Molecular Diversity, Structures And Functions Of Antimicrobial Peptides From Skin Of Rana Nigrovittata

Numerous studies have proved that the amphibian skin is a treasure abundant in bioactive substances. Antimicrobial peptides(AMPs) are an important component of the natural defence of most living organisms, and An impressive number of AMPs have been found in amphibian skin secretions. In this thesis, The molecular diversity, structures and functions of antimicrobial peptides from skin secretions of a ranid frog, Rana nigrovittata was studied.Two novel antimicrobial peptides which belong to two different antimicrobial families were isolated from the skin secretions of Rana nigrovittata by a two-step protocol including Sephadex G-50 and RP-HPLC, and they were named Nigroain-A1, Rugosin-RN1,respectively. As most AMPs from ranid frogs, the two peptides with antimicrobial activity share a conserved disulfide-bridged heptapeptide segment at the C-terminal, and are cationic because of several basic amino acid residues.By molecular cloning, 167 cDNA sequences (Genebank mumbers,EU136401-EU136567)of codeing different antimicrobial peptides precursors were obtained from a single individual skin of Rana nigrovittata. All these 167 cDNA sequences encode 45 novel antimicrobial peptides belonging to 15 divergent families, 21 of which belong to four of the known antimicrobial families previously identified in the skins of other species of Ranid frogs: Rugosin-RN,Gaegurin-6-RN,Temporin-RN and Brevinin-2-RN. The other 24 peptides show little structural similarity towards other known antimicrobial peptides and so are classified into 11 new families: Nigroain-A,-B, -C, -D, -E, -F, -G, -H, -I, -J, -L. The cDNA-encoding precursors of the 15 antimicrobial peptide families code for a single copy of the mature antimicrobial peptide and they share similar N-terminal signal and propiece peptide domain, followed by a markedly different C-terminal domain corresponding to the mature AMPs. All this novel antimicrobial peptides can be roughly placed into one of two groups: Linear peptides and Annular peptides contain a single intramolecular disulfide bond, and the latter is predominant, which is consistent with the known antimicrobial families previously identified in the skins of Ranid frogs. Our results suggest that point mutations, as well as insertion, deletion, and "shuffling" of oligonucleotide sequences can be responsible for the patterns of molecular diversification of antimicrobial peptides from skin of Rana nigrovittata. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides suggests that the corresponding genes form a multigene family originating from a common ancestor,which is one of the reason of molecular diversity of amphibian AMPs.These structural diverse peptides exhibit multiple biological function. They have a broad spectrum of antimicrobial properties, exhibiting against Gram-positive and Gram-negative bacteria including their resistant strains, as well as fungi. Most of this novel peptides can induce And quite a few possess intensive antioxidant activities in terms of free radical scavenging activity and reducing power. we also found a muli-functional antimicrobial peptide with serine protease inhibitor and hemagglutination properties. Moreover, some of them are hemolytic peptides. Especially, some antimicrobial peptide-like peptides which have devoid of antimicrobial activity however can provoke mast cell degranulation or have anti-histamine release activity, as well as antioxidant activity. The individual mutation of amini acid residues has conspicuous influence on the antimicrobial, hemolytic and antioxidant properties.A muli-functional peptide from the skin of Rana nigrovittata, Nigroain-A1,which is abundant in the skin secretions of this ranid frog, only against Gram-positive bacterium {Staphylococcus aureus), but its ability to quickly agglutinate Gram-positive, Gram-negative bacteria and fungi was observed. The minimum agglutinative concentration of rabbit red cells is 15μg/ml. The inhibition constant to trypsin is 5.26×10^-6 M. This multi-functional peptide may provide an effective defense against invading microbes and predators in the amphibian Rana nigrovittata. The precursor structural similarity of Nigroain-A1 with other antimicrobial peptides and the functional similarity of three bioactive compounds which are all direct defensive molecules against microorganisms or pests suggest that these three functional molecules might share a common ancestor. Nigroain-A1,as the multi-functional vector, play an important role in studying gene formation and evolution,structure-function relationship of bioactive substances and novel clinical drugs.