Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): Use in assessing gene expression during the moult cycle

Abstract

Trypsin is the most abundant protease in Crustacea. This enzyme was purified from the digestive gland of Penaeus vannamei, revealing three major isoforms (molecular weights 31–32 kDa) and several minor components. Five cDNAs encoding five isoforms of trypsin were detected by two successive screenings of an amplified cDNA library from the digestive gland of P. vannamei. The longest isolated and sequenced cDNA encoded a preproenzyme of 255 amino acids containing a putative precursor peptide of 14 residues and a highly hydrophobic signal sequence of 14 amino acids. Amino acid sequence alignments revealed a high degree of identity between the trypsin from P. vannamei and that from crayfish (74%) and an equal level of sequence similarity to that from mammals and insects (≈40). Dot blot hybridization and subsequent analysis of the variation in trypsin-specific activities revealed that mRNA expression is at a maximum during early premoult (D 1), declining sharply in late premoult (D 2–D 3). The specific activity of trypsin also followed this pattern, suggesting the regulation of trypsin biosynthesis is, at least in part, transcriptional. The characterization of trypsin cDNA from P. vannamei provides the first description of a putative zymogen sequence in a crustacean species, enabling us to elucidate the regulatory mechanism of trypsin synthesis in these important marine organisms.