Sequence variations in monotreme Gcg genes are present in regions known to be important for the regulation of protein function

Gcg, Glp-1r, Gip and Dpp-4 genomic sequences were identified in the platypus genome database. At the beginning of this study there were no reported transcript sequences for these genes and some of the genomic sequences were incomplete. In this study for the first time cDNA sequences encoding platypus and echidna preproglucagon (encoded by the GCG gene) were identified (Supplementary Table S2). Phylogenetic analysis of the preproglucagon amino acid sequence in vertebrates revealed expected tree topology but also highlights sequence divergence in the monotreme lineage (Fig. 1b). The proglucagon peptide (Fig. 1a) includes a signal peptide (SP), glicentin-related polypeptide (GRPP), glucagon (GCG), intermediate peptides-1 and -2 (IP-1 and IP-2), glucagon like peptides 1 and 2 (GLP-1 and GLP-2), and is proteolytically processed into the mature glucagon-like peptides. While therian mammal GCG genes encode identical GLP-1 protein sequences, there are significant changes in the platypus Gcg ortholog. Importantly the inferred sequence of the platypus GLP-1 peptide (pGLP-1) differs in 11 of the 30 amino acids (37%) compared to human GLP-1 (hGLP-1, Fig. 1a, as reported previously8). A smaller difference was seen between platypus and human GLP-2 (30%) or glucagon peptides (20%) (Supplementary Fig. S1). Notably we also discovered specific changes in the DPP-4 cleavage site in pGLP-1 (pGLP-1, Ala8 to Ser) (Fig. 1a) and in the platypus GIP peptide (Supplementary Fig. S1, Ala2 to Ser), whereas there was no change to the cleavage sites in platypus glucagon or GLP-2 (Supplementary Fig. S1). To investigate if this change is also present in the echidna GLP-1 (eGLP-1) we cloned the echidna Gcg (eGcg) transcript and found a different amino acid at residue eight (Ala8 to Phe) in the eGLP-1 DPP-4 cleavage site (Fig. 1a), as well as a total of 17 differences from hGLP-1 of the 30 amino acids (57% changed). We also saw differences in echidna GLP-2 (pGLP-2) from hGLP-2 (45% changed), and in echidna glucagon from human glucagon (17% changed) (Supplementary Fig. S2). However, there was no change to the DPP-4 cleavage sites in both peptides. Remarkably, GLP-1 peptide sequence comparisons revealed a total of 12 differences between the two monotreme sequences (i.e. 40% of the sequence), indicating not only divergence from other mammals but major divergence within the monotremes, which separated only 17–48 Million years ago22 (Fig. 1b). These results raise fundamental questions about stability and potency of monotreme GLP-1.

Figure 1 Identification and characterisation of monotreme Gcg genes and GLP-1 peptides. (a) Alignment of human, platypus, echidna and gila monster (Ex-4) GLP-1 sequences highlights hGLP-1 residues involved in hGLP-1R binding (underlined)26,27,28,29. The DPP-4 target site is highlighted in blue. Residues identical to those of hGLP-1are boxed (grey). (b) Phylogenetic reconstruction (Neighbor Joining, MEGA4) based on preproglucagon amino acid sequences of nine vertebrate species. The preproglucagon peptide domains: signal peptide (SP), glicentin-related polypeptide (GRPP), glucagon (GCG), intermediate peptides-1 and -2 (IP-1 and IP-2), glucagon like peptides 1 and -2 (GLP-1 and GLP-2). Full size image

Monotreme Gcg and Dpp-4 exhibit similar tissue expression patterns to other mammals, but both genes are also expressed in venom

Expression analysis showed that platypus and echidna Gcg, Glp-1r and Dpp-4 tissue expression is similar to other mammals (Fig. 2)23,24, suggesting they play a similar role in monotremes. Surprisingly, both Gcg and Dpp-4 genes are also expressed in platypus and echidna venom (Fig. 2). In monotremes it is the same Gcg gene encoding GLP-1 that is expressed in gut and venom. This is in contrast to Heloderma suspectum, where the Gcg is expressed in gut and Exendin-4 in venom15.

Figure 2 Expression of platypus and echidna Gcg, Dpp-4 and Glp-1r in different tissues assessed by RT-PCR. (a) RT-PCR amplified Gcg, (b) DPP-4 and GLP-1R showing expression in a range of tissues including venom gland. Beta actin was used as a positive control. These gel photos have been cropped as indicated. Full size image

Monotreme GLP-1 peptides are resistant to cleavage by human DPP-4 and human serum components but are cleaved in platypus and echidna sera, independently of DPP-4 activity

Sequence alignments between human, platypus and echidna GLP-1 proteins (Fig. 1) revealed substitutions at the known DPP-4 cleavage site (Ala8 in human) to Ser or Phe in platypus and echidna respectively, predicting that DPP-4 cleavage could be affected. To test if the specific amino acid changes at the DPP-4 cleavage site in pGLP-1 and eGLP-1 result in resistance to degradation, we compared their cleavage to that of human GLP-1 (hGLP-1) and the DPP-4 resistant exendin-4 (Ex-4) (Fig. 3). Incubation of the peptides with purified human DPP-4 resulted in rapid degradation of hGLP-1 (50% reduction of intact peptide within 1 hour) but not Ex-4. Significantly, both echidna and platypus GLP-1 were not degraded by human DPP-4 (Fig. 3a), confirming that monotreme GLP-1 is resistant to DPP-4 cleavage. Next we investigated stability in human serum. In albumin-depleted human serum, platypus and echidna GLP-1 and Ex-4 remained stable whereas hGLP-1 was rapidly degraded (Fig. 3b). To investigate if monotremes employ a different way to break down GLP-1, we measured cleavage in platypus and echidna serum. Surprisingly, degradation of platypus and echidna GLP-1 was observed when incubated in platypus and echidna sera (Fig. 3c,d). Degradation was slower than for hGLP-1 but clearly measurable, with less than 50% uncleaved pGLP-1 and eGLP-1 remaining after 11 hours of incubation (Table 1). Interestingly, Ex-4 was cleaved slowly in echidna serum but remained intact in platypus and human sera.

Table 1 Percentage of intact peptides remaining after 11 h incubation in each serum. Full size table

Figure 3 Degradation of hGLP-1 (orange circles), pGLP-1 (pink squares), eGLP-1 (green triangles) and Ex-4 (turquoise inverted triangles) at different incubation times by purified human DPP-4 enzyme (a), human serum (b), platypus serum (c) and echidna serum (d) determined by measuring the area under the curve of the intact peptide following rHPLC analysis. All values represent means ± S.E.M. (n = 3). ***Statistically significant, P < 0.001 peptide remaining at last time point compared with starting peptide concentration. Full size image

DPP-4 is not the only enzyme that can degrade GLP-1. Human neural endopeptidase (NEP24.11), for example also cleaves GLP-1 but utilizes different target sites within the peptide25. To further investigate whether monotremes evolved a DPP-4 independent pathway to degrade GLP-1, we firstly confirmed the presence of DPP-4 in platypus and echidna sera using a synthetic DPP-4 peptide as a substrate and a DPP-4 inhibitor to prevent this cleavage (Supplementary Fig. S3), thus showing that cleavage was due to DPP-4. Despite the presence of DPP-4 inhibitor we were able to detect cleavage of both monotreme GLP-1 peptides (Supplementary Fig. S4), showing that a different enzyme(s) must be responsible for their cleavage in monotreme sera. To gain further insight into the mechanism of degradation we used mass spectrometry to analyse the pGLP-1 and eGLP-1 cleavage products. We saw cleavage products that suggested trypsin or chymotrypsin-like activity with cleavage after basic and hydrophobic residues (Fig. 4). Together this supports the idea that in monotremes a DPP-4 independent system has evolved to regulate GLP-1 half-life and activity.

Figure 4 Amino acid sequences of cleavage products predicted by MALDI-Mass spectrometry. Degradation of hGLP-1, pGLP-1 and eGLP-1 in echidna serum for seven hours was monitored by RP-HPLC, fragments were collected and analysed by mass spectometry. Known cleavage sites of DPP-4 and NEP24.11 are shown above the hGLP-1 sequence by red and green arrows, respectively. Identified cleavage sites in pGLP-1 and eGLP-1 are highlighted by blue arrows. Full size image

Monotreme GLP-1 peptides bind with lower affinity to the GLP-1 receptor than human GLP-1

We then asked how changes in pGLP-1 and eGLP-1 affect binding and activation of the GLP-1 receptor (GLP-1R). All of the known key hGLP-1 residues (underlined in Fig. 1a) involved in binding to the human GLP-1R (hGLP-1R) core and four of the six C-terminal residues (excepting Ala25 to Thr and Val33 to Leu) involved in binding to the hGLP-1R N-terminal domain are conserved in pGLP-126,27,28. Interestingly, in echidna GLP-1 there is less conservation of the receptor binding residues with additional changes at the N-terminal receptor binding Phe12 (conservatively substituted to Tyr) and Asp15 (changed to Glu) residues. hGLP-1 Gly22, involved in kinking of the helix, is a Glu in the extended Ex-4 helix, leading to different modes of interaction with the GLP-1R29. The same substitution is seen in the monotreme GLP-1 sequences (Fig. 1a). The platypus GLP-1R amino acid sequence (deduced in this study from a sequenced cDNA transcript) is similar to the hGLP-1R (76% identity compared to hGLP-1R), including conservation of the residues important for ligand binding (Fig. 5). The pattern of pGLP-1R expression (Fig. 2b) is also similar to other mammals30. Receptor binding assays on hGLP-1R overexpressing cells showed that, compared to hGLP-1 and Ex-4 both platypus and echidna GLP-1 peptides have lower affinity for the human receptor (Fig. 6a, Table 2). hGLP-1 has an almost identical affinity for the platypus GLP-1R (pGLP-1R) and the human receptor, but unexpectedly both monotreme GLP-1 peptides had a significantly lower affinity than hGLP-1 for the pGLP-1R (Fig. 6b, Table 2). For both receptors monotreme GLP-1 peptides were equipotent with the GLP-1R agonist oxyntomodulin (OXM).

Table 2 Characterisation of the binding to human and platypus GLP-1R. Full size table

Figure 5 Schematic diagram of the pGLP-1R highlighting residues equivalent to hGLP-1R important for structure and function. Cysteine residues involved in disulphide bonds, denoted by dashed lines, are highlighted in yellow. Residues vital for ligand recognition and binding are highlighted in blue12,29,52. Residues important for receptor signalling through interaction with Gs-proteins are highlighted in green boxes29. Residues different between hGLP-1R and pGLP-1R are highlighted in red. Where a residue is marked blue or green but is different to hGLP-1 it is highlighted by a red circle. The putative signal peptide cleavage site is depicted with a black arrow. Full size image

Figure 6 hGLP-1R and pGLP-1R binding and hGLP-1R signalling. Characterisation of the binding of hGLP-1 (orange circles), pGLP-1 (pink squares), eGLP-1 (green triangles), Ex-4 (green turquoise inverted triangles) and OXM (blue diamonds) in competition with radiolabeled 125I-hGLP-1 (7–36) in FlpInCHO cells stably expressing hGLP-1R (a) or pGLP-1R (b). Data are normalized to the maximum 125I-hGLP-1 (7-36) of each data set. cAMP accumulation (c), Ca2+ mobilization (d) and ERK1/2 phosphorylation (e) was measured using FlpInCHO cells stably expressing hGLP-1R. Data were normalized to the maximal response induced by 100 μM forsklin (cAMP), 100 μM ATP (Ca2+) or 10% FBS (ERK1/2) respectively. Data were analysed with a three-parameter logistic equation as described previously47. (f ) Glucose-induced insulin release stimulated with or without 100 nM each peptide in the presence of either 2.8 mM or 20 mM of glucose. (g) Webs of bias generated to quantify and compare signalling bias as described in the methods section. Data are presented on a log scale. All values are the means ± S.E.M from at least three independent experiments performed in triplicate. *Statistically significant at P < 0.05 versus negative control group without peptide, ***Statistically significant at P < 0.001 versus negative control group without peptide. Full size image

Monotreme GLP-1 peptides are less potent in their activation of the GLP-1 receptor

We then investigated if this difference in affinities translates into a difference in activation of the GLP-1 receptor. As expected monotreme GLP-1 peptides showed significantly less potency than hGLP-1 in assays measuring cAMP accumulation, Ca2+ mobilization and ERK1/2 phosphorylation acting through both human and platypus receptors (Fig. 6c–e). eGLP-1 showed a markedly lower potency at both human and platypus receptors that was even lower than OXM. Differences in the structure of monotreme GLP-1 peptides compared with hGLP-1 could account for the lower affinity for the receptor. Circular dichroism spectroscopy (CD) on Ex-4 and hGLP-1 yielded results similar to previously published data26 and showed that all peptides utilized were folded correctly (Supplementary Fig. S5). All peptides retained significant helical content, although pGLP-1 had more and eGLP-1 had slightly less than hGLP-1. As has been seen with Ex-426, a difference in helical content can result in a different mode of interaction with GLP-1R.

Monotreme GLP-1 elicits a different signalling cascade compared to hGLP-1 when binding to the GLP-1 receptor

Closer examination of potencies in receptor activation revealed differential signalling bias for monotreme GLP-1 peptides in comparison to that elicited by hGLP-1. Distinct signalling bias arising through activation of the GLP-1R by different ligands (including OXM) has recently been established and may, at least in part, underlie differences in the physiological profile of naturally occurring ligands of the GLP-1R12. Indicators used to determine the signalling profile of peptides include cAMP and intracellular Ca2+ mobilisation, which are involved in promotion of insulin release, and pERK1/2 that is part of the mitogenic signalling pathways activated via the GLP-1R12. Intriguingly, both the platypus and echidna GLP-1 peptides displayed a distinct pattern of signalling in comparison to hGLP-1 and the clinically approved mimetic Ex-4, which was apparent at both the human and platypus GLP-1 receptors (Fig. 6, Supplementary Figs S6 and S7, Table 3). The signalling profile of the monotreme GLP-1 peptides closely matched that of OXM with a bias towards pERK1/2, and to a lesser extent iCa2+, relative to cAMP (Fig. 6, Supplementary Figs S6 and S7, Table 3), although the bias towards calcium mobilisation was less apparent for the pGLP-1 at the human receptors (Fig. 6). These observations suggest that monotreme GLP-1 peptides may have gained new as yet undefined functions.

Table 3 Characterisation of activation of human and platypus GLP-1R. Full size table

Platypus GLP-1 stimulates insulin release in mouse islet cells

Ultimately, the signal cascade triggered by incretins results in the release of insulin from pancreatic islet cells. We investigated the ability of pGLP-1 to stimulate insulin release from isolated mouse islets. Results showed that 100 nM pGLP-1 can stimulate insulin release in vitro similar to hGLP-1 (Fig. 6f). It appears at least at mouse islet GLP-1R that pGLP-1 would act with classical incretin function to promote insulin release, although whether this is the primary function in the platypus remains to be proven.