Purification and cDNA cloning of the antimicrobial peptide apMolluscidin from the pen shell, Atrina pectinata

摘要

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C-18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys-Lys and Lys-Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 mu g/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 mu g/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full-length apMolluscidin cDNA contained 812 base pairs (bp), including a 5'-untranslated region (UTR) of 82 bp, a 3'-UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue-specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence.