Author response: Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes. eLife digest Insulin is a hormone critical for maintaining healthy blood sugar levels in humans. When the insulin system becomes faulty, blood sugar levels become too high, which can lead to diabetes. At the moment, the only effective treatment for one of the major types of diabetes are daily insulin injections. However, designing fast-acting insulin drugs has remained a challenge. Insulin molecules form clusters (so-called hexamers) that first have to dissolve in the body to activate the insulin receptor, which plays a key role in regulating the blood sugar levels throughout the body. This can take time and can therefore delay the blood-sugar control. In 2015, researchers discovered that the fish-hunting cone snail Conus geographus uses a specific type of insulin to capture its prey – fish. The cone snail releases insulin into the surrounding water and then engulfs its victim with its mouth. This induces dangerously low blood sugar levels in the fish and so makes them an easy target. Unlike the human version, the snail insulin does not cluster, and despite structural differences, can bind to the human insulin receptor. Now, Ahorukomeye, Disotuar et al. – including some of the authors involved in the previous study – wanted to find out whether other fish-hunting cone snails also make insulins and if they differed from the one previously discovered in C. geographus. The insulin molecules were extracted and analyzed, and the results showed that the three cone snail species had different versions of insulin – but none of them formed clusters. Ahorukomeye, Disotuar et al. further revealed that the snail insulins could bind to the human insulin receptors and could also reverse high blood sugar levels in fish and mouse models of the disease. This research may help guide future studies looking into developing fast-acting insulin drugs for diabetic patients. A next step will be to fully understand how snail insulins can be active at the human receptor without forming clusters. Cone snails solved this problem millions of years ago and by understanding how they have done this, researchers are hoping to redesign current diabetic therapeutics. Since the snail insulins do not form clusters and should act faster than currently available insulin drugs, they may lead to better or new diabetes treatments. Introduction Insulin is a pancreatic hormone that is critical for glucose homeostasis. Secretion of insulin from pancreatic β-cells triggers the uptake of blood glucose into a variety of tissues, including the liver, skeletal muscle and adipose cells. Impairment of insulin secretion and/or insensitivity to the insulin produced can lead to the disease diabetes mellitus. To date, three major types of diabetes have been recognized: Type one diabetes (T1D) or autoimmune diabetes, Type two diabetes (T2D) and gestational diabetes mellitus (GDM). Daily insulin injections are the only effective treatment for patients with T1D, late-stage T2D and some with GDM (Mack and Tomich, 2017). A major limitation of insulin therapy is its delayed action. The native hormone (consisting of an A and B chain connected by disulfide bonds) oligomerizes into a stable hexamer comprising three insulin dimers held together by two central zinc ions (Adams et al., 1969). Following subcutaneous injection, the hexamer has to dissociate into the dimer, then monomer, in order to activate the insulin receptor. Hexamer-to-monomer conversion is a slow process that can lead to a significant delay in glucose control. This limitation has spurred efforts to design insulin analogues with reduced dimerization (and thus oligomerization) rates (Owens, 2002). However, despite decades of research the best fast-acting insulin formulations are not truly monomeric and still require 15–90 min to effectively lower blood glucose (Elleri et al., 2011); this is because the region involved in dimerization of the insulin molecule, the C-terminus of the insulin B chain, is also of critical importance for receptor activation (Menting et al., 2013). Until now, removing this region of the B chain in order to generate a fast-acting analog could not be achieved without a near complete loss of biological activity (Bao et al., 1997; De Meyts et al., 1978). Novel insights for potentially solving this long-standing problem recently came from our discovery of an insulin peptide found in the venom of the fish-hunting cone snail, Conus geographus (Safavi-Hemami et al., 2015). C. geographus belongs to a large genus of predatory marine snails that use their complex venoms for prey capture, defense and competitive interactions (Olivera, 1997). C. geographus insulin (Con-Ins G1) was shown to rapidly induce insulin shock (dangerously low blood sugar) in its fish prey (Safavi-Hemami et al., 2015; Robinson and Safavi-Hemami, 2016). Remarkably, Con-Ins G1 lacks the region of the B chain that is critical for both, dimerization and receptor engagement in human