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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
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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
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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
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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
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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
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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). The AKH precursor transcripts are present in
112 head/thorax region of pupae and adult females and in the abdomen of males (Kaufmann et al.,
113 2009). Expression of AKHR was shown in all life stages (Kaufmann et al., 2009), but similar
114 to the ACPR and ACP transcripts, AKHR transcript variants were found to be significantly
115 enriched in early adult stages (Oryan et al., 2018). On the tissue level, highest expression in
116 adults was found in thorax and abdomen with low levels detected in ovaries (Kaufmann et al.,
117 2009). Similar expression profiles were determined recently by Oryan et al. (2018) using RT-
118 qPCR: expression in head, thoracic ganglia, accessory reproductive tissues and carcass of
119 adult females as well as in abdominal ganglia and specific enrichment in the carcass and
120 reproductive organs of adult males. Similarly, CRZR was mostly expressed during later
121 development, including pupal and adult stages. In terms of tissue expression profiles, this
122 was examined in adult stages where CRZR was found in the heads and thoracic ganglia of
123 both sexes as well as in ovary, abdominal ganglia and Malpighian tubules in females and in
124 the testes and carcass of males (Oryan et al., 2018).
125 Thus, although quite a substantial body of information on the three signalling systems in A.
126 aegypti is known, we lack knowledge of how the various ligands interact with their cognate
127 receptor. For instance, which amino acid residue in the ligand is specifically important for
128 this interaction? By replacing each residue successively with simple amino acids such as
129 glycine or alanine one can probe into the importance of amino acid side chains and their
130 characteristics in relation to facilitating receptor specificity. Such structure-activity studies
131 have been conducted on the AKH system in a number of insects and a crustacean either by
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132 measuring physiological actions in vivo (Gäde and Hayes, 1995; Ziegler et al., 1998) or in a
133 cellular mammalian expression system in vitro (Caers et al., 2012; Marchal et al., 2018;
134 Marco et al., 2017). In conjunction with nuclear magnetic resonance (NMR) data on the
135 secondary structure of AKHs (Nair et al., 2001; Zubrzycki and Gäde, 1994) and the
136 knowledge of the receptor sequence, molecular dynamic methods can devise models how the
137 ligand is interacting with its receptor (Jackson et al., 2018; Mugumbate et al., 2013).
138 Since no such data is available for any ACP system, the objective of the current study was to
139 fill this knowledge gap by determining critical amino acids of the ACP neuropeptide
140 necessary for activation of its cognate receptor, ACPR. We therefore designed a series of
141 analogs based on the endogenous A. aegypti ACP sequence and screened them using an in
142 vitro receptor assay system to determine the crucial residues for ACPR activation. Given the
143 closer evolutionary relationship between the ACP and AKH systems in arthropods (Hansen et
144 al., 2010; Hauser and Grimmelikhuijzen, 2014; Tian et al., 2016; Zandawala et al., 2018), a
145 second aim of the current study was to advance our knowledge and understanding of critical
146 residues and properties of uniquely positioned amino acids necessary for the specificity of the
147 AKH ligand to its receptor, AKHR-IA. Here, we took advantage of naturally occurring AKHs
148 from other insects to examine amino acid substitutions and determine the consequences onto
149 AKHR-IA activation using the same in vitro heterologous assay. Collectively, determining
150 indispensable amino acid residues of these two GnRH-related mosquito neuropeptides that
151 are necessary for activation of their prospective receptors will help clarify how these
152 evolutionarily-related systems uphold specific signalling networks avoiding cross activation
153 onto the structurally related, but functionally distinct signalling system.
154 155
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156 Materials and Methods 157 158 ACP and AKH Receptor Expression Construct Preparation
159 A mammalian expression construct containing the ACPR-I ORF (hereafter denoted as
160 ACPR) with Kozak translation initiation sequence had been previously prepared in
161 pcDNA3.1+ (Wahedi and Paluzzi, 2018); however, this earlier study revealed that coupling of
162 the ACPR with calcium mobilization in the heterologous expression system was not very
163 strong, even following application of high concentrations of the native ACP ligand.
164 Therefore, to improve the bioluminescent signal linked to calcium mobilization following
165 receptor activation, which was required in order to provide greater signal to noise ratio when
166 testing ACP analogs with substitutions leading to intermediate levels of activation of the
167 ACPR, we created a new mammalian expression construct using the dual promoter vector,
168 pBudCE4.1. Specifically, we utilized a cloning strategy as reported previously (Paluzzi et al.,
169 2015) whereby the ACPR was inserted into the multiple cloning site (MCS) downstream of
170 the CMV promoter in pBudCE4.1 using the sense primer SalI-KozakACPR, 5’-
171 GGTCGACGCCACCATGTATCTTTCGG-3’ and anti-sense primer XbaI-ACPR, 5’-
172 TCTAGATTATCATCGCCAGCCACC-3’, which directionally inserted the ACPR within the
173 MCS downstream of the CMV promoter. Secondly, the murine homolog (Gα15) of the
174 human promiscuous G protein, Gα16, which indiscriminately couples a wide variety of
175 GPCRs to calcium signaling (Offermanns and Simon, 1995) was inserted into the MCS
176 downstream of the EF1-α promoter in pBudCE4.1 using the sense primer NotI-Gα15, 5’-
177 GCGGCCGCCACCATGGCCCGGTCCCTGAC-3’ and anti-sense primer BglII-Gα15, 5’-
178 AGATCTTCACAGCAGGTTGATCTCGTCCAG-3’, which directionally inserted the
179 murine Gα15 within the MCS downstream of the EF1-α promoter.
