Purification, Characterization, Molecular Cloning, Structures And Functions Of Antimicrobial Peptides From Skin Secretions Of Fejervarya Cancrivora

Amphibian skin glands are rich resources for bioactive substances of complex functions to protect host resist the invasion of harmful environmental factors. Up to date, many biologically active proteins and peptides have been purified and identified from the skin of frog. The crab-eating frog, Fejervarya cancrivora, is the unique species of amphibian that tolerates saline waters, so its physiological regulatory mechanism and biological active substances may be different from the freshwater frog. However, there are no bioactive peptides or proteins from sea-frog reported. In order to investigate the innate immune system of amphibians inhabiting brackish water and freshwater, the molecular diversity, structures and functions of antimicrobial peptides from skin secretions of Fejervarya cancrivora (from mangrove swamps in Hainan Province of China) was studied.Three antimicrobial peptides with the amino acid sequence of GSAQPYKQLHKVVNWDPYG, RVCSAIPLPICH and RVCMAIPLPLCH were isolated from skin secretions of the crab-eating frog through Sephadex G-50 gel filtration chromatography and RP-HPLC purification, which is the first report on antimicrobial peptide from sea amphibians. One of the peptides named cancrin is composed of 19 amino acid residues without cysteine and rich in alkaline and heterocyclic amino acids with positive charge. By BLAST search, cancrin had no significant similarity to any known peptides, so it is a novel family of antimicrobial peptides. Two other antimicrobial peptides, tigerinin-RC1 and-RC2 share significant structural similarity with tigerinins found in the skin of Indian frog, Rana tigerina(Synonym:Hoplobatrachus tigerinus). These two novel 12-residue antimicrobial peptides differ with each other only in one or two amino acid with the tigerinin-2 antimicrobial peptide which was only found in Rana tigerina. These peptides with net positive charges, which we have named as tigerinin-RC1 and-RC2 respectively, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring.From the skin cDNA library of F. cancrivora,22 positive clones were screened and sequenced by the special primers of antimicrobial peptide from a single individual skin cDNA library of F.cancrivora. Among those,17 different cDNA sequences of encoding different antimicrobial peptides precursors were obtained, and 4 different mature peptides due to synonymous mutation. These peptides belonging to 2 different families of cancrin and tigerinin-RC are named as tigerinin-RC1 and -RC2, cancrin Al and A2, respectively. The current work was also the first report of precursor and cDNA cloning of the tigerinin antimicrobial peptide family.Cancrin precursors are composed of 68 amino acid residues, and Tigerinin-RC precursors are composed of 71 amino acid residues. The overall structures of the precursors are quite similar to other amphibian antimicrobial peptide precursors, comprising a signal peptide sequence, an N-terminal spacer peptide region containing several aspartic and glutamic acid residues, and the mature antimicrobial peptide with net positive charges at the C-terminus of the precursor. In addition, they share similar di-basic enzymatic processing site (-KR-) between spacer peptides and mature peptides. Maybe because of the evolusion choice pressure by environment, the mature peptides of those two families are very conservative, which are different with other antimicrobial peptide families of Rana genus. Such phenomena suggested these two antimicrobial peptide families play an important role in the innate defense of brackish water-living amphibian.Cancrin, tigerinin-RC 1 and-RC 2 exhibited antimicrobial activity against the tested strains. Among the tested strains, E. coli (ATCC25922) was the one not sensitive to cancrin, but the antibiotic activity of cancrin was proved to be lethal for the sensitive strains like Staphylococcus aureus, Bacillus subtilis and Candida albicans. Among all the peptides used, cancrin was the most effective one to S. aureus. Tigerinin-RC1 and -RC2 also showed strongest antimicrobial abilities against Pseudomonas aeruginosa beside the above strains. Cancrin couldn't induce mast cell degranulation and histamine release and had little hemolytic activity on red blood cells. They also had no trypsin/chromotrypsin inhibitory activity and hemagglutination properties. On the other hand, they can scavenge ROS with antioxidant properties which maybe have a role in inhibiting viral infection in vivo.The current results confirmed that both amphibians inhabiting freshwater and brackish water share the same antimicrobial peptide family of tigerinin. The crab-eating frog also has evolved a novel antimicrobial peptide family of cancrin with particular intensive defense mechanism to resist the halophilic and psychrophilic marine organisms which can forbear with high pressure and low nutrition. According to the similarity of amphibian antimicrobial peptide precursors sequences among cancrin, tigerinin-RC and the other Rana frog, those antimicrobial peptide may be processed from a unique family of precursor polypeptides which produce various peptides and functions during long-term natural selection. The researches of mechanism of peptide formation and evolusion and the relationships between biological activities and conformation of antimicrobial peptide are very important for pharmacologists to discover a number of novel candidates of anti-inflammatory agents.