Solstad RG et al. (2016), Novel Antimicrobial Peptides EeCentrocins 1, 2 ...

ECB-ART-44590

PLoS One 2016 Mar 04;113:e0151820. doi: 10.1371/journal.pone.0151820.

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Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications.

Solstad RG , Li C , Isaksson J , Johansen J , Svenson J , Stensvåg K , Haug T .

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The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring.

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Genes referenced: LOC100887844

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	Fig 1. The Edible sea urchin, E. esculentus.Image credit: Runar Gjerp Solstad.
	Fig 2. RP-HPLC chromatogram showing the separation of AMPs in the 40% SPE fraction of E. esculentus coelomocytes.The peptides were separated using a preparative C18 column using a flow rate of 8 ml/min, and an optimised HPLC gradient protocol of 0.05% TFA/ACN in 0.05% TFA/H2O for 60 min. One-minute fractions were collected and tested for antibacterial activity. Antibacterial fractions are marked with a black line under the chromatogram and peak fractions selected for further analysis are marked with arrows.
	Fig 3. Multiple sequence alignment of Ee4835 and Ee5024 with homologues in S. droebachiensis and S. purpuratus.In the aligned sequences, grey indicates identical amino acids. The predicted signal peptides, propeptides and mature peptides are marked with curly brackets. Gaps are inserted to maximise similarity. In the top row, accession numbers are given in parentheses, the mature peptide sequences are presented in the bottom rows.
	Fig 4. Multiple sequence alignments of Ee5922 with homologues in S. droebachiensis and S. purpuratus.In the aligned sequences, grey indicates identical amino acids. The predicted signal peptides, propeptides and mature peptides are marked with curly brackets. Gaps are inserted to maximise similarity. In the top row accession numbers are given in parentheses, the mature peptide sequences are presented in the bottom rows.
	Fig 5. Evolutionary relationships of A) centrocins and B) strongylocins identified in E. esculentus, S. droebachiensis and S. purpuratus. The evolutionary history was inferred using the Neighbour-joining method [39] and the optimal trees are shown. The percentage of replicate trees in which the proteins clustered together during the bootstrap test (500 replicates) is given next to the nodes [55]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [40]. Accession numbers are given in parentheses.
	Fig 6. Characterisation of the post-translational modifications of EeCentrocin 1 HC.High-resolution mass spectra showing the m/z isotope cluster corresponding to the [M+H]+ ions of A) Main peptide fragment from a tryptic digest of native EeCentrocin 1, B) Synthesised fragment GWBrWBrR, and C) Calculated isotope distribution of [GWBrWBrR+H]+. The identical and distinctive distributions (A, B and C) of the singly charged isotopes indicate the presence of two Br-Trp residues in EeCentrocin 1.
	Fig 7. Carbon chemical shifts of Br-Trp in native peptides correlated with synthetic 5-Br-Trp and 6-Br-Trp.The chemical shifts are less well correlated for the 5-Br reference compound (left, 5.36 ppm average error) than those of the 6-Br reference compound (right, 1.55 ppm average error). Each data point in the figure represents a carbon of the indole of Trp.
	Fig 8. Structures of peptides discovered in E. esculentus.The two top structures are the heterodimeric EeCentrocins 1 and 2. The disulphide bonds connecting the HC and LC are indicated with “\|”. The lower sequence is the primary structure of EeStrongylocin 2. All Trp substitutions are assigned to the 6 position of the indole.
	Fig 9. Haemolytic activity of synthetic analogues of EeCentrocin 1 and 2.The different synthetic peptide analogues of EeCentrocin 1 and 2 display minor haemolytic activity in concentrations up to 12.5 μM. EeCentrocin 2 HC displays the highest haemolytic activity (56.4% haemolysis at 100 μM).

References [+] :

Beauregard, The detection and isolation of a novel antimicrobial peptide from the echinoderm, Cucumaria frondosa. 2001, Pubmed, Echinobase