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180 The AKHR mammalian expression construct used in the current study, specifically AKHR-
181 IA which was the receptor isoform that exhibited the greatest sensitivity to its native AKH
182 ligand, was prepared previously in pcDNA3.1+ (Oryan et al., 2018) and coupled strongly with
183 calcium signaling in the heterologous system so there was no need utilize the dual promoter
184 vector containing the promiscuous Gα15 as was necessary for ACPR. For each construct, an
185 overnight liquid culture of clonal recombinant bacteria containing the appropriate plasmid
186 construct was grown in antibiotic-containing LB media and used to isolate midiprep DNA
187 using the PureLink Midiprep kit (Life Technologies, Burlington, ON). Midiprep samples
188 were then quantified and sent for sequencing as described previously (Oryan et al., 2018;
189 Wahedi and Paluzzi, 2018).
190
191 Cell culture, transfections, and calcium bioluminescence reporter assay
192 Functional activation of the AedaeACPR and AedaeAKHR-IA receptors was carried out
193 following a previously described mammalian cell culture system involving a Chinese hamster
194 ovary (CHO)-K1 cell line stably expressing aequorin (Paluzzi et al., 2012). Cells were grown
195 in DMEM:F12 media containing 10% heat-inactivated fetal bovine serum (Wisent, St. Bruno,
196 QC), 200μg/mL Geneticin, 1x antimycotic-antibiotic to approximately 90% confluency, and
197 were transiently transfected using either Lipofectamine LTX with Plus Reagent or
198 Lipofectamine 3000 transfection systems (Invitrogen, Burlington, ON) following
199 manufacturer recommended DNA to transfection reagent ratios. Cells were detached from the
200 culture flasks at 48-hours post-transfection using 5mM EDTA in Dulbecco’s PBS. Cells were
201 then prepared for the receptor functional assay following a procedure described previously
202 (Wahedi and Paluzzi, 2018). Stocks of peptide analogs of ACP and AKH were used for
203 preparation of serial dilutions all of which were prepared in assay media (0.1% BSA in
204 DMEM:F12) and loaded in quadruplicates into 96-well white luminescence plates (Greiner
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205 Bio-One, Germany). Cells were loaded with an automatic injector into each well of the plate
206 containing different peptide analogs at various concentrations as well as negative control
207 (assay media alone) and positive control wells (50µM ATP). Immediately after injection of
208 the cells, luminescence was measured for 20 seconds using a Synergy 2 Multi-Mode
209 Microplate Reader (BioTek, Winooski, VT, USA). Calculations, including determination of
210 EC50 values, were conducted in GraphPad Prism 7.02 (GraphPad Software, San Diego, USA)
211 from dose-response curves from 3-4 independent biological replicates.
212 Synthetic Peptides and Analogs
213 Peptide analogs based on the native ACP sequence were designed and synthesized by Pepmic
214 Co., Ltd. (Suzhou, China) at a purity of over 90%. All synthetic peptides were initially
215 prepared as stock solutions at a concentration of 1mM in dimethyl sulfoxide. All stocks of
216 arthropod AKH analogs were prepared similarly to the ACP synthetic peptide analogs and
217 were commercially synthesized by either Pepmic Co., Ltd. (Suzhou, China) or by Synpeptide
218 Co. (Shanghai, China) at a purity of over 90%. Specific sequence information for each ACP
219 and AKH peptide analog used in this study is provided in Tables 1 and 2, respectively.
220 221
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222 Results 223 ACP synthetic analog activity on mosquito ACPR 224 Analogs of native ACP with single alanine substitutions at each amino acid were synthesized
225 and evaluated by examining their functional activation of the A. aegypti ACP receptor
226 (ACPR) expressed using a heterologous system. Carboxy-terminal amidation of ACP was
227 found to be critical for bioactivity since the free acid ACP analog elicited no activation of
228 ACPR (Figure 1A) detectable up to the highest tested concentration of 10uM (Table 1). In
229 addition, analogs replacing either of the two aromatic residues of ACP, including the
230 phenylalanine in the fourth position or tryptophan in the eighth position, were similarly
231 inactive on the ACPR (Figure 1A) with no detectable response by any of the tested
232 concentrations (Table 1). Further examination of single residue alanine substitutions revealed
233 that the next most critical residue for retaining ACPR functional activation was the polar
234 threonine in the third position, which upon replacement with alanine resulted in a ~551-fold
235 reduced activity compared to native ACP (Figure 1A, Table 1). A comparable reduction of
236 activity, approximately 411-fold, was measured with an ACP analog that had the first residue,
237 the blocked pyroglutamate in the native ACP, substituted by alanine. If the substitution,
238 however, was also made with a blocked compound, N-Acetyl-alanine instead of pGlu, the
239 analog was 10-times more active as the non-blocked one, thus the reduction in activity was
240 only about 44-fold (Figure 1A). Interestingly, substitutions with analogs containing alanine at
241 the second (valine), fifth (serine), sixth (arginine) and seventh (aspartic acid) positions, had
242 noticeably less impact on the reduction of activity (Figure 1B), by ~70-fold, ~45-fold, ~23-
243 fold, and ~16-fold, respectively. Unexpectedly, the analog containing an alanine substitution
244 for the asparagine in position nine resulted in a more potent ACPR activation, leading to a
245 ~5-fold greater activity compared to the native ACP peptide (Figure 1B). C-terminally
246 truncated analogs, which lacked either the naturally occurring alanine residue in the tenth
247 residue position or the last two C-terminal residues including asparagine and alanine in
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248 positions nine and ten, were almost completely inactive, demonstrating ~434-fold and ~410-
249 fold reduced ACPR activation compared to native ACP (Figure 1C). A C-terminally
250 extended analog ending with a glycine on the C-terminus elicited a small decrease of activity
251 by about ~6-fold (Figure 1C). Lastly, the internally truncated ACP analog lacking a valine in
252 the second position also was without any effect, similar to the two aromatic residue
253 substituted analogs or the non-amidated ACP analog (Figure 1C).
254 Activity of natural arthropod AKH analogs on mosquito AKHR-IA 255 Taking advantage of the variety of natural AKH analogs in arthropods, we examined a subset
256 of insect AKH peptides (see Table 2) with unique substitutions compared to the endogenous
257 mosquito AKH peptide and determined their activity on the AKH receptor, A. aegypti
258 AKHR-IA. From this analysis, the least effective analog tested was the C-terminally
259 truncated peptide, Lacol-AKH-7mer, whose activity was nearly completely abolished with
260 only ~7% AKHR-IA activation at the highest tested concentration (10μM), which represents
261 a reduced efficacy of at least five orders of magnitude (Figure 2A). The next least active
262 AKH analog was the cricket peptide, Grybi-AKH, which has five residues substituted over
263 the length of the octapeptide compared to the mosquito AKH (see Table 2). Specifically,
264 leucine and threonine in positions two and three are replaced by valine and asparagine,
265 respectively. Further, threonine, proline and serine in positions five to seven are replaced
266 with serine, threonine and glycine, respectively. As a result of these combined substitutions,
267 Grybi-AKH activity on the A. aegypti AKHR-IA was compromised by over three orders of
268 magnitude (1032-fold reduced activity) compared to the endogenous mosquito AKH (Table 2
269 and Figure 2A). We next tested the AKH from the dragonfly, Libellula auripennis, which
270 allowed us to isolate a subset of the substitutions of Grybi-AKH, specifically positions two
271 and three alone. The activity of Libau-AKH was improved nearly two-fold over Grybi-AKH;
272 however, the activity of this peptide on the A. aegypti AKHR-IA was still reduced by 548-
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273 fold compared to Aedae-AKH (Table 2). The next AKH analog examined was that from the
274 firebug Pyrrhocoris apterus (Pyrap-AKH), which has two substitutions in positions three and
275 seven, both asparagine, for the natural threonine and serine, respectively, found in A. aegypti
276 AKH. The activity of Pyrap-AKH was found to be improved compared to Libau-AKH and
277 Grybi-AKH by 1.6 and 3-fold, respectively. However, these substitutions resulted in the
278 activity of Pyrap-AKH on the A. aegypti AKHR-IA being compromised by 337-fold relative
279 to the native AKH (Figure 2A). To better understand the importance of N-terminal residues,
280 we next tested a second AKH from Odonata, this analog from Erythemis simplicicollis
281 (Erysi-AKH) where the second position valine found in Libau-AKH is instead leucine, which
282 is conserved with the A. aegytpi AKH. Thus, the activity of this AKH analog having only a
283 single amino acid substitution in the third position (threonine to asparagine; see Table 2)
284 resulted in Erysi-AKH eliciting activity on the A. aegypti AKHR-IA which was 310-fold less
285 compared to native mosquito AKH (Figure 2A). When testing an AKH analog having a
286 single equivalent substitution in only position seven (serine to asparagine), as is naturally
287 available in the American cockroach peptide Peram-CAH-II, this substitution was completely
288 tolerated since effectively no difference in the activity of this analog on the AKHR-IA
289 relative to the A. aegypti AKH was detected (Figure 2B). Indeed, we could next verify the
290 criticalness of the third position threonine residue by examining the activity of another
291 cockroach AKH analog, V2-Peram-CAH-II (a synthetic analog and not naturally occuring in
292 P. americana), which has a second position substitution of valine for leucine along with the
293 seventh position substitution of asparagine for serine (Table 2). The activity of V2-Peram-
294 CAH-II was found to be much improved, albeit still 69-fold less effective on the AKHR-IA
295 compared to the native A. aegypti AKH (Figure 2B). Since it was demonstrated that the C-
296 terminal tryptophan was fundamental for bioactivty, with truncated analogs having little or no
297 activity (see above for Lacol-AKH-7mer), we were interested to examine if extension of the
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298 C-terminus may also equally impact the activity of the AKH analog on the mosquito A.
299 aegypti AKHR-IA. To do this, we utilized the AKH analog from the domestic silkmoth,
300 Bombyx mori (Bommo-AKH), which contains an extended C-terminus comprised of a
301 glycine and glutamine residue (resulting in a decapeptide). Additionally, Bommo-AKH has a
302 seventh position substitution of glycine for serine; however, considering the extension on the
303 C-terminus as well as the substitution in position seven, this analog was surprinsingly quite
304 tolerated with only 11-fold reduced activity on the A. aegypti AKHR-IA (Figure 2B). Finally,
305 examining naturally occuring analogs of this peptide family containing single substitutions in
306 the C-terminal region, but sparing the eighth critical tryptophan residue, the findings
307 demonstrate that these changes are quite tolerated. It was noted above that the cockroach
308 AKH, Peram-CAH-II, which has a seventh position serine substituted with asparagine, had
309 near identical efficacy to the native mosquito AKH. Similarly, an AKH analog from the
310 sphingid moth, Hippotion eson (Hipes-AKH-I), which has a serine substitution for proline in
311 the sixth position, also was well tolerated with only a 8-fold reduced activity on the A.
312 aegypti AKHR-IA (Figure 2B). Lastly, using the AKH analog from the black horse fly,
313 Tabanus atratus (Tabat-AKH), the results demonstrated that another substitution in the
314 seventh position, in this case a glycine for the naturally occurring serine in Aedae-AKH, is
315 indeed well tolerated since the activity of this analog matched and partially exceeded (~30%
316 improved efficacy) the activity of the native mosquito AKH (see Table 2; Figure 2B).
