Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) fromMytilus 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.

Myticalins: A Novel Multigenic Family of Linear, Cationic Antimicrobial Peptides from Marine Mussels (Mytilus spp.)

“…These results suggest that considerable interindividual variability might occur in the inventory of myticalin genes across mussels, to the point that each individual could present a unique gene set, similarly to what has previously been reported for other AMPs in Mytilus spp. [ 5 , 39 ]. However, it remains to be established whether this variation is the result of extreme allelic variability linked to high heterozygosity [ 37 ], widespread genetic introgression within the M. edulis species complex [ 40 ] or whether it is linked to genome-wide phenomena of presence/absence variation, which have been previously described in oyster big defensins [ 41 ].…”

“…The analysis of mussel transcriptome data suggested that, in contrast with most AMPs described so far in mussels [ 5 , 6 , 7 ], myticalins are preferentially expressed in the gills. Indeed, a number of myticalin transcripts could be de novo assembled to their full length using gill-derived RNA-seq.…”

“…Primers were designed to assess the expression level of myticalins in the main tissues of adult unchallenged mussel specimens (hemolymph, digestive gland, posterior abductor muscle, inner mantle, mantle edge, gills and foot). Total RNA, extracted from 30 individuals of roughly the same size (5–7 cm) all sampled in the Gulf of Trieste (Italy), was quantified, pooled and reverse transcribed into cDNA like previously described [ 5 ].…”

“…Gene expression levels, representing the mean plus standard deviation of three technical replicates, were calculated using the Δ C t method and normalized across tissues based on the housekeeping gene elongation factor 1 alpha, as described elsewhere [ 5 , 87 ].…”

“…These AMPs are all expressed at high levels in hemocytes, circulating cells that are considered to be major players in molluscan immunity, and are all characterized by multiple disulfide bridges which ensure a precisely folded, stable structure for the mature peptides that are compact, cationic and amphipathic in nature. Although more recent reports have provided evidence for the presence of AMPs in other mussel tissues [ 5 , 6 , 7 ], a finding in line with the emerging role of mucosal immunity in bivalves [ 8 , 9 ], all currently known mussel AMPs can be placed within the cysteine-rich category based on the conventional structural classification of AMPs [ 10 ].…”