Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

Abstract

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

Keywords: antimicrobial peptide; bivalve mollusk; mussel; toxin; transcriptome.

Fig. 1.—

Sequence variability of MgCRP-I sequences; variability index W is plotted in the upper panel, whereas the sequence consensus, obtained with Weblogo (http://weblogo.berkeley.edu, last accessed July 24, 2015) is shown in the lower panel. Only positions covered by at least 50% sequences in the global alignment of MgCRP-I peptides are shown. Sites under positive selection are indicated by an asterisk.

Fig. 2.—

Exon/intron structure of the complete coding regions of the MgCRP-I 13, 14, 28, 45 and multi-MgCRP-I 2 genes (A) and corresponding organization of the encoded peptide precursors (B). The positions of the signal peptide, pro-region and mature peptide regions are highlighted, and each cysteine-rich module is marked by a box.

Fig. 3.—

Maximum-likelihood tree obtained with the MgCRP-I peptides based on the alignment of the signal peptide region only. Only bootstraps values greater than 75 are shown. Some sequences were not considered in this analysis as their N-terminal region was incomplete (see table 2). Arrows indicate two MgCRP-I-like peptides with a disrupted cysteine array (MgCRP-I 12 and 23), marking an unconventional mature region.

Fig. 4.—

Codon usage for the Arg residue responsible of the pro-peptide cleavage site and for the six cysteine residues engaged in disulfide bridges, calculated on Mytilus galloprovincialis MgCRP-I peptides. The probabilities of finding the observed codon biases were calculated assuming a binomial distribution and the codon usage (a priori probabilities) inferred from the transcriptome published by Gerdol et al. (2014) (75.1–24.9% for TGT-TGC, encoding Cys, 49.3–16.3–13.3–12.5–4.7–3.8% for AGA-AGG-CGA-CGT-CGG-CGC, encoding Arg). Significant (P < 0.05) and highly significant (P < 0.01) deviations from the expected distributions (P < 0.01) are marked by * and **, respectively. NS, not significant.

Fig. 5.—

Maximum-likelihood tree obtained with the signal peptide of MgCRP-I, the orthologous sequences from other mussel species, and all the CRPs mined from UniProtKB/Swiss-Prot (see Materials and Methods). Peptides from pancrustaceans, conoideans, spiders, scorpions, nematodes, chordates, plants, fungi, viruses, and mussels are shown. Mussel CRP-I peptides are highlighted in a gray background.

Fig. 6.—

Gene expression of 17 selected MgCRP-I genes in six tissues (HE, hemolymph; DG, digestive gland; IM, inner mantle; MR, mantle rim; FO, foot; GI, gills; AM, posterior adductor muscle); primers for MgCRP-I 3 also target MgCRP-I 25, primers for MgCRP-I 10 also target MgCRP-I 26. Bars represent the expression relative to EF-1 alpha; results are mean ± standard deviation of three replicates. MgCRP-I sequences are divided into three panels based on their expression level: (A)—genes with maximum relative expression value comprised between 0.05 and 0.25, (B)—genes with maximum relative expression value comprised between 0.004 and 0.01, (C)—with maximum relative expression value lower than 0.003. (D) A schematic representation of a Mytilus galloprovincialis anatomical features, highlighting the sampled tissues.