cgMolluscidin

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 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 μg/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 μg/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.

A 5.5 kDa antimicrobial peptide consisting of 55 amino acids, cgMolluscidin, was purified from the acidified gill extract of the Pacific oyster, Crassostrea gigas, by ion-exchange and C18 reversed-phase high performance liquid chromatography. By comparing the N-terminal amino acid sequences and the molecular weight of this peptide with those of other known antimicrobial peptides, it has been revealed that this peptide had no homology with any known peptides. cgMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria, including Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus (minimal effective concentrations [MECs]; 1.3-31.3 μg/mL), and Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Vibrio parahaemolyticus ([MECs]; 0.4-2.3 μg/mL), without hemolytic activity. However, cgMolluscidin did not show any significant activity against Candida albicans. The deduced amino acid sequence of the cgMolluscidin showed no hit in public protein databases, while the nucleotide sequence had a 99% homology (E value = 0) with only the unknown ESTs sequenced by C. gigas EST project. Tissue distribution of the cgMolluscidin mRNA suggests that it is constitutively expressed as a mature form in a non-tissue-specific manner. The cgMolluscidin mRNA expression level was significantly up-regulated at 12 h (2.8-fold) post injection with Vibrio sp. This peptide is highly basic and contains several dibasic residue repeats including Lysine-Lysine or Lysine-Arginine in the sequence, but may not form an ordered structure. These results suggest that cgMolluscidin might be an oyster-specific novel antimicrobial peptide.

A 4.7 kDa antimicrobial peptide was purified from the acidified gill extract of the Abalone, Haliotis discus, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). Comparison of the amino acid sequences and molecular weight of this peptide with those of other known antimicrobial peptides revealed that this antimicrobial peptide have high sequence homology with that of cgMolluscidin and was designated hdMolluscidin. hdMolluscidin is composed of 46 amino acid residues containing several dibasic residue repeats like KK or K-R. hdMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis and Staphylococcus aureus (minimal effective concentrations [MECs]; 0.8-19.0 μg/mL) and Gram-negative bacteria including Aeromonas hydrophila, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, and Vibrio parahemolyticus ([MECs]; 1.0-4.0 μg/mL) without hemolytic activity. However, hdMolluscidin did not show any significant activity against Candida albicans. The secondary structural prediction suggested that hdMolluscidin might not form an ordered or an amphipathic structure. hdMolluscidin did not show membrane permeabilization or leakage ability. The full-length hdMolluscidin cDNA contained 566-bp, including a 5'-untranslated region (UTR) of 63-bp, a 3'-UTR of 359-bp, and an open reading frame of 144-bp encoding 47 amino acids (containing Met). cDNA study of hdMolluscidin suggests that it is expressed as a mature peptide. Our results indicate that hdMolluscidin could relate to the innate immune defenses in abalone and it may not act directly on bacterial membrane.