Examination of genetic polymorphisms in outbred wild-living species provides insights into the evolution of complex systems. In higher vertebrates, the proopiomelanocortin (POMC) precursor gives rise to α-, β-, and γ-melanocyte-stimulating hormones (MSH), which are involved in numerous physiological aspects. Genetic defects in POMC are linked to metabolic disorders in humans and animals. In the present study, we undertook an evolutionary genetics approach complemented with biochemistry to investigate the functional consequences of genetic polymorphisms in the POMC system of free-living outbred barn owl species (family Tytonidae) at the molecular level. Our phylogenetic studies revealed a striking correlation between a loss-of-function H9P mutation in the β-MSH receptor-binding motif and an extension of a poly-serine stretch in γ3-MSH to ≥7 residues that arose in the barn owl group 6-8 MYA ago. We found that extension of the poly-serine stretches in the γ-MSH locus affects POMC precursor processing, increasing g3-MSH production at the expense of g2-MSH and resulting in an overall reduction of g-MSH signaling, which may be part of a negative feedback mechanism. Extension of the γ3-MSH poly-serine stretches ≥7 further markedly increases peptide hormone stability in plasma, which is conserved in humans, and is likely relevant to its endocrine function. In sum, our phylogenetic analysis of POMC in wild living owls uncovered a H9P β-MSH mutation subsequent to serine extension in γ3-MSH to 7 residues, which was then followed by further serine extension. The linked MSH mutations highlight the genetic plasticity enabled by the modular design of the POMC gene.

Figure 1 Alignment 5 mitochondrial genes from different species of Tytonidae

Alignment of sequences of 5 mitochondrial genes for one representative individual per species from the family of Tytonidae to reconstruct the phylogenetic tree published in Uva, V., Packert, M., Cibois, A., Fumagalli, L., & Roulin, A. (2018). Comprehensive molecular phylogeny of barn owls and relatives (Family: Tytonidae), and their six major Pleistocene radiations. Mol Phylogenet Evol, 125, 127-137.

Roulin_Tyto_ReducedEditedAlignmentTytonidae_mitochondrial_genes_190104.nex

Figure 1 Alignment 2 nuclear genes from different species of Tytonidae

Alignment of sequences of 2 nuclear genes for one representative individual per species from the family of Tytonidae to reconstruct the phylogenetic tree published in Uva, V., Packert, M., Cibois, A., Fumagalli, L., & Roulin, A. (2018). Comprehensive molecular phylogeny of barn owls and relatives (Family: Tytonidae), and their six major Pleistocene radiations. Mol Phylogenet Evol, 125, 127-137.

Roulin_Tyto_ReducedEditedAlignmentTytonidae_nuclear_genes_190104.nex

Figure 2D Tyto alba / Strix aluco beta-MSH dose-response signaling on MC3R

Luninescence measurement to assess cAMP signaling induced by MCR stimulation, exel files

Low_Tyto_Fig2D_bMSH_SA_TA_MC3R_DR_exel_190104.xlsx

Figure 2D Tyto alba / Strix aluco beta-MSH dose-response curve

Dose response graph, prism

Low_Tyto_Fig2D_bMSH_SA_TA_MC3R_DR_prism_190104.pzfx

Figure 2E Dose response curves Strix aluco/Tyto alba beta-MSH variants on MC3R

Luminescence measurement to assess cAMP signaling after MC3R stimulation, exel

Low_Tyto_Fig2E_bMSHvariants_SA_TA_MC3R_DR_exel_190104.xlsx

Figure 2E Graph dose response curves Strix aluco/Tyto alba beta-MSH variants on MC3R

Graph dose response curves, prism

Low_Tyto_Fig2E_bMSHvariants_SA_TA_MC3R_DR_prism_190104.pzfx

Figure 2F Graph dose response curves Strix aluco/Tyto alba beta-MSH variants on MC4R

