role but is known for many additional functions such as 79 involvement in the (1) release of pre-ecdysis and ecdysis triggering hormones, (2) reduction 80 of silk spinning rates in the silk moth, (3) pigmentation events (darkening) of the epidermis in 81 locusts during gregarization and (4) regulation of caste identity in an ant species (Gospocic et 4 bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 82 al., 2017; Kim et al., 2004; Tanaka et al., 2002; Tawfik et al., 1999). The functional role of 83 ACP is less clear. All previous studies had not found a clear-cut function for this peptide until 84 work by Zhou and colleagues claimed that ACP in the cricket Gryllus bimaculatus regulates 85 the concentration of carbohydrates and lipids in the haemolymph (Zhou et al., 2018). 86 Experiments in the current study have been conducted with the mosquito species Aedes 87 aegypti for a number of reasons. Primarily because of being such an infamous disease vector 88 for pathogens such as Yellow and Dengue fever, Chikungunya and, as latest addition, Zika 89 arboviruses, all of which summarily are responsible for affecting approximately 50-100 90 million people per year, with the majority of these attributed to Dengue virus infection (Baud 91 et al., 2017; Price et al., 2015; Saiz et al., 2016). Secondly, knowledge on the interaction of 92 the ligands with their respective receptor are thought to be very helpful for drug research 93 using specific GPCRs as targets for development of selective biorational insecticides 94 (Audsley and Down, 2015; Hill et al., 2018; Verlinden et al., 2014). Another reason was that 95 each of the three neuropeptide systems has already been partially investigated, thus, this 96 study could build on previous research data. With respect to the peptides, these were first 97 predicted from the genomic work on A. aegypti (Nene et al., 2007). The presence of mature 98 AKH and corazonin, but not of ACP, was shown by direct mass profiling (Predel et al., 99 2010). The AKH and ACP precursors have been cloned (Kaufmann et al., 2009) as have one 100 CRZ receptor (CRZR) and various variants of the AKHR and ACPR (Kaufmann et al., 2009; 101 Oryan et al., 2018; Wahedi and Paluzzi, 2018). Each receptor is very selective and responds 102 only to its cognate peptide, thus there are indeed three completely separate and independently 103 working neuroendocrine systems active in this mosquito species. 104 With respect to the ACP system in A. aegypti, expression studies revealed transcripts of the 105 major ACPR form as well as the ACP precursor were enriched during development in adults, 106 specifically during day one and four after eclosion (Kaufmann et al., 2009; Wahedi and 5 bioRxiv preprint doi: https://doi.org/10.1101/699645; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 107 Paluzzi, 2018). On the tissue level, ACP precursor transcripts are mainly present in the 108 head/thorax region (Kaufmann et al., 2009) and, more specifically, within the brain and 109 thoracic ganglia (Wahedi and Paluzzi, 2018) and the ACPR transcripts are mainly detected in 110 the central nervous system with significant enrichment in the abdominal ganglia, particularly 111 in males (Wahedi and Paluzzi, 2018).