Maximin S2

Antimicrobial peptides secreted by the skin of many amphibians play an important role in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA sequences, each of which encodes a precursor peptide that can give rise to two kinds of antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating the diversity of maximins and maximin Hs. The gene trees based on different domain regions revealed that domain shuffling or gene conversion among these genes might have happened frequently.

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys16 to Gly16 mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys16 to Gly16 mutant (with MICs>100 microM). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys16 to Gly16 mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 microM, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments.

It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin-H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 1-8, 10, 11, maximin H1, 3-5, 7, 9, 10, 12, 15, 16) from B. maxima skin secretions. Fifty-nine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and -negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains.