| Antimicrobial peptides (AMPs) are a kind of small molecules revealing wide broad antibacterial, antivirus and antitumor effects and many advantages over traditional antibiotics and have been considered as a source of novel pharmaceutical candidates. However, AMPs are still far away from clinical application because many problems are required to be solved such as screening or design of effective molecules, technics for artificial manufacture, clinical test and application procedure etc. The solution of these problems relies on the discoveries in antibiotic mechanisms, gene structure and in vitro expression, metabolic pattern etc.Northeast brown frog (Rana dybowskii) which is an endemic and artificially breedable species in northeast China is a rich reservoir of antimicrobial peptides. In this study, cDNAs of antimicrobial peptides of Rana dybowskii skin was cloned and sequenced by using RT-PCR technique, and chemical properties of matured peptides of each cDNA was predicted. Highly conserved upper primers were desinged based on the 5' sequences of signal peptide of Rana species from amplifying multiple cDNAs of antimicrobial peptides together with polyT lower primer.Totally 65 different antimicrobial peptide cDNA sequences were obtained from a single individual, 60 of which encode 49 propeptides and 24 mature peptides, indicating a high diversity of antimicrobial peptides in skin of Rana dybowskii. The propeptides are composed of signal peptide, acidic propiece and mature peptide region with a maturation cutting site KR. The signal peptide and acidic peptide are very conservative across all propeptides, while the mature peptides are variable. These peptides could be cataloged into seven families based on amino acids of mature peptides, respectively named chensirin-3, dybowsin-1, dybowsin-2, dybowsin-3, amurin-3, chensirin-1 and chensirin-2. Family chensirin-1, chensirin-2 and chensirin-3 have high homology with AMPs from Rana chensinensis. Family amurin-3 has high homology with amurin-3 from Rana amurensis. The rest three families are novel peptides with a homology below 35% with already reported peptides. Seven AMP families composed of 13 to 37 amino acid residues are rich in Lys, Gly, Leu and some hydrophobic amino acids. Their molecular weight ranges from 1407 to 3732. These AMPs are alkaline and cationic with theoretical isoelectric points among 8.75 to 10.07 and charge scores between +2 and +5 except acidic chensirin-1 family without charge. Family chensirin-3,dybowsin-1,chensirin-1 and chensirin-2 are amphiphilic, while Family dybowsin-2, dybowsin-3 and amurin-3 are solely hydrophobic. Family chensirin-3, dybowsin-1, dybowsin-2 and dybowsin-3 have a featuring Rana box structure. The prediction of secondary structure showed that Family chensirin-2 and dybowsin-2 are extended coils, and the rest families have one or two a-helix where polar amino acids are located on one side of the helix wheel and apolar amino acids on the other side, attaching an amphiphilic property to the whole helix. The analysis of cDNA sequences indicate that genes of AMPs are of multiple gene families, each of which contains multiple gene loci. The acidic propiece region and mature peptide region of cDNAs belonging to a same family may be variable in length, indicating post-transcriptional alternative editing. Therefore, extremely high diversity of antimicrobial peptides cDNAs in an individual frog may be resulted from both gene duplication and post-transcriptional alternative editing.
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