Graph dose response curves, prism

Low_Tyto_Fig2F_bMSHvariants_SA_TA_MC4R_DR_prism_190104.pzfx

Figure 2F Dose response curves Strix aluco/Tyto alba beta-MSH variants on MC4R

Luminescence measurement to assess cAMP signaling after MC4R stimulation, exel

Low_Tyto_Fig2F_bMSHvariants_SA_TA_MC4R_DR_exel_190104.xlsx

Figure 2G Beta-MSH P9/H9 competition assay on MC3R

Luminescence measurement to assess cAMP signaling after MC3R stimulation, exel

Low_Tyto_Fig2G_TA_bMSHH9_bMSHP9_comp_MC3R_exel_190104.xlsx

Figure 2G Graph beta-MSH P9/H9 competition assay on MC3R

Graph beta-MSH competition assay on MC3R, prism

Low_Tyto_Fig2G_TA_bMSHH9_bMSHP9_comp_MC3R_prism_190104.pzfx

Figure 2H Beta-MSH P9/H9 competition assay on MC4R

Luminescence measurement to assess cAMP signaling after MC4R stimulation, exel

Low_Tyto_Fig2H_TA_bMSHH9_bMSHP9_comp_MC4R_exel_190104.xlsx

Figure 2H Graph beta-MSH P9/H9 competition assay on MC4R

Graph beta-MSH competition assay on MC4R

Low_Tyto_Fig2H_TA_bMSHH9_bMSHP9_comp_MC4R_prism_190104.pzfx

Figure 3B Cleavage analysis of Strix aluco and Tyto alba POMC variants by PC1 and PC2 in Western blot

Western blot tiff images

Figures 4B-F Western blot time courses of POMC cleavage by PC1 and PC2

Western blot tiff images

Figure 4G Western blot exposures of Figures 4B-F for densitometric quantification of gamma3-MSH steady state levels

Western blot tiff images

Western blot exposures of Figures 4B-F for densitometric quantification of gamma3-MSH steady state levels .zip

Figure 4G Densitometric quantification of gamma3-MSH steady state levels after PC digest of Tyto alba POMC variants

Densitometric quantification of Western blots, exel

Low_Tyto_Fig4G_Westernblots4B-F_densitometric_quantification_exel_190104.xlsx

Figure 4G Graph steady state levels of Tyto alba POMC with varying numbers of serine residues

Graph gamma3-MSH steady state levels, prism

Low_Tyto_Fig4G_Westernblots4B-F_densitometric_quantification_prism_190104.pzfx

Figure 5A Signaling of synthetic Tyto alba gamma3-MSH peptides with varying serine numbers on MC3R

Luminescence measurements to establish cAMP signaling dose response curves of Tyto alba synthetic gamma3-MSH variants on MC3R and determine inverse EC50.

Exel files and Prism Graph files are provided.

Figure 5C Detection of recombinant Tyto alba pro-gamma-MSH fragment in cell supernatants by Western blot

Western blot tiff images

Figure 5D Quantification of Tyto alba recombinant pro-gamma-MSH fragment in Western blot

Densitometric quantification of pro-gamma-MSH expression in Western blot, exel

Low_Tyto_Fig5D_TA_progMSHfragment_quantification_Western_blot_exel_190104.xlsx

Figure 5D Graph quantification of Tyto alba recombinant pro-gamma-MSH fragment in Western blot

Graph densitometric quantification of pro-gamma-MSH in Westernblot for 3 transfection series, prism

Low_Tyto_Fig5D_TA_progMSHfragment_quantification_Western_blot_prism_190104.pzfx

Figure 5E Signaling of recombinant Tyto alba pro-gamma-MSH fragments on MC3R relative to N-terminal gamma-2 MSH fragment

Luminescence measurements to establish dose response curves for cAMP signaliing of recombinant N-terminal POMC fragments on MC3R, exel

Low_Tyto_Fig5E_TA_progMSHfragment_signaling_MC3R_DR_norm_cl2fragment_exel_190104.xlsx

Figure 5E Graph dose response curves of recombinant Tyto alba pro-gamma-MSH fragments on MC3R

Graphs dose response curves for cAMP signaling of recombinant N-terminal POMC fragments, prism

Low_Tyto_Fig5E_TA_progMSHfragment_signaling_MC3R_DR_ norm_cl2fragment_prism_190104.pzfx

Figure 5F Signaling activity of pro-gamma-MSH fragments with varying serine repeat numbers on MC3R relative to N-terminal gamma2-MSH fragment

Inverse relative EC50 calculated from 3 independent dose-response curves in Figure 5F, exel

Low_Tyto_Fig5F_1:relativeEC50_TAprogMSHfragment_variants_norm_cl2fragment_exel_190104.xlsx

Figure 5F Graph signaling activity of pro-gamma-MSH fragments with varying serine repeat numbers

Bar diagram for inverse EC50 of recombinant pro-gamma-MSH fragments relative to N-terminal gamma-2-MSH fragment, prism

Low_Tyto_Fig5F_1:relativeEC50_TAprogMSHfragment_variants_norm_cl2fragment_prism_190104.pzfx

Figure 6A Plama degradation of Tyto alba gamma3-MSH synthetic peptide variants

Luminescence measurements to determine the residual signaling activity of synthetic gamma3-MSH peptides on MC3R after plasma incubation for various times, exel