317
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318 Table 1. Primary structure of the synthetic ACP analogs designed and tested on the A. 319 aegypti ACP receptor (AedaeACPR). Using heterologous expression of ACPR, the half 320 maximal effective concentration for each of the analogs was determined as well as the 321 corresponding reduction in activity, highlighting how a particular residue substitution or other 322 modification is tolerated in this system. ND = represents no determined EC50 value due to 323 minimal or no detectable activity of the analog when tested up to 10μM. 324 Fold-reduction Activity on A. in activity Peptide/Analog: Primary structure of analog: aegypti ACPR relative to (EC ) 50 AedaeACP AedaeACP pQVTFSRDWNAamide 2.394E-08 1.0 AedaeACP (A1) AVTFSRDWNAamide 1.576E-06 411.1 AedaeACP (A2) pQATFSRDWNAamide 7.052E-07 69.8 AedaeACP (A3) pQVAFSRDWNAamide 2.26E-05 550.8 AedaeACP (A4) pQVTASRDWNAamide ND >1000 AedaeACP (A5) pQVTFARDWNAamide 1.038E-06 44.9 AedaeACP (A6) pQVTFSADWNAamide 6.872E-07 23.3 AedaeACP (A7) pQVTFSRAWNAamide 3.863E-07 15.9 AedaeACP (A8) pQVTFSRDANAamide ND >1000 AedaeACP (A9) pQVTFSRDWAAamide 4.96E-09 0.2 AedaeACP (-OH) pQVTFSRDWNA-OH ND >1000 AedaeACP (A1b) Acetyl-AVTFSRDWNAamide 5.272E-07 43.6 AedaeACP (1-9) pQVTFSRDWNamide 4.153E-06 434.2 AedaeACP (1-8) pQVTFSRDWamide 2.156E-06 410.1 AedaeACP (1,3-10) pQTFSRDWNAamide ND >1000 AedaeACP (+G) pQVTFSRDWNAGamide 1.113E-07 5.6 325 326
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327 Figure 1.
A1 A1b A3 A ACP A4 A8 OH
1001.00
0.75 75
0.50 50 Response 0.25 25
Normalized Luminescent 0.00 0 -10 -9 -8 -7 -6 -5 Peptide concentration (log M)
ACP A2 A5 B A6 A7 A9
1001.00
0.75 75
0.50 50 Response 0.25 25
Normalized Luminescent 0.00 0 -10-9-8-7-6-5 Peptide concentration (log M)
1-8 1-9 C ACP 1,3-10 +G
1001.00
0.75 75
0.50 50 Response 0.25 25
Normalized Luminescent 0.00 0 -10-9-8-7-6-5 Peptide concentration (log M) 328
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329 Table 2. Primary structure of select naturally occurring AKH analogs from insects and their 330 activity on the A. aegypti AKHR-IA receptor (AKHR-IA). Using heterologous expression of 331 AKHR-IA, the half maximal effective concentration for each of the analogs was determined 332 as well as the corresponding reduction in activity, highlighting how a particular single residue 333 substitution, a combination of substitutions or other modifications are tolerated in this system. 334 ND = represents no determined EC50 value due to minimal or no detectable activity of the 335 analog when tested up to 10μM. 336 Activity on Fold-reduction in Primary structure of A. aegypti Peptide/Analog: Species origin: activity relative to analog: AKHR-IA Aedae-AKH-I (EC50) Aedae-AKH-I Aedes aegypti pQLTFTPSWamide 2.159E-09 1.0 Lacol-AKH-7mer Laconobia oleracea pQLTFTSS-amide ND >10,000 Grybi-AKH Gryllus bimaculatus pQVNFSTGWamide 2.23E-06 1031.5 Libau-AKH Libellula auripennis pQVNFTPSWamide 1.18E-06 547.9 Pyrap-AKH Pyrrhocoris apterus pQLNFTPNWamide 7.269E-07 336.7 Erythemis Erysi-AKH simplicicollis pQLNFTPSWamide 6.69E-07 309.9 *Periplaneta V2-PeramCAH-II americana pQVTFTPNWamide 1.485E-07 68.8 Bommo-AKH Bombyx mori pQLTFTPGWGQamide 2.362E-08 10.9 Hipes-AKH-I Hippotion eson pQLTFTSSWamide 1.647E-08 7.6 Periplaneta Peram-CAH-II americana pQLTFTPNWamide 2.226E-09 1.0 Tabat-AKH Tabanus atratus pQLTFTPGWamide 1.522E-09 0.7 * Denotes a synthetic analog that does not occur in Periplaneta americana.
337
338 339
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340 Figure 2.