insulin (Safavi-Hemami et al., 2015; Menting et al., 2016). Despite this, Con-Ins G1 is a potent agonist of the human insulin receptor (Menting et al., 2016). Structure-function studies provided a rationale for this conundrum: two residues within the B chain of Con-Ins G1 act as surrogates for the missing C-terminus of the B chain of human insulin (Menting et al., 2016). Here, we demonstrate that fish-hunting cone snails have evolved a diverse set of B chain minimized insulins as part of their complex predation strategy. Remarkably, these insulins activate the vertebrate insulin receptor (in fish, mouse and human) but residues previously identified to serve as surrogates for the loss of the B region in Con-Ins G1 are variable. Molecular dynamics simulations reveal the modus operandi of these unique molecules. Our findings suggest the evolution of diverse molecular mechanisms of insulin receptor activation, providing a set of solutions to potentially solve a long-standing problem of designing truly monomeric, fast-acting insulin analogs for the treatment of diabetes. Results Identification and analysis of venom insulin sequences We investigated three species of fish-hunting cone snails native to the Central Philippines: C. geographus and C. tulipa from the Gastridium clade and C. kinoshitai from the Afonsoconus clade (Figure 1A). Reverse transcription PCR (RT-PCR) combined with whole transcriptome sequencing led to the identification of several distinct insulin sequences in the two cone snail species C. geographus and C. tulipa (Safavi-Hemami et al., 2015). Of these sequences, the venom insulin Con-Ins G1 shared highest similarity with fish insulin and was previously selected for functional and structural characterization (Safavi-Hemami et al., 2015; Menting et al., 2016). Here, we synthesized and functionally characterized four additional venom insulins from these species with various degrees of sequence divergence: Con-Ins G3 from C. geographus and Con-Ins T1A, T1B and T2 from C. tulipa. Con-Ins G1 and G3 differ by 32 residues across the entire precursor (22 residues within the A and B chain, Figure 1—figure supplement 1) and likely represent paralogs originated by gene duplication (Safavi-Hemami et al., 2016). Con-Ins T1A and T1B only differ by three residues across their entire precursor sequence and likely represent allelic variants of the same gene locus. Con-Ins T2 differs at 15 positions across the precursor (13 positions within the A and B chain) and likely originates from a distinct gene locus (Figure 1—figure supplement 1). In addition to these sequences, we performed RT-PCR and whole transcriptome sequencing of the venom gland of C. kinoshitai leading to the identification of two new insulin sequences, named Con-Ins K1 and Con-Ins K2. The two C. kinoshitai insulin sequences differ at 24 positions, 22 of which fall within the A and B chain (Figure 1—figure supplement 1). Compared to insulins from C. geographus and C. tulipa, the C-peptide regions of Con-Ins K1 and Con-Ins K2 are shorter suggesting an insertion/deletion event of 14 amino acids (14 codons, 42 nucleotides) during the evolution of these peptides. Figure 1 with 2 supplements see all Download asset Open asset Species and insulin sequences analysed in this study. (A) Cone snail species analyzed in this study and their collection sites in the Philippines. (B) Alignment of the A and B chains of venom insulins with insulin from human and zebrafish. Amino acid numbering for venom insulins was adapted from human insulin (shown on top of sequences). Residues in venom insulins that differ from those in Con-Ins G1 are shown in blue. The sequence logo shows conservation/variability at each position in venom insulins (generated using Geneious vs 11.1.2 software). Cysteines are shown in bold and the disulfide connectivity is depicted as connecting lines between cysteines. Post-translational modifications were identified for Con-Ins G1 and Con-Ins G3 (8) and predicted for all other insulins. Modifications are provided in their single letter code. y: y-carboxylated glutamate; O: hydroxyproline, *: C-terminal amidation. Residues of human insulin involved in binding to sites 1 and 2 of the insulin receptor are indicated by a single (site 1) or double (site 2) green rectangle below the hIns sequence (De Meyts, 2015). B chain region involved in dimerization and receptor activation in human and fish insulin and positions of residues previously suggested to compensate for the loss of this region in venom insulins are highlighted in gray. (C) The endogenous cone snail signaling insulin was sequenced from the circumoesophageal nerve ring of C. geographus (Safavi-Hemami et al., 2016). Additional disulfide bond missing in venom insulins is shown as dashed lines between cysteines. Image of C.tulipa: courtesy of Jason Biggs. As evident by a highly conserved N-terminal signal sequence, all venom insulins belong to the same gene superfamily of venom gland-specific insulins (Safavi-Hemami et al., 