Low_Tyto_Fig6A_TASAg3MSH_synthetic_peptide_variants_plasma_degradation_TAMC3R_exel_190104.xlsx

Figure 6A Graph plama degradation of Tyto alba gamma3-MSH synthetic peptide variants

Graph degradation curves of Tyto alba gamma3-MSH synthetic peptides in plasma, prism

Low_Tyto_Fig6A_TASAg3MSH_synthetic_peptide_variants_plasma_degradation_TAMC3R_prism_190104.pzfx

Figure 6B Half-lives of Tyto alba gamma3-MSH synthetic peptide variants in plasma

Bar diagram half-lives Tyto alba synthetic gamma3-MSH variants, prism

Low_Tyto_Fig6B_TASAg3MSH_synthetic_peptide_variants_plasma_half-lives_prism_190104.pzfx

Figure 6C Degradation of Tyto alba gamma3-MSH synthetic peptide variants in water

Luminescence measurements to determine the residual signaling activity of gamma3-MSH synthetic peptide variants on MC3R after variuos incubation times in water, exel

Low_Tyto_Fig6C_TASAg3MSH_synthetic_peptide_variants_water_degradation_exel_190104.xlsx

Figure 6C Graph degradation of Tyto alba gamma3-MSH synthetic peptide variants in water

Degradation curves of Tyto alba gamma3-MSH synthetic peptide variants in water, prism

Low_Tyto_Fig6C_TASAg3MSH_synthetic_peptide_variants_water_degradation_prism_190104.pzfx

Figure 6D Competition assay between Tyto alba beta-MSH P9 and gamma-MSH on MC3R

Luminescence measurement to determine the cAMP signaling activity on MC3R, exel

Low_Tyto_Fig6D_comp_TAbMSHP9_gMSH_MC3R_exel_190104.xlsx

Figure 6D Graph competition assay between Tyto alba beta-MSH P9 and gamma-MSH on MC3R

Dose response graph for MC3R competition assay, prism

Low_Tyto_Fig6D_comp_TAbMSHP9_gMSH_MC3R_prism_190104.pzfx

Figure 6E Competition assay between Tyto alba beta-MSH P9 and gamma-MSH on MC4R

Luminescence measurements for MC4R signaling in competition assay between Tyto alba beta-MSH P9 and gamma-MSH, exel

Low_Tyto_Fig6E_comp_TAbMSHP9_gMSH_MC4R_exel_190104.xlsx

Figure 6E Graph competition assay between Tyto alba beta-MSH P9 and gamma-MSH on MC4R

Dose response graph for competition between Tyto alba beta-MSH P9 and gamma-MSH on MC4R, prism

Low_Tyto_Fig6E_comp_TAbMSHP9_gMSH_MC4R_prism_190104.pzfx

Figures 6F-6K Determination of human beta and gamma MSH peptide concentration

UV absorption measurement of peptide stock solutions, exel

Low_Tyto_Fig6F-6K_determination human peptide conc_exel_190104.xlsx

Figure 6F Dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC3R

Luminescence measurement to establish dose-response curves of h beta-MSH P9 and h beta-MSH H9 cAMP signaling on h MC3R, exel

Low_Tyto_Fig6F_hbMSHP9_hbMSHH9_hMC3R_DR_exel_190104.xlsx

Figure 6F Graph dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC3R

Graph dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC3R with EC50 calculations, prism

Low_Tyto_Fig6F_hbMSHP9_hbMSHH9_hMC3R_DR_prism_190104.pzfx

Figure 6G Dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC4R

Luminescence measurement to establish dose-response curves of h beta-MSH P9 and h beta-MSH H9 cAMP signaling on h MC4R, exel

Low_Tyto_Fig6G_hbMSHP9_hbMSHH9_hMC4R_DR_exel_190104.xlsx

Figure 6G Graph dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC4R

Graph dose response curves of h beta-MSH P9 and h beta-MSH H9 signaling on h MC4R and EC50 calculations, prism

Low_Tyto_Fig6G_hbMSHP9_hbMSHH9_hMC4R_DR_prism_190104.pzfx

Figure 6H Dose-response curves for h beta-MSH variants on h MC3R

Luminescence measurement to establish dose-response curves for h beta-MSH variant cAMP signaling on h MC3R, exel

Low_Tyto_Fig6H_hbMSH_variants_hMC3R_DR_exel_190104.xlsx

Figure 6H Graph dose-response curves for h beta-MSH variants on h MC3R

Graph dose-response curves for h beta-MSH variants on h MC3R with EC50 calculations, prism