A Aedae AKH Libau AKH Lacol AKH 7-mer Pyrap AKH Grybi-AKH Erysi AKH
100
75
50 Response 25
Normalized Luminescent Normalized 0 -11 -10 -9 -8 -7 -6 -5 Peptide concentration (log M)
B Aedae AKH Hipes AKH-I V2 Peram CAH-II Peram CAH-II Bommo AKH Ta ba t A KH
100
75
50 Response 25
Normalized Luminescent 0 -11 -10 -9 -8 -7 -6 -5 Peptide concentration (log M) 341 342 343
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344 Discussion 345 In light of the high fidelity of the three GnRH-related systems (Hansen et al., 2010; Oryan et
346 al., 2018; Wahedi and Paluzzi, 2018), the current study set out to examine for the first time
347 the ligand structure-activity relationship for an insect ACP receptor. This is of utmost
348 importance in order to gain insight on the specific structural features of the ACP system
349 given that extensive studies have been carried out previously on AKH receptors in a variety
350 of species using natural and synthetic analogs of this neuropeptide family (Caers et al., 2012;
351 Fox and Reynolds, 1991; Gäde et al., 2000; Gäde, 1992; Gäde, 1993; Gäde et al., 2016;
352 Keeley et al., 1991; Lee and Goldsworthy, 1996; Lee et al., 1996; Lee et al., 1997; Marco and
353 Gäde, 2015; Marco and Gäde, 2019b; Poulos et al., 1994; Velentza et al., 2000; Ziegler et al.,
354 1991; Ziegler et al., 1998). By examining the activity of a variety of ACP analogs on the
355 activation of the A. aegytpi ACP receptor (ACPR), including single alanine substitutions
356 along with truncated or extended analogs, a few observations can be highlighted. Firstly, the
357 overall charge of the peptide does not appear to play a role with regards to its influence on
358 receptor activation since substitution of the basic (arginine) or acidic (aspartic acid) residues
359 did not lead to a highly detrimental effect. Specifically, one would assume that the ACP
360 receptor may prefer a neutral ligand given that the native ACP has no net charge (i.e. is
361 neutral) since it has both a single basic and acidic residue. However, the modified analogs
362 which replace the basic (position 6) or acidic (position 7) residues with alanine, create
363 negatively or positively charged molecules, respectively, with both being quite active and so
364 neither proved detrimental substitutions. Secondly, with the exception of the aromatic
365 tryptophan residue in the eighth position, the C-terminal residues of ACP do not appear to be
366 highly important since alanine substitutions in positions five through seven only marginally
367 impacted activity between ~16 to 45-fold whereas alanine substitution in position nine
368 resulted in this analog having five-fold increased activity. Amidation of the C-terminal
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369 residue was critical since the free acid analog exhibited no activity; however, it is noteworthy
370 that the C-terminally extended peptide having an amidated glycine was also quite effective
371 with ACPR activation compromised by only 6-fold. Previous studies examining free acid
372 analogs of AKH (including both in vitro and in vivo assays) have demonstrated that in some
373 cases the absence of a blocked C-terminus is less critical (Caers et al., 2012; Caers et al.,
374 2016; Lee et al., 1996), whereas other investigations have revealed the presence of an
375 amidated C-terminus to be very important with poor or no responses with analogs lacking this
376 feature (Gäde and Hayes, 1995; Gäde, 1990). These observed differences in response to key
377 structural elements, including the normally amidated C-terminus of AKH, may reflect the
378 occurrence of inter- and intra-species receptor subtypes which may be differentially sensitive
379 to these modifications (Caers et al., 2016; Gäde and Hayes, 1995), and could also point to the
380 well documented phenomenon that some species have AKH receptors that are more
381 promiscuous whereas others are very strict in the structural characteristics of their particular
382 ligand (Caers et al., 2012; Gäde, 1990; Marchal et al., 2018). This latter scenario is more in-
383 line with observations for the A. aegypti ACP system where only a single functional receptor
384 variant occurs (Wahedi and Paluzzi, 2018), which we show herein does not tolerate the
385 absence of the C-terminal amidation and is thus of high importance for the ACP system.
386 Thirdly, both aromatic residues were essential for activity on the ACPR while N-terminal
387 residues of the peptide, including mainly threonine and pyroglutamic acid, were also highly
388 critical since alanine substitutions were not tolerated leading to significantly reduced activity
389 of these analogs relative to the native ACP peptide. However, alternatively blocking this
390 alanine substitution on the N-terminus through acetylation improved the activity of this
391 analog by 10-fold. Similar observations have been made with AKH analogs tested using both
392 in vivo and in vitro assays whereby complete removal of the N-terminal pyroglutamate
393 abolishes activity of the AKH analog whereas the alternatively blocked N-terminus (i.e. N-
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394 Acetyl-Ala), or even simply glutamate or glutamine that can spontantenously undergo
395 cyclization (Schilling et al., 2008), were shown to have improved activity, albeit lower than
396 the native AKH (Caers et al., 2016; Gäde and Hayes, 1995; Marco and Gäde, 2019b). Lastly,
397 although alanine substitution for valine in the second position led to a relatively minor effect
398 on ACPR activation (~70-fold reduced activity), the internally truncated analog omitting
399 valine within the N-terminal region elicited no activation of the ACPR, indicating the spacing
400 between the more critical residues, namely pyroglutamate and polar threonine, is essential for
401 ACP peptide activity on the ACPR. Thus, perhaps in common with AKH structural features,
402 the switch between adjacent hydrophobic and hydrophilic residues is necessary in order to
403 properly ‘fit’ and bind with its particular receptor (Gäde and Hayes, 1995) so that not only is
404 the ACP analog shorter by one amino acid (a nonapeptide), but moreover, the entire structural
405 configuration of the peptide is disrupted and the whole sequence is no longer in sync with its
406 receptor. This corroborates with studies that have examined the conservation of the ACP
407 primary structures across insects from different orders (including Diptera, Lepidoptera,
408 Coleoptera, Hymenoptera and Hemiptera) as well as from various species within the same
409 order (seventeen species of Coleoptera), where the N-terminal pentamer sequence, when the
410 ACP system is present, is nearly completely conserved across these various species with a
411 consensus motif of pQVTFS-, whereas significant sequence variability occurs within the C-
412 terminus of the ACP sequence with deca-, nona- and even dodecapeptides reported (Hansen
413 et al., 2010; Veenstra, 2019).