2016). Sequence variability is highest within the A and B chain while the signal sequence is conserved (Figure 1—figure supplement 2). This juxtaposition between conserved and hypervariable regions is a common characteristic of cone snail venom toxins (Woodward et al., 1990). Cleavage of the A and B chains and post-translational modifications were predicted from precursor sequences based on mass spectrometric sequence information available for venom insulins from C. geographus (Safavi-Hemami et al., 2015). Unlike the endogenous insulins used by cone snails for insulin signaling (Safavi-Hemami et al., 2016), all seven venom insulins exhibit the cysteine framework of vertebrate insulin (with 4 and 2 cysteines in the A and B chain, respectively). Critically, all seven sequences lack the C-terminal region of the B chain involved in dimerization and receptor activation of human insulin (Figure 1B). Insulins from C. geographus and C. tulipa share more sequence similarity to one another than insulins from C. kinoshitai, consistent with the close phylogenetic relationship of these two species (Puillandre et al., 2014). Con-Ins K1 and K2 have longer A chain C-termini and B chain N-termini and differ in nearly every position from Con-Ins G1 (Figure 1B). Notably, venom insulins are distinct from the endogenous signaling insulin expressed in the circumoesophageal nerve ring of cone snails (Figure 1C) (Safavi-Hemami et al., 2016). This is also evident by the presence of a distinct signal sequence (and distinct 5’ and 3’ untranslated regions) between the venom insulin gene family and their endogenous homologs (data not shown). All sequences were deposited to GenBank (see Material and methods section for accession numbers). Overall, venom insulin sequences exhibit pronounced sequence divergence with very few conserved amino acids. The few relatively well conserved amino acids include the first four residues in the A chain, Gly8, Ser9 and Leu18 in the B chain and all six cysteines (see Sequence Logo in Figure 1). Strikingly, the two residues (TyrB15 and TyrB20) previously identified to serve as surrogates in Con-Ins G1 for the missing B chain C-terminus of human insulin are only moderately conserved (TyrB15) or hypervariable (TyrB20). Peptide synthesis Since its first chemical synthesis in 1963 (Meienhofer et al., 1963), insulin has remained challenging to synthesize with the correct intra- and intermolecular disulfide bonds. Here, all novel insulin sequences were successfully synthesized using Fmoc peptide chemistry. Con-Ins G3 and Con-Ins T2 were synthesized using procedures similar to those previously applied to Con-Ins G1 (11). To simplify peptide synthesis for Con-Ins T1A, Con-Ins T1B, Con-Ins K1 and Con-Ins K2, these peptides were synthesized using a selenocysteine replacement strategy in which the intra-molecular disulfide bond in the A-chain is substituted by a diselenide (Sec-Sec) bond (Safavi-Hemami et al., 2015). We previously reported that there is no difference in activity between Con-Ins G1 containing a diselenide bond and native Con-Ins G1 (11) and similar observations have been made for other peptides (Walewska et al., 2009; Muttenthaler et al., 2010). Several different methods were applied for the formation of the first inter-molecular disulfide bridge (i.e., using DMSO, Cu ions and pre-activation of the B-chain with DTNP as recently described by others (Liu et al., 2014)). These methods allowed the accumulation of the desired intermediate product containing the first intermolecular disulfide bond in yields of up to 50%. When forming the second intermolecular disulfide bridge between the A and B chain in the presence of iodine we observed the formation of a small amount of an additional disulfide isomer for some venom insulins. This was most pronounced for Con-Ins K1, where two products with the same mass were isolated in almost equimolar ratio. When synthesized with a disulfide instead of a diselenide bond only one final product was observed that was selected for subsequent functional analysis. Venom insulins reduce blood glucose in a zebrafish model of diabetes The presence of insulins with structural similarity to vertebrate insulin in the venoms of these fish-hunting species strongly suggested that these compounds are used to induce hypoglycemic shock in fish prey. Indeed, behavioral observations of C. geographus and C. tulipa have demonstrated that these species prey on fish and release compounds into the water prior to prey capture (Olivera et al., 2015) (Figure 1A). While no behavioral data is available for C. kinoshitai, phylogenetic analysis places this species within fish-hunters (the majority of cone snail species prey on marine worms and some prey on snails) (Olivera et al., 2015). In order to determine whether venom insulins are capable of lowering blood glucose in fish via activation of the insulin receptor, cone snail insulins were tested in the streptozotocin (STZ)-induced model of diabetes in zebrafish (Safavi-Hemami et al., 2015). Animals were first rendered hyperglycemic through i.p. injection of the β-cell poison STZ (1.5 g/kg) (Olsen et al., 2010), and the effects of subsequent injection of each venom insulin were examined. Following STZ treatment, blood glucose levels were significantly elevated from 65.9 ± 4.8 mg/dL (n = 11) to 393.3 ± 10.2 mg/dL (n = 7). Administration of venom insulins at 65 ng peptide/g body weight significantly lowered blood glucose levels for all venom insulins tested (Figure 2) with values ranging from 77.8 ± 39.8 mg/dL for Con-Ins T1A (p<0.0001, n = 5) to 199.2 ± 39.8 mg/dL for Con-Ins T2 (p<0.0067, n = 5). For comparison, 65 ng of human insulin (hIns)/g body weight reduces blood glucose to 92.0 ± 17.4 mg/dL (Safavi-Hemami et al., 2015). This demonstrates that venom insulins are capable of binding to and activating the fish insulin receptor, supporting their biological role in inducing insulin shock in fish prey. Figure 2 Download asset Open asset Venom insulins significantly lower blood glucose in a zebrafish model of T1D. Insulin activity was determined using the streptozotocin (STZ)-induced model of hyperglycemia. Hyperglycemia was successfully reversed on administration of 65 ng/g of each venom insulin with most significant reduction of blood glucose observed for Con-Ins T1A (p<0.0001, n = 5). Data for human Ins (65 ng/g, n = 6) was replotted from (Safavi-Hemami et al., 2015). Data were analyzed in Graphpad Prism software (version 7.0) using unpaired t tests with Welch’s correction. Plotted data represent individual fish (black circles), median values (horizontal black bars), first and third quartile (boxes) and minimum and maximum values (error bars). Raw blood glucose values are provided in Source File 1 (Figure 2—source data 1.Zebrafish.STZ.Assay). Figure 2—source data 1 Zebrafish.STZ.Assay: Data plotted in Figure 2. https://cdn.elifesciences.org/articles/41574/elife-41574-fig2-data1-v2.xlsx Download elife-41574-fig2-data1-v2.xlsx Venom insulins bind to the human insulin receptor In order to determine if venom insulins were capable of binding to the human insulin receptor (hIR) the affinity of these compounds for the B isoform of hIR (hIR-B) was determined in competition assays based on the displacement of europium-labeled hIns from solubilized immunocaptured receptors (Denley et al., 2004). All seven venom insulins bound to the hIR with affinities ranging from IC50 9.92 nM (7.75–12.68 nM; 95% CI) for the most potent insulin Con-Ins T1A to IC50 275.2 nM (162.2–487.6 nM; 95 % CI) for Con-Ins K2 (n = 2 biological replicates with three technical replicates each; the IC50 indicates half-maximal inhibitory concentration; Figure 3). For comparison, the affinity of hIns is ~20 fold higher than the most potent venom insulin Con-Ins T1A [hIns IC50 0.44 nM (0.40–0.49 nM; 95 % CI)]. Figure 3 Download asset Open asset Competition binding analysis to hIR-B of venom insulins (n = 2 independent measurements, each comprising three technical replicates). Results are expressed as a percentage of binding in the absence of competing ligand (%B/B0). Plotted values represent means ± s.d. Curve fitting was performed using Prism seven using nonlinear regression (one-site) analysis. IC50 values indicate half-maximal inhibitory concentration and are provided on the right (95% confidence intervals (CI)). Plotted values are provided in Source File 2 (Figure 3—source data 1.IR-B.binding). Figure 3—source data 1 IR-B.binding: Data plotted in Figure 3. https://cdn.elifesciences.org/articles/41574/elife-41574-fig3-data1-v2.binding.xlsx Download elife-41574-fig3-data1-v2.binding.xlsx Binding of venom insulins to the hIR induces downstream insulin signaling The ability of venom insulins to activate insulin receptor signaling receptor binding was determined using an of from of mouse As previously Con-Ins G1 the human insulins receptor, at lower to human insulin of Con-Ins G1 nM; 95% nM; of human insulin nM; 95% in which the indicates half-maximal effective (Figure and The other insulin identified from C. geographus, Con-Ins was significantly potent at the hIR-B with an of 95% In all three insulins identified from C. tulipa were active at the hIR-B with to Con-Ins G1 of Con-Ins T1A 95% nM; of Con-Ins T1B 95% nM; of Con-Ins T2 95% (n = 4 technical Figure and C. kinoshitai insulins showed activity with an of nM % for Con-Ins K1 and nM % for Con-Ins K2 (Figure and The ability of Con-Ins T1A and Con-Ins G1 to induce hIR-B with downstream receptor activation (Con-Ins T1A and Con-Ins G1 were and potent in these assays to Figure supplement 1). Overall, the ability of venom insulins to bind to the hIR with downstream signaling activity some can be These include that Con-Ins T2 and Con-Ins K1 have higher and Con-Ins G3 lower activation than be from their receptor binding These observations were not further in the current study but could indicate signaling of some venom insulins receptor binding and/or receptor Figure 4 with 1 supplement see all Download asset Open asset insulin receptor activation insulin receptor isoform B (hIR-B) activation analysis of venom insulins from (A) C. geographus, (B) C. tulipa and (C) C. Plotted values represent means ± Curve fitting was performed using Prism seven using nonlinear regression (one-site) analysis. between the most active venom insulin from each species (Con-Ins T1A, Con-Ins G1 and Con-Ins to native human insulin black and the human peptide insulin analog values and 95% confidence intervals for all insulins tested in this study. Plotted values are provided in Source File 3 (Figure data Figure data 1 Data plotted in Figure Download As out venom insulins lack the region of the B-chain that is known to be for receptor activation for The ability of the venom insulins to induce downstream signaling was to a B-chain analog of peptide insulin (Figure and Of the seven venom insulins had significantly higher activity at the hIR-B than suggesting the presence of structural that receptor activation despite the lack of the B-chain C-terminal The venom insulins Con-Ins G1, Con-Ins T1A and Con-Ins K1 reduce blood glucose in a mouse model of diabetes on their activity the hIR-B and ability to lower blood glucose in fish, the most active venom insulin from each species was tested in the mouse model of T1D. Animals were rendered hyperglycemic through i.p. injection of glucose was the of prior to administration of human insulin (n = biological and venom insulins (n = 3 biological replicates for each venom their to lower activity at the hIR-B human insulin, venom insulins were at the effective concentration reported for human insulin body et al., 2014). At this concentration Con-Ins G1, Con-Ins T1A and Con-Ins K1 effectively reversed every 15 min injection the of min (Figure 5). the in blood with Con-Ins T1A were at min (black Following this, Con-Ins T1A was also at the same concentration of human insulin to 1 body At this Con-Ins T1A effectively lowered blood glucose from ± mg/dL to ± mg/dL min (Figure with hIR-B binding and activation Con-Ins K1 was effective at lowering blood glucose at the concentration of human insulin (Figure When tested at to body Con-Ins K1 showed a similar in to human Figure Download asset Open asset The three venom insulins Con-Ins G1, Con-Ins T1A and Con-Ins K1 lower blood glucose in a mouse model of T1D. Insulin activity was determined using the streptozotocin (STZ)-induced model of hyperglycemia. of STZ into as evident by a high of blood glucose prior to insulin Hyperglycemia was successfully reversed on administration of 1 of human insulin and a of Con-Ins G1 and Con-Ins its high with Con-Ins T1A were at min (black and Con-Ins T1A was also at the same concentration as human Compared to Con-Ins G1 and Con-Ins T1A, Con-Ins K1 was potent at and reduced blood glucose from mg/dL to At Con-Ins K1 showed similar effects to human insulin at This is consistent with its lower at the Plotted data represent (n = 3). Plotted values are provided in Source File 4 (Figure data Figure data 1 Data plotted in Figure Download Molecular of Con-Ins T1A and Con-Ins K1 residues critical for insulin receptor activation In order to an understanding of the and of the most potent venom insulins identified from each species with the insulin receptor, we a model of Con-Ins T1A and Con-Ins K1 based on the of Con-Ins G1, in complex with the human insulin (hIR) of the first and the C-terminal residues of the hIR (Menting et al., Menting et al., (Figure top The model of Con-Ins T1A was stable throughout the molecular dynamics with of residues and the residues with the of the N-terminal region of the B chain, which a high of The by the of and with the observed for the residues in the of Con-Ins G1 (11). The of and are in position in the model of Con-Ins T1A bound to the hIR (Figure a consistent with these residues as a for the of the These positions are to positions previously identified in the model of G1 is by in Con-Ins T1A (Figure residues that differ between Con-Ins T1A and G1 differ at the of and the B chains (shown in in Figure Residues and of T1A, and in Con-Ins G1, bind on the of In the model of Con-Ins K1 bound to the residues at positions and to a role in receptor However, residues in Con-Ins G1 and T1A at these positions are by in Con-Ins and likely the longer C-terminus of the A chain for a of the C-terminal A chain the (Figure The significant sequence divergence across Con-Ins K1 is and in maintaining interactions with for models are provided as in the supporting information (Figure Figure data Figure data Figure data 3). Figure Download asset Open asset molecular model of Con-Ins G1 Con-Ins T1A and Con-Ins K1 in the of the binding of the human insulin receptor. A and B chains are shown in and hIns residues (in their are in black in the top of venom insulins are shown below Molecular models how the chains of and (shown in sequence below may act as surrogates for the absence of hIns by with Residues that differ between Con-Ins G1 and Con-Ins T1A are shown in