Identifying exoskeleton proteins in the blue crab from an expressed sequence tag (EST) library

Abstract

A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including discovery of genes for cuticular (exoskeletal) proteins, some of which may regulate mineralization. One of the expression libraries sequenced was from the hypodermis (the epithelium depositing the cuticle). RNAs used for cDNA synthesis were pooled from arthrodial and mid-dorsal hypodermis at both pre-ecdysis and post-ecdysis. This ensured representation from both calcifying and non-calcifying regions and from layers of cuticle deposited both before and after ecdysis. The EST database was mined for cuticular protein sequences in three ways. First, we searched for sequences coding for known cuticle-specific motifs like the Rebers-Riddiford chitin-binding sequence and a motif known only from proteins extracted from mineralized exoskeletons of other decapods. Second, we checked the associated annotations in the EST project for similarity to known cuticular proteins, often from insects. Third, BLAST was used to search the EST data for significant homology to published cuticular protein sequences from other crustaceans. In all, the database contains at least 73 contigs or singlets representing transcripts of cuticular proteins. Forty-five of these distribute among ten clusters of very similar transcripts, possibly representing alternative splicing or recent gene duplications. The rest share less similarity. We have obtained complete sequences for 25 of the transcripts, have produced phylogenetics trees comparing them with similar proteins from insects and other crustaceans, and have determined expression patterns across the molt in calcifying versus non-calcifying cuticle. The combination of homology analysis and gene expression analysis allows us to infer putative functions in cuticle synthesis and calcification.