414 Considering the resultant activation of the mosquito AKH receptor in response to the various
415 mostly naturally-occurring insect AKH analogs examined in the current study, these findings
416 indicate that these bioanalogs can be grouped into distinct categories based on their
417 determined potencies on the A. aegypti AKHR-IA. The first group includes natural or
418 synthetic AKH analogs having substitutions in critical positions, including the absence of the
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419 C-terminal tryptophan that occurs in the heptapeptide, Lacol-AKH-7mer, which is not
420 tolerated whatsoever since activity is completely abolished and is therefore of greatest
421 importance for AKH receptor activation. Observations correlating with these findings have
422 been made in various studies utilizing in vitro heterologous assays as well as in vivo
423 bioassays monitoring lipid- and/or carbohydrate-mobilizing actions of AKH analogs.
424 Specifically, removal or replacement of the critical aromatics that are archetypal features of
425 AKH neuropeptides found in positions four and eight demonstrates the absolute requirement
426 of these features for receptor activation in heterologous systems or in vivo biological activity
427 (Gäde, 1992; Gäde, 1993; Marco and Gäde, 2015; Marco and Gäde, 2019b).
428 The second group in the current study includes analogs that were similar, but had a higher
429 efficacy, to the tested C-terminally truncated peptide lacking the eighth position tryptophan.
430 In particular, additional residues of major importance were identified in both the amino and
431 carboxyl region of the octapeptide, since Grybi-AKH, which has substitions in positions 2-3
432 as well as 5-7 compared to the native A. aegypti AKH, demonstrated a large reduction in
433 activity by over three orders of magnitude. Interestingly, this implies that despite Grybi-
434 AKH retaining the prototypical features of an AKH, including the pyroglutamic acid in the
435 first position, phenylalanine in position four, tryptophan in position eight and an amidated C-
436 terminus, the A. aegypti AKH receptor is quite specific compared to other insect AKH
437 receptors including, for example, the locust, Schistocerca gregaria or the pea aphid,
438 Acyrthosiphon pisum, whose receptors responded similarly to both the mosquito AKH and
439 Grybi-AKH (Marchal et al., 2018). Similar findings were reported for the AKH receptor
440 from another dipteran, namely the fruit fly D. melanogaster, which is also highly selective
441 and was not strongly responsive to non-dipteran AKH analogs (Marchal et al., 2018). In
442 order to determine whether the substitutions in positions 2-3 versus 5-7 were of greatest
443 importance, we examined the activity of a natural AKH analog from L. auripennis (Libau-
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444 AKH), whose sequence matches that of native A. aegypti AKH except for positions 2-3
445 (valine and asparagine) that are instead shared with Grybi-AKH. This allowed us to identify
446 that the substitutions in this N-terminal region are much more critical and are not well
447 tolerated by the AKH receptor system in A. aegypti since this analog diplayed only a
448 marginal improvement (i.e. nearly two-fold) compared to Grybi-AKH. This finding is
449 corroborated by recent analysis of select dipteran AKH analogs on the A. aegypti AKH
450 receptor, which showed that amino acid changes at positions five (serine in both Glossina
451 morsitans AKHs and in D. melanogaster AKH compared to threonine in A. aegypti AKH) or
452 seven (glycine in G. morsitans AKH-I and aspartic acid in D. melanogaster AKH compared
453 to serine in A. aegypti AKH) are indeed well tolerated with these dipteran AKH analogs
454 being similarly active to the endogenous mosquito AKH (Marchal et al., 2018). It was also
455 previously shown in other dipteran AKH systems where structure-activity relationships were
456 investigated, that the least tolerated substitutions were localized to the N-terminal region,
457 with substitutions to residues in positions two through five from the N-terminus proving
458 crucial for activation of D. melanogaster and A. gambiae AKH receptors, which the authors
459 described was due to this core region of the peptide forming a predicted β -strand necessary
460 for receptor interaction (Caers et al., 2012). Comparatively, although only fruit fly AKH
461 analogs were tested, substitutions to position seven alone and positions six plus seven for the
462 D. melanogaster and A. gambiae AKH receptors, respectively, were far more tolerated with
463 little or no influence on the activation of the corresponding receptor (Caers et al., 2012). This
464 supports our conclusions that the C-terminal region of the AKH octapeptide, excluding the
465 critical tryptophan in the eighth position, is not as critical for activity. As a putative reason for
466 this, it is argued that the side chains of a subset of these amino acids may be buried in the
467 assumed β-turn formed by the residues in the C-terminal region of AKH neuropeptides
468 (Caers et al., 2012; Gäde, 1992; Gäde and Hayes, 1995), which is now supported by
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469 molecular modeling and nuclear magnetic resonance spectroscopy studies that have
470 confirmed the β-turn configuration in the AKH secondary structure (Jackson et al., 2014;
471 Nair et al., 2000; Nair et al., 2001; Zubrzycki and Gäde, 1994).
472 Interestingly, structure-activity studies on the two tsetse fly (G. morsitans) AKH receptors
473 using synthetic analogs of the pair of endogenous AKHs identified the most critical sites
474 being the aromatic residues in positions four and eight, but the next most important features
475 included positions 1-3 from the N-terminus with activity of these analogs eliciting only 20-
476 50% activity compared to the two endogenous AKH peptides (Caers et al., 2016). On the
477 other hand, the region of the G. morsitans AKH diplaying increased permissiveness for
478 substitutions included positions six and seven, where glycine substitutions of these sites
479 resulted in these analogs having between 65-97% activity compared to the efficacy of the
480 native AKHs. Interestingly, however, substitutions in the fifth position of the octapeptide
481 were not tolerated, since activity of these analogs was reduced to only 11-13% that of the
482 natural AKHs, which nearly matches the effect of substitutions in the aromatic residue sites at
483 positions four and eight (Caers et al., 2016). Supporting the notion that substitutions in
484 positions 5-7 are more permissive in the mosquito AKH receptor system, we examined the
485 activity of two additional natural AKH analogs, which in comparison to A. aegypti AKH,
486 have substitutions in either the third position alone or combined with a substitution of the
487 seventh position residue. In particular, the firebug P. apterus AKH (Pyrap-AKH) has the
488 third position threonine found in A. aegypti AKH replaced with asparagine while the seventh
489 position serine is also replaced by asparagine, resulting with this analog having ~337-fold
490 reduced activity relative to the native mosquito AKH. Next, by comparing the tolerance of
491 these two substitutions in Pyrap-AKH with that occurring in the AKH from E. simplicicollis
492 (Erysi-AKH), which has only a single substitution in the third position with threonine
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493 replaced by asparagine, reveals this AKH analog has similar activity as Pyrap-AKH with
494 ~310-fold reduced activity relative to the endogenous A. aegypti AKH.
495 The third group includes analogs with activities similar to, or in some cases, stronger than the
496 endogenous A. aegypti AKH that contained substitutions or insertions in the C-terminal
497 region, while sparing the critical tryptophan residue. Specifically, we next tested two natural
498 AKH analogs with substitutions in the C-terminal region of the peptide, maintaining the core
499 5-6 residues on the N-terminus. Specifically, the domestic silkmoth AKH (Bommo-AKH),
500 has a glycine substitution in an equivalent location to the serine in position seven and also has
501 an extended C-terminus containing glycine and glutamine that follows tryptophan in the
502 eighth position, forming a decapeptide. Despite these changes within the C-terminal region,
503 this AKH analog retained strong activity on the A. aegypti AKHR-IA. Similarly, analysis of
504 Hipes-AKH-I from the hawk moth, Hippotion eson, which along with another member of the
505 genus, each produce the greatest number of AKH analogs so far reported from a single insect
506 (Gäde et al., 2013), revealed this analog to be very active, with only a marginal reduction in
507 activity on the mosquito AKHR-IA. Notably, Hipes-AKH-I shares an identical sequence to
508 the Lacol-AKH-7mer with the one exception that the latter lacks the terminal tryptophan
509 residue. This indicates that serine for proline substitution in the sixth position is in fact well
510 tolerated and is not the root cause of the abolished activity of the Lacol-AKH-7mer, which
511 instead is most certainly due to the absence of the C-terminal amphipathic tryptophan residue
512 with its large indole side chain.
513 Considering the resulting activity of these various AKH analogs, the findings support that
514 substitutions in positions 5-7 are more permissive, whereas comparatively, substitutions in
515 positions 2-3 are not well tolerated by the A. aegypti AKH receptor system. Therefore, given
516 the importance of substitutions in positions 2-3, we aimed to discern which of these two sites
517 were the most critical for eliciting AKHR-IA functional activation. To achieve this aim, we
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518 tested a synthetic analog of a cockroach P. americana AKH, V2-PeramCAH-II, which has a
519 second position valine substituted for the naturally occurring leucine in A. aegypti AKH but
520 also contains a substitution in the seventh position with asparagine replacing serine. The
521 results from analysis of this analog confirm the valine substitution for leucine in the second
522 position is far better tolerated compared to the asparagine substitution for threonine in the
523 third position, since V2-Peram-CAH-II displayed nearly five-fold improved activity on the A.
524 aegypti AKHR-IA compared to Pyrap-AKH (which has asparagine substitutions in both
525 positions three and seven). Thus, although both of these residues are important for functional
526 activation of the mosquito AKHR-IA, substitutions of the second position proved this site to
527 be the most critical. One additional matter to consider is the seventh position substitution,
528 which is an asparagine in Pyrap-AKH, V2-Peram-CAH-II and is the only substitution in the
529 natural AKH analog, Peram-CAH-II. The activity of Peram-CAH-II was equivalent to the
530 native mosquito AKH, which confirms that this seventh position is quite permissive to
531 substitutions and, more importantly, that the markedly reduced activity of the AKH analogs
532 Pyrap-AKH and V2-Peram-CAH-II is solely a result of the substitutions in the second and
533 third positions. Finally, to our surprise, the survey of natural AKH analogs revealed that
534 Tabat-AKH, which has a single glycine substitution in position seven replacing the natural
535 serine of A. aegypti AKH, may serve as a potential lead substance for design of a
536 superagonist since this natural AKH analog elicited 30% improved activity relative to the
537 native mosquito AKH. It appears that the more flexible and non-polar Gly residue allows the
538 ligand a tighter binding to the receptor than the polar and neutral Ser. This is particularly
539 interesting given the permissiveness of substitutions in the vicinity of the C-terminus
540 (excluding the eighth position tryptophan) established in the current study and previously for
541 the A. aegypti AKHR-IA (Marchal et al., 2018) as well as homologous receptors from other
542 dipterans (Caers et al., 2012; Marchal et al., 2018), which may allow a greater variety of lead
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543 compounds to be designed and tested that could interfere with this neuropeptide system and
544 perturb its normal functioning vital for mobilizing energy substrates in insects.
545 Integrating the novel data from the present study analyzing structural characteristics of the
546 two most recently diverged GnRH-related family members in arthropods, we can infer with
547 some degree of confidence how these ligands act only upon their respective receptors. The
548 ACP sequence in many insect species often contain charged residues in positions 6, 7 or 9,
549 which in instances where only a single basic or acidic residue occurs, provide an overall
550 charge to the peptide (Hansen et al., 2010; Veenstra, 2019). However, our results revealed
551 that the overall charge of the ACP analog did not largely compromise activity since
552 substitutions of these charged residues with alanine were quite tolerated by the ACPR.
553 Comparatively, AKH receptors do not like charges associated with their ligands since no
554 basic residues occur and only a few natural AKH family members containg an acidic residue
555 (e.g. aspartic acid at position 7), which have been shown to be not well tolerated by AKH
556 receptors in L. migratoria and P. americana (Gäde, 1991; Gäde, 1993; Gäde et al., 1990).
557 Another feature differentiating the AKH and ACP peptides in A. aegypti is the presence of an
558 alternating pattern of hydrophobic and hydrophilic residues over the entire length of the
559 AKH, which is shared with other AKH family members (Gäde and Hayes, 1995), whereas
560 the ACP contains a motif of three hydrophilic residues at its core residues 5-7, which may
561 lead to incompatability with the AKHR-IA. Additionally, clearly the valine substitution in
562 position 2 is not tolerated well by the A. aegypti AKHR-IA, and so this residue which is
563 present in position 2 of the ACP sequence may also contribute towards the inactivity of this
564 peptide on the AKHR-IA. On the other hand, since the AKH peptide from B. mori (a
565 decapeptide) was quite active on the A. aegypti AKHR-IA, the longer length of ACP is
566 unlikely to be the cause of its inactivity on the AKHR-IA.
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567 Our findings also set the framework towards understanding why the A. aegypti AKH has no
568 activity on the ACP receptor. As we saw with the AKHR-IA, substitution of valine for
569 leucine in position 2 led to nearly a 70-fold reduction in receptor activity. On the other hand,
570 the removal of valine and its replacement with alanine in the ACP sequence led to a similar
571 70-fold reduction in activity on the ACPR. Thus, the presence of valine in position two is
572 needed for optimal activity for ACPR whereas its absence in the same position (but on the
573 AKH peptide) is necessary for optimal activation of AKHR-IA. Again, as discussed earlier,
574 the importance of charged residues for optimal ACPR activity that are often found in insect
575 ACPs (Hansen et al., 2010; Veenstra, 2019), whereas charged residues are unusually found
576 on AKH peptides and are not well tolerated by AKH receptors that have been studied either
577 in vivo or in vitro (Gäde, 1991; Gäde, 1993; Gäde et al., 1990). Finally, one clear finding
578 from these studies is that ACP analogs that are too short (nonapeptide or octapeptide), even if
579 they contain all the hallmark features, as demonstrated by the C-terminally truncated analogs
580 for example, fail to activate ACPR. Thus, the length of the ligand is most relevant and the
581 binding pocket of ACPR requires at least a decapeptide leaving the A. aegypti AKH, an
582 octapeptide, too short for ACPR binding and activation.
583 584
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585 Author Contributions
586 GG and JP designed the synthetic analogs and wrote the manuscript. AW performed all the
587 experiments and AW, JP and GG analyzed the data. All authors have contributed towards the
588 revisions and have granted approval of the final manuscript submitted for publication.
589 Funding
590 This work was funded by a Natural Sciences and Engineering Research Council of Canada
591 (NSERC) Discovery Grant and an Ontario Ministry of Research, Innovation and Science
592 Early Researcher Award to JP and Incentive Grant from the National Research Foundation
593 (Pretoria, South Africa; grant number 85768 [IFR13020116790] to GG) and by the
594 University of Cape Town (block grant to GG).
595 Conflict of Interest Statement
596 The authors declare that the research was conducted in the absence of any commercial or
597 financial relationships that could be construed as a potential conflict of interest.
598 599
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600 Figure captions 601
602 Figure 1. Dose–response curves of the native A. aegypti ACP along with synthetic analogs
603 that contain single substitutions or other modifications, which were tested for their activity on
604 the ACP receptor (ACPR) using a cell-based bioluminescent assay. (A) Single amino acid
605 substitutions with alanine or modifications to the normally blocked N- or C-termini resulting
606 in a significant reduction to their activity on the ACPR. (B) Single amino acid substitutions
607 with alanine to the native ACP sequence having minimal effects to the activity of the tested
608 analogs indicating these substitutions are well tolerated. (C) Analogs with internal or terminal
609 truncations or extension (i.e. +Gly analog) relative to the native ACP sequence conferring
610 significant reductions to their activity on the ACPR. The half maximal effective
611 concentration (EC50) for each analog along with the corresponding change in activity relative
612 to native A. aegypti ACP is provided in Table 1. Luminescence is plotted relative to the
613 maximal response achieved when 10-5M Aedae-ACP was applied to the ACPR. Data
614 represent mean +/- standard error of three independent biological replicates.
615
616 Figure 2. Dose–response curves of the native A. aegypti AKH along with mostly naturally
617 occurring analogs from a variety of insects that contain substitutions or other modifications,
618 which were tested for their activity on the AKHR-IA receptor using a cell-based
619 bioluminescent assay. (A) Naturally occurring insect AKH analogs having critical
620 substitutions that are not well tolerated by the AKHR-IA, with moderately compromised
621 activity relative to the native A. aegypti AKH. (B) Naturally occurring insect AKH analogs,
622 or a synthetic analog (V2-Peram-CAH-II), having substitutions that are well tolerated by the
623 AKHR-IA, with marginally compromised or slightly improved activity relative to the native
624 A. aegypti AKH. The half maximal effective concentration (EC50) for each analog along with
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625 the corresponding change in activity relative to native A. aegypti AKH is provided in Table 2.
626 Luminescence is plotted relative to the maximal response achieved when 10-5M Aedae-AKH
627 was applied to the AKHR-IA. Data represent mean +/- standard error of three independent
628 biological replicates.
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629 References:
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