Short macrocyclic peptides in sponge genomes

Abstract

Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.

Keywords: RIPPs; circular peptide; cyclic peptide; metazoan biosynthesis; sponges.

Fig. 1.

Structures of marine sponge derived PRMPs and two common enzymatic mechanisms of peptide macrocyclization. NRPS: nonribosomal peptide synthetase. TE: thioesterase domain responsible for macrocyclizing some nonribosomal peptides. RiPPs: ribosomally synthesized and post-translationally modified peptides. RSII and RSIII: recognition sequences that are used by protease such as PagA and PagG in bacteria to N-C cyclize short peptides.

Fig. 2.

Sequence logos of (A) identified signal peptides, (B) recognition sequences, and (C) core sequences from PRMPS precursor peptides. The core sequences in the box for S. carteri are identical to those of known sponge PRMPs, the core sequences in the box for A. corrugata are experimentally identified cyclic peptides by MS² sequencing, which was examplified by the identification of cyclic (VYPYKPP). Starting from each of the three different dipeptide fragments, ^NPro-Val^C (1, MS2 m/z 197), ^NPro-Pro^C (2, MS2 m/z 195), and ^NPro-Tyr^C (3, MS2 m/z 261), the same cyclic sequence, cyclo(VYPYKPP), was recovered.

Fig. 3.

Relative transcriptional abundance of the top 13 transcribed cores and their correlation with the corresponding counts detected in LC-MS analysis. Each dot represents a core peptide sequence (SI Appendix, Table S2).

Fig. 4.

Architecture of multicore-containing RiPP precursor peptides. (A) General architecture of sponge PRMP precursor peptides. (B) For comparison, representative multicore-containing RiPPs from fungal (Aspergillus), bacterial (Prochloron), and plant (Oldenlandia) kingdoms, and a neuropeptide from animals.

Fig. 5.

PRMP biosynthetic genes in the A. corrugata genome. (A) PRMP precursor distribution in A. corrugata contigs. The red ORFs represent PRMP precursor genes, gray ORFs represent other genes. (B) Diagram of PRMP precursor peptide gene architecture in A. corrugata. Repetitive sequences are also observed in introns (SI Appendix, Fig. S3). (C) The core peptides found in the intact precursor peptides from genome assembly. Each different color represents a single precursor peptide. The numbers on the x-axis indicate the counts for each core peptide in the corresponding precursor peptide.

Fig. 6.

3D plot of bins for A. corrugata metagenome. Each point represents a contig in the metagenome. They are plotted on the two dimensions that result from dimension-reduction by BH-tSNE, versus the GC content of the contig in the third dimension. The two circled bins are comprised of bacterial contigs, while the light green dots represent contigs from the sponge genome. The sponge contigs containing PRMP precursor genes are shown in red.