Identification Of Cathelicidins, Structural And Functional Characterization Of CATH_BRALE, The Defense Molecule In The Ancient Salmonoid, Brachymystax Lenok

Brachymystax lenok, animal of second class protection, is thick-lipped lenok belonging to salmonidae Brachymystax, and mainly live in mountain streams with low temperature water pure water.It’s worth noting that there is only one in China, mainly distributed in northeast and qinling area of our country. Cathelicidins, as the host defense genes,are a family of multi-functional antimicrobial peptides exclusively found in mammals,and had been found almost all kinds of vertebrates.They possess broad spectrum antimicrobial activity, not only against Gram-positive bacteria, Gram-negtive bacteria, fungi and viruses, but also many antibiotic-resisted clinical bacteria. In addition, they possess many other biological activities, such as immune cell chemotaxