Barrettides: A Peptide Family Specifically Produced by the Deep-Sea Sponge Geodia barretti

Abstract

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC₅₀ 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Figure 1

Identification of barrettides across different G. barretti samples. Full black circles: barrettide sequence found in respective sequencing data, open gray circles: barrettide m/z found in metabolic profile of the same sample. Dashed gray circles: m/z isobaric with barrettide B. The corresponding amino acid sequences are shown to the right, and amino acid differences to barrettide A are highlighted with gray shading. Gb07 did not yield any hits due to the quality filtering process removing most of the assembled transcripts.

Figure 2

Left: Selected ion mass chromatograms for the m/z 1080 [M + 3H]³⁺ of barrettide C. The synthetic peptide (yellow) has the same retention time (t_R) as the m/z observed in the native chemical profile (turquoise). Likewise, the native chemical extract spiked with synthetic barrettide C (red) only shows one peak at the same t_R. Right: Isotopic patterns show a signal increase in the spiked native extract both in absolute terms and relative to the intensities of barrettide A ([M + 3H]³⁺ 1071) and B ([M + 3H]³⁺ 1076).

Figure 3

Solution NMR structure of barrettide C and comparison to barrettide A. (A) Superimposed structural ensemble of barrettide C in stick format, with disulfide bonds in yellow. (B) Superimposition of barrettide C (dark blue β-sheet, light blue backbone, and purple α-helix) and barrettide A (gray β-sheet and backbone) in ribbon format with ball-and-stick disulfide bonds in yellow. This superimposition highlights the conserved β-hairpin structure of the barrettides. (C) Surface representation of barrettide C. Colors are used to denote residue side chain properties: negatively charged (red), positively charged (blue), hydrophobic (green), disulfide (yellow), and polar (white). Individual amino acids are also labeled for orientation.

Figure 4

(Left) Larval settlement after 3 days of incubation with barrettide C. No mortality was observed. The experiments were performed in triplicate with n = 20 (±2) larvae per assay. Statistical evaluation was performed using a χ² test comparing treatments to control. (Right) Dose–response curve of barnacle larval settlement after 3 days of incubation with barrettide C. The IC₅₀ is indicated as a dashed line.