Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus

Abstract

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Figure 1. Phenylseptin peptides present on H. punctatus skin secretion.

Chromatographic profile of H. punctatus crude extract and Phenylseptin isolation. (A) The crude extract was eluted with 0.1% TFA (Solvent A) and 95% acetonitrile containing 0.1% TFA (Solvent B), under a linear gradient of solvent B for 120 minutes in a semi-preparative C18 column. The identified Phenylseptin peptides ‘a’ and ‘b’ (B and D, respectively) were further purified by reverse-phase polystyrene/divinyl benzene chromatography using a 5 RPC ST 4.6 mm/150 mm column under optimized gradient of acetonitrile at a flow rate of 1.0 mL·min-1. After these two separation steps, final purification of the peptides ‘a’ and ‘b’ (C and E, respectively) was obtained using Ultra Fast Liquid Chromatography using a Shimpack-XR-ODS column under a linear gradient of acetonitrile at a flow rate of 0.4 mL·min-1. In all steps of purification the absorbance was measured at 216 nm, and when necessary at 254 or 280 nm.

Figure 2. UFLC analysis and molecular mass determination of Phenylseptin mixture.

(A) The accurate molecular masses and purity of Phes peptides were determined by MALDI-TOF/MS and the observed molecular mass was 1954.2 Da for both molecules. (B) Analytical chromatographic profile of natural (black line) and synthetic L-Phes (red dash line) and D-Phes (blue dash line). The two peptides were mixed in similar molar concentrations and load into an Ultra Fast Liquid Chromatography using a Shimpack-XR-ODS column under a linear gradient of acetonitrile at a flow rate of 0.4 mL·min-1. The two distinct fractions eluted around 11 and 12 minutes corresponded to L-Phes and D-Phes, respectively.

Figure 3. One single gene encoding Phes peptide.

(A) Nucleotide sequences of clone encoding precursor of selected Phenylseptin peptides. The putative signal peptide (in box), acidic spacer (dash underline) and mature peptide (bold underline), C-terminal codon for Glycine (blue underline) and stop codon (asterisk) are indicated. The nucleotide sequences were deposited in the NCBI Nucleotide Sequence Database under HQ012497 annotated accession code. (B) Predicted amino acid sequence alignment of Phenylseptin with previously sequenced Hylidae peptides aurein, ranateurin, brevinin and gaegurin. Sequence alignments were done using CLUSTAL W software and were edited with the BIOEDIT software.

Figure 4. Structural studies on L- and D- Phenylseptin peptide isomers by Ion Mobility Mass Spectrometry (IM-MS) and Nuclear Magnetic Resonance (NMR).

(A) L-Phes and D-Phes were individually analyzed showing that each one (M+3H⁺ = 652.04 m/z) can assume at least two major conformations with distinct amounts of each type. L-Phes conformations at 10.45 and 12.89 ms and D-Phes conformations at 10.80 and 12.72 ms. D-Phes has its major conformation at 10.80 ms. Experiments were performed on a Synapt HDMS instrument (Quadrupole Ion Mobility High-Definition mass spectrometry – Waters Co. MA, USA) equipped with nano-electrospray ionization. All spectra were acquired with a direct infusion of 1 µL·min-1 of in a range m/z 300 up to 2000. Precursor charge state: 3. Tolerance: 0.1 Da. (B) and (C) The 20 lowest-energy structures for both peptides. The hydrophobic residues are represented in gold yellow, the hydrophilic residues in green. (D) The lowest-energy Phenylseptins showing Phenylalanine enatiomerization, the aromatic phenylalanine are in dark red and (E) The alignment of lowest-energy L- and D-Phes structure in the presence of 60% TFE viewed along the helix axis and from the side.

Figure 5. Phenylseptin Phe-Phe-Phe motif.

Phenylseptin N-terminal sequence was compared to other peptides previously described on the literature as being bitter. Sequence alignments were edited with the BIOEDIT software. On the right side a bitterness scale based on Kim, et al., 2006 analysis.

Figure 6. Trpm5 dependent bitter substances.

Short-term (10 min) (A) caffeine vs. water, (B) theophylline and (C) theobromine two-bottle preference tests. Trpm5 knockout (“KO”) mice displayed indifference to all choices. (D) The bitterness of Phenylseptin peptides is dependent on Trpm5 regulation. Short-term (10 min) (E) L-Phes vs. water, (F) D-Phes vs. water and (3) L-Phes.1 vs. water two-bottle preference tests. Trpm5 knockout (“KO”) mice displayed indifference to all choices. Values displayed as preference ratios for water. (G) Amino acid organization on peptide structure is fundamental to gustatory perception. Short-term (10 min) L-Phes mixture vs. water and D-Phes mixture vs. water. Wild-type and Trpm5 knockout (“KO”) mice were equally indifferent to all choices. Values displayed as preference ratios for water. Values displayed as preference ratios for water. Post-hoc (Bonferroni-corrected) one sample t-test against indifference ratio of 0.5: *p<0.05; **p<0.04; all other comparisons p>0.05. Red line denotes level of indifference (preference ratio of 0.5).