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Cited by 108Diabetes Treatment and ManagementPancreatic function and diabetesChemical Synthesis and Analysis

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Peptide lipidation stabilizes structure to enhance biological function

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Ward, Brian P., Ottaway, Nickki L., Perez-Tilve, Diego, Ma, Dejian, Gelfanov, Vasily M., Tschöp, Matthias H., DiMarchi, Richard D.·2013·Molecular Metabolism
DOI 10.1016/j.molmet.2013.08.008PMC PMC3854995
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Abstract

Medicines that decrease body weight and restore nutrient tolerance could improve human diabetes and obesity treatment outcomes. We developed lipid-acylated glucagon analogs that are co-agonists for the glucagon and glucagon-like peptide 1 receptors, and stimulate weight loss and plasma glucose lowering in pre-diabetic obese mice. Our studies identified lipid acylation (lipidation) can increase and balance in vitro potencies of select glucagon analogs for the two aforementioned receptors in a lipidation site-dependent manner. A general capacity for lipidation to enhance the secondary structure of glucagon analogs was recognized, and the energetics of this effect quantified. The molecular structure of a lipid-acylated glucagon analog in water was also characterized. These results support that lipidation can modify biological activity through thermodynamically-favorable intramolecular interactions which stabilize structure. This establishes use of lipidation to achieve specific pharmacology and implicates similar endogenous post-translational modifications as physiological tools capable of refining biological action in means previously underappreciated.

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Figures

Image 1
CC-BY-NC-SA · From the PDF
Figure 1 In vitro characterization of lipidated Aib2,16a analogs. (A) GCGR activation by lipidated Aib2,16a analogs in engineered HEK293 cells ( N ¼ 2, SD shown). Data are fit to a doseresponse function to provide the EC50 values. N , number of independent data points. SD, standard deviation. (B) The GLP-1R response measured using the same assay format and lipidated peptides as panel (A).
Figure 1 In vitro characterization of lipidated Aib2,16a analogs. (A) GCGR activation by lipidated Aib2,16a analogs in engineered HEK293 cells ( N ¼ 2, SD shown). Data are fit to a doseresponse function to provide the EC50 values. N , number of independent data points. SD, standard deviation. (B) The GLP-1R response measured using the same assay format and lipidated peptides as panel (A).CC-BY-NC-SA · From the PDF
Image 3
CC-BY-NC-SA · From the PDF
Figure 3 CD spectra and thermal denaturation of lipidated Aib2,16a analogs. (A) CD spectra ( N ¼ 3) of lipidated Aib2,16a analogs (10 m M) in water (pH 7.4) at 298 K. [], mean residue ellipticity. (B) CD spectra of Lys13-Glu-Glu-C16 ( 12 ) (10 m M) compared to Aib2,16a ( 2 ) (10 m M) in water (pH 7.4) or 90% TFE/10% 0.01 N NaOH (pH 11) at 298 K. (C) Reversible thermal denaturation of Aib2,16a and lipidated Aib2,16a analogs in water (pH 7.4) ( N ¼ 2 from consecutive experiments using the same sample, SD shown). Data are fit to a two-state folding equilibrium model where for the lipidated peptides the fit was performed as two individual transitions separated at 323 K (denoted by an * on the temperature axis) in each instance [13]. Thermodynamic parameters (Table S3) were obtained from the fit and used to calculate the helical free energy at 310 K ( G (310 K)), which was determined to be 2.8 kcal/mol, 1.9 kcal/mol, 2.5 kcal/mol, 3.5 kcal/mol and 3.8 kcal/mol for peptides 2 , 20 , 21 , 12 and 8 , respectively. These values were used to calculate G Helix relative to 2 in Table 1B. (D) A second view of the data in panel (C) showing only peptides 20 and 21 .
Figure 3 CD spectra and thermal denaturation of lipidated Aib2,16a analogs. (A) CD spectra ( N ¼ 3) of lipidated Aib2,16a analogs (10 m M) in water (pH 7.4) at 298 K. [], mean residue ellipticity. (B) CD spectra of Lys13-Glu-Glu-C16 ( 12 ) (10 m M) compared to Aib2,16a ( 2 ) (10 m M) in water (pH 7.4) or 90% TFE/10% 0.01 N NaOH (pH 11) at 298 K. (C) Reversible thermal denaturation of Aib2,16a and lipidated Aib2,16a analogs in water (pH 7.4) ( N ¼ 2 from consecutive experiments using the same sample, SD shown). Data are fit to a two-state folding equilibrium model where for the lipidated peptides the fit was performed as two individual transitions separated at 323 K (denoted by an * on the temperature axis) in each instance [13]. Thermodynamic parameters (Table S3) were obtained from the fit and used to calculate the helical free energy at 310 K ( G (310 K)), which was determined to be 2.8 kcal/mol, 1.9 kcal/mol, 2.5 kcal/mol, 3.5 kcal/mol and 3.8 kcal/mol for peptides 2 , 20 , 21 , 12 and 8 , respectively. These values were used to calculate G Helix relative to 2 in Table 1B. (D) A second view of the data in panel (C) showing only peptides 20 and 21 .CC-BY-NC-SA · From the PDF
Figure 4 1 H NMR-based structural model of Aib2,16a Lys13-Glu-Glu-C16 ( 12 ) in water. (A) Bar graph displaying the number of interresidue NOE connectivities per amino acid residue identified through the water NMR experiments. (B) One of the 10 lowest energy conformers from a 100 structure calculation in xplor-NIH [17] using the NOESY chemical shift resonance assignments as structural distance restraints is shown with the NOE interactions between C16 (H C4 and H C16 , respectively) and residues Gln3 (H N ) or Phe6 (H and H) indicated by dotted lines. *, N-terminus. (C) Structure calculation parameters. Twelve additional hydrogen bond restraints were used for the calculation corresponding to the six helical NOEs assigned (Figure S3A) [17,18]. (D) Overlay of the 10 lowest energy conformers from the calculation. A single conformer is shown in cartoon form and the other nine as ribbons.
Figure 4 1 H NMR-based structural model of Aib2,16a Lys13-Glu-Glu-C16 ( 12 ) in water. (A) Bar graph displaying the number of interresidue NOE connectivities per amino acid residue identified through the water NMR experiments. (B) One of the 10 lowest energy conformers from a 100 structure calculation in xplor-NIH [17] using the NOESY chemical shift resonance assignments as structural distance restraints is shown with the NOE interactions between C16 (H C4 and H C16 , respectively) and residues Gln3 (H N ) or Phe6 (H and H) indicated by dotted lines. *, N-terminus. (C) Structure calculation parameters. Twelve additional hydrogen bond restraints were used for the calculation corresponding to the six helical NOEs assigned (Figure S3A) [17,18]. (D) Overlay of the 10 lowest energy conformers from the calculation. A single conformer is shown in cartoon form and the other nine as ribbons.CC-BY-NC-SA · From the PDF
Figure 5 1 H NMR-based structural model of Aib2,16a Lys13-Glu-Glu-C16 ( 12 ) in d -TFE. (A) Bar graph displaying the number of interresidue NOE connectivities per amino acid residue identified through the d -TFE NMR experiments. (B) One of the 10 lowest energy conformers from a 100 structure calculation in xplor-NIH [17] using the NOESY chemical shift resonance assignments as structural distance restraints is shown with the NOE interactions between C16 and residues Leu14 (H C14 -H), Arg17 (H C15 -H) or Arg18 (H C15 -H) indicated by dotted lines. *N-terminus. (C) Structure calculation parameters. (D) Overlay of the 10 lowest energy conformers from the calculation. A single conformer is shown in cartoon form and the other nine as ribbons.
Figure 5 1 H NMR-based structural model of Aib2,16a Lys13-Glu-Glu-C16 ( 12 ) in d -TFE. (A) Bar graph displaying the number of interresidue NOE connectivities per amino acid residue identified through the d -TFE NMR experiments. (B) One of the 10 lowest energy conformers from a 100 structure calculation in xplor-NIH [17] using the NOESY chemical shift resonance assignments as structural distance restraints is shown with the NOE interactions between C16 and residues Leu14 (H C14 -H), Arg17 (H C15 -H) or Arg18 (H C15 -H) indicated by dotted lines. *N-terminus. (C) Structure calculation parameters. (D) Overlay of the 10 lowest energy conformers from the calculation. A single conformer is shown in cartoon form and the other nine as ribbons.CC-BY-NC-SA · From the PDF
Table 1: Summary of lipidated glucagon-based peptides. (A) Important peptide sequences for this study. The differences of Aib2,16a and GLP-1 with the glucagon sequence are underlined. The N- and C-terminal hydrophobic helical faces in all three peptides consist of residues 6, 10 and 13, and residues 22, 23, 25 and 26, respectively [23]. X, aminoisobutyric acid (Aib). (B) Master table for peptides used in this study. EC50 values were obtained from experiments in Figure 1A and B for peptides 1 , 2 , 3 , 8 , 15 , 20 and 21 . For peptides 4 , 5 , 6 , 10 , 11 , 12 , 13 , 14 , 16 , 17 and 19 , the EC50 values were obtained from Figures S5 and S6, and normalized against standards from Figure 1A and B. As reference, liraglutide ( 9 ) has in vitro potency equal to GLP-1 ( 3 ) at the GLP-1R [2]. G GLP-1R was calculated using Gibbs equation [14]. G Helix was calculated using thermodynamic parameters (Table S3) obtained from the thermal denaturation experiments in Figure 3C. The aqueous and 90% TFE helical content relative to 2 was determined by dividing the 222 nm CD signal for each indicated peptide by the equivalent signal for Aib2,16a ( 2 ) (Figures 4A and S7A-C). PBS solubility was measured by ultraviolet absorption spectroscopy. Self-aggregation tendency was determined by dynamic light scattering experiments in 0.01 N sodium hydroxide (Figure S7D). Peptides were classified as strong if they aggregated immediately after sample preparation, moderate if aggregated after a single 24-h freeze cycle, weak if aggregated after 1 month of freezing or no apparent if they had no detected aggregation throughout these trials. No. peptide number. [], mean residue ellipticity. Calc. pI, calculated isoelectric point. (C) Chemical structure of a Lys-Glu-Glu-C16 side chain at neutral pH.
Table 1: Summary of lipidated glucagon-based peptides. (A) Important peptide sequences for this study. The differences of Aib2,16a and GLP-1 with the glucagon sequence are underlined. The N- and C-terminal hydrophobic helical faces in all three peptides consist of residues 6, 10 and 13, and residues 22, 23, 25 and 26, respectively [23]. X, aminoisobutyric acid (Aib). (B) Master table for peptides used in this study. EC50 values were obtained from experiments in Figure 1A and B for peptides 1 , 2 , 3 , 8 , 15 , 20 and 21 . For peptides 4 , 5 , 6 , 10 , 11 , 12 , 13 , 14 , 16 , 17 and 19 , the EC50 values were obtained from Figures S5 and S6, and normalized against standards from Figure 1A and B. As reference, liraglutide ( 9 ) has in vitro potency equal to GLP-1 ( 3 ) at the GLP-1R [2]. G GLP-1R was calculated using Gibbs equation [14]. G Helix was calculated using thermodynamic parameters (Table S3) obtained from the thermal denaturation experiments in Figure 3C. The aqueous and 90% TFE helical content relative to 2 was determined by dividing the 222 nm CD signal for each indicated peptide by the equivalent signal for Aib2,16a ( 2 ) (Figures 4A and S7A-C). PBS solubility was measured by ultraviolet absorption spectroscopy. Self-aggregation tendency was determined by dynamic light scattering experiments in 0.01 N sodium hydroxide (Figure S7D). Peptides were classified as strong if they aggregated immediately after sample preparation, moderate if aggregated after a single 24-h freeze cycle, weak if aggregated after 1 month of freezing or no apparent if they had no detected aggregation throughout these trials. No. peptide number. [], mean residue ellipticity. Calc. pI, calculated isoelectric point. (C) Chemical structure of a Lys-Glu-Glu-C16 side chain at neutral pH.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Ward, B. P., Ottaway, N. L., Perez-Tilve, D., Ma, D., Gelfanov, V. M., Tschöp, M. H., & DiMarchi, R. D.
Affiliations
  • Indiana University Bloomington (US)
  • University of Cincinnati (US)
  • Helmholtz Munich (DE)
Corresponding author
Ward, Brian P.
ORCID
Venue
Molecular Metabolism
Publisher
Elsevier BV
Published
2013-11
Volume
2
Issue
4
Pages
468-479
ISSN
2212-8778 (ISSN-L)
DOI
10.1016/j.molmet.2013.08.008
PMC
PMC3854995
OpenAlex
W2047944726
Cited by
101
References
43
Citations by year
2026: 6 · 2025: 5 · 2024: 7 · 2023: 5 · 2022: 12 · 2021: 10 · 2020: 9 · 2019: 10 · 2018: 10 · 2017: 9 · 2016: 8 · 2015: 6 · 2014: 4
FWCI
4.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Metabolism (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Site cant be reached (submittedVersion, cc-by-nc-nd)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diabetes Treatment and Management
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Pancreatic function and diabetes
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
glucagon receptor, GLP-1 receptor, weight loss, blood glucose reduction, obesity, diabetes mellitus, prediabetes, intramolecular interactions, post-translational modification, structure-activity relationship

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