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.
Table 1.
Peptide fractions with corresponding retention times (min) obtained by preparative RP-HPLC, monoisotopic masses (Da) of the native peptides obtained by HR-MS, and the partial amino acid sequences obtained by Edman degradation analysis.
X denotes unidentified amino acids.
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.
Table 2.
Antimicrobial activities of native EeCentrocin 1, EeStrongylocin 2 and synthesised analogues of EeCentrocin 1 and 2.
LC = light chain, HC = heavy chain, HC-diBr = peptide with two brominated Trp residues.
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).