Establishing a reference array for the CS-αβ superfamily of defensive peptides.

Background: "Invertebrate defensins" belong to the cysteine-stabilized alpha-beta (CS-αβ), also known as the scorpion toxin-like, superfamily. Some other peptides belonging to this superfamily of defensive peptides are indistinguishable from "defensins," but have been assigned other names, making it unclear what, if any, criteria must be met to qualify as an "invertebrate defensin." In addition, there are other groups of defensins in invertebrates and vertebrates that are considered to be evolutionarily unrelated to those in the CS-αβ superfamily. This complicates analyses and discussions of this peptide group. This paper investigates the criteria for classifying a peptide as an invertebrate defensin, suggests a reference cysteine array that may be helpful in discussing peptides in this superfamily, and proposes that the superfamily (rather than the name "defensin") is the appropriate context for studying the evolution of invertebrate defensins with the CS-αβ fold.

Methods: CS-αβ superfamily sequences were identified from previous literature and BLAST searches of public databases. Sequences were retrieved from databases, and the relevant motifs were identified and used to create a conceptual alignment to a ten-cysteine reference array. Amino acid sequences were aligned in MEGA6 with manual adjustments to ensure accurate alignment of cysteines. Phylogenetic analyses were performed in MEGA6 (maximum likelihood) and MrBayes (Bayesian).

Results: Across invertebrate taxa, the term "defensin" is not consistently applied based on number of cysteines, cysteine spacing pattern, spectrum of antimicrobial activity, or phylogenetic relationship. The analyses failed to reveal any criteria that unify "invertebrate defensins" and differentiate them from other defensive peptides in the CS-αβ superfamily. Sequences from various groups within the CS-αβ superfamily of defensive peptides can be described by a ten-cysteine reference array that aligns their defining structural motifs.

Conclusions: The proposed ten-cysteine reference array can be used in addition to current nomenclature to compare sequences in the CS-αβ superfamily and clarify their features relative to one another. This will facilitate analysis and discussion of "invertebrate defensins" in an appropriate evolutionary context, rather than relying on nomenclature.