Low_Tyto_Fig6H_hbMSH_variants_hMC3R_DR_prism_190104.pzfx

Figure 6I Dose-response curves for h beta-MSH variants on h MC4R

Luminescence measurement to establish dose-response curves for h beta-MSH variant cAMP signaling on h MC4R, exel

Low_Tyto_Fig6I_hbMSH_variants_hMC4R_DR_exel_190104.xlsx

Figure 6I Graph dose-response curves for h beta-MSH variants on h MC4R

Graph dose-response curves for h beta-MSH variants on h MC4R with EC50 calculations, prism

Low_Tyto_Fig6I_hbMSH_variants_hMC4R_DR_prism_190104.pzfx

Figure 6J Dose-response curves for h gamma-MSH variants on h MC3R

Luminescence measurement to establish dose-response curves of h gamma-MSH variant cAMP signaling on h MC3R, exel

Low_Tyto_Fig6J_hgMSH_variants_hMC3R_DR_exel_190104.xlsx

Figure 6J Graph dose-response curves for h gamma-MSH variants on h MC3R

Graph dose-response curves for h gamma-MSH variants on h MC3R with EC50 calculations, prism

Low_Tyto_Fig6J_hgMSH_variants_hMC3R_DR_prism_190104.pzfx

Figure 6K Dose-response curves for h gamma-MSH variants on h MC4R

Luminescence measurement to establish dose-response curves of h gamma-MSH variant cAMP signaling on h MC4R, exel

Low_Tyto_Fig6K_hgMSH_variants_hMC4R_DR_exel_190104.xlsx

Figure 6K Graph dose-response curves for h gamma-MSH variants on h MC4R

Graph dose-response curves for h gamma-MSH variants on h MC4R with EC50 calculations, prism

Low_Tyto_Fig6K_hgMSH_variants_hMC4R_DR_prism_190104.pzfx

Figure 6L Plasma degradation of human gamma-MSH variants

Luminescence measurements to establish the residual cAMP signaling activities of human gamma-MSH variants on MC3R after various times of incubation in plasma, exel

Low_Tyto_Fig6L_hgMSH_variants_plasma_degradation_hMC3R_exel_190104.xlsx

Figure 6L Graph plasma degradation of human gamma-MSH variants

Graph plasma degradation curves of human gamma-MSH variants with half-life calculations, prism

Low_Tyto_Fig6L_hgMSH_variants_plasma_degradation_hMC3R_prism_190104.pzfx

Figure 6M Determination of non-MC3R signaling of human gamma-MSH variants and their plasma breakdown products

Luminescence measurements to assess non-MC3R signaling of human gamma-MSH variants and their plasma breakdown products, exel

Low_Tyto_Fig6M_hgMSH_variants_plasma_degradation_noMC3R_exel_190104.xlsx

Figure 6M Graph non-MC3R signaling of human gamma-MSH variants and their plasma breakdown products

Graph non-MC3R signaling of human gamma-MSH variants and their plasma breakdown products established with HEK cells expressing GloSensor reporter plasmid but not MC3R plasmid, prism

Low_Tyto_Fig6M_hgMSH_variants_plasma_degradation_noMC3R_prism_190104.pzfx

Figures S1A S1B Deglycosylation of TA POMC 18S and TA POMC 3S with Endo H and PNGase F

Western blot tiff image files

Figure S2 POMC, PCSK1 and PCSK2 expression in different tissues of Tyto alba

Real time PCR data normalized to 2 reference genes to show the relative expression of POMC, PCSK1 and PCSK2 in different tissues of one individual of Tyto alba alba, exel

Roulin_Tyto_qPCR_Tyto_alba_tissue_samples_POMC_PCSK1_PCSK2_exel_190104.xlsx

Figure S2 Graph POMC, PCSK1 and PCSK2 expression in different tissues of Tyto alba

Graph POMC, PCSK1 and PCSK2 expression in different tissues of Tyto alba, prism

Roulin_Tyto_qPCR_Tyto_alba_tissue_samples_POMC_PCSK1_PCSK2_prism_190104.pzfx

Figure S3A Detection of the expression of myc-tagged PC1 and PC2 in Western blot

Western blot tiff images

Figure S3B pH dependence of Tyto alba PC1 and PC2 activity

Fluorescence measurements to determine the in vitro activity of recombinant Tyto alba PC1 and PC2 in supernatants of transfected cells, exel

Low_Tyto_FigS3B_TAPC1_TAPC2_activity_pH_dependence_exel_190104.xlsx

Figure S3B Graph pH dependence of Tyto alba PC1 and PC2 activity

Graphs pH dependence of Tyto alba PC1 and PC2 activity, prism

Low_Tyto_FigS3B_TAPC1_TAPC2_activity_pH_dependence_prism_190104.pzfx

Figures S3C, S3D Calcium and 7B2 chaperone dependence of Tyto alba PC1 and PC2 activity

Fluorescence measurements to determine the in vitro activity of recombinant Tyto alba PC1 and PC2 in supernatants of transfected cells, exel

Low_Tyto_FigS3C_FigS3D_TAPC1_TAPC2_activity_EDTA_7B2_exel_190104.xlsx

Figures S3C, S3D Graph calcium and 7B2 chaperone dependence of Tyto alba PC1 and PC2 activity

Graph calcium and 7B2 chaperone dependence of Tyto alba PC1 and PC2 activity, prism

Low_Tyto_FigS3C_FigS3D_TAPC1_TAPC2_activity_EDTA_7B2_prism_190104.pzfx

Figure S4B Detection of Strix aluco N-terminal POMC ladder fragments in cell-lysates by Western blot

Western blot tiff images

Figure S4C Detection of Strix aluco N-terminal POMC ladder fragments in cell-supernatants by Western blot

Western blot tiff images

Figure S4D Deglycosylation of Strix aluco N-terminal POMC ladder fragment L3 (pro-gamma-MSH) by PNGase F

Western blot tiff images

Figure S4E Identification of Strix aluco POMC fragments after PC1 and PC2 digest using the Strix aluco POMC ladder

Western blot tiff images

Figure S4F Deglycosylation of Tyto alba N-terminal POMC ladder fragment L3 (pro-gamma-MSH) by PNGase F

Western blot tiff images

Figures S5A, S5B Detection of PC1 and PC2 expression in cell-lysates from experiments in Figure 3B

Western blot tiff images

Figure S6A Detection of POMC, PC1 and PC2 expression in cell-lysates from experiments in Figures 4B-F

Western blot tiff images

Figure S6B Secretion of fully glycosylated Tyto alba and Strix aluco POMC forms for all variants with different numbers of serine residues

Western blot tiff images

Figure S7A Non-MC3R signaling of SA/TA gamma2-MSH, SA gamma3-MSH 3S and TA gamma3-MSH 5S and their plasma degradation products

Luminescence measurements to determine the residual cAMP signaling activity of SA/TA gamma2-MSH, SA gamma3-MSH 3S and TA gamma3-MSH 5S on non-MC3R but GloSensor transfected cells after various times of incubation in plasma, exel

Low_Tyto_FigS7A_SATAg2MSH_SAg3MSH3S_TAg3MSH5S_plasma_degradation_noMC3R_exel_190104.xlsx

Figure S7A Non-MC3R signaling of SA/TA gamma2-MSH, TA gamma3-MSH 13S and TA gamma3-MSH 18S and their plasma degradation products

Luminescence measurements to determine the residual cAMP signaling activity of SA/TA gamma2-MSH, TA gamma3-MSH 13S and TA gamma3-MSH 18S on non-MC3R but GloSensor transfected cells after various times of incubation in plasma, exel

Low_Tyto_FigS7A_SATAg2MSH_TAg3MSH13S_TAg3MSH18S_plasma_degradation_noMC3R_exel_190104.xlsx

Figure S7A Graphs for non-MC3R signaling of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH 18S, and their plasma degradation products

Graphs for non-MC3R signaling of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH 18S, and their plasma degradation products, prism

Low_Tyto_FigS7A_SATAg2MSH_SAg3MSH3S_TAg3MSH5S_TAg3MSH13S_TAg3MSH18S_plasma_degradation_noMC3R_prism_190104.pzfx

Figure S7B Degradation time course of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH18S in CSF

Luminescence measurements to determine the residual cAMP signaling activity of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH18S on MC3R after various times of incubation in CSF, exel

Low_Tyto_FigS7B_SATAg2MSH_SAg3MSH3S_TAg3MSH5S_TAg3MSH13S_TAg3MSH18S_CSF_degradation_TAMC3R_exel_190104.xlsx

Figure S7B Graph degradation time course of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH18S in CSF

Graph degradation time course of SA/TA gamma2-MSH, SA gamma3-MSH 3S, TA gamma3-MSH 5S, TA gamma3-MSH 13S and TA gamma3-MSH18S in CSF, prism

Low_Tyto_FigS7B_SATAg2MSH_SAg3MSH3S_TAg3MSH5S_TAg3MSH13S_TAg3MSH18S_CSF_degradation_TAMC3R_prism_190104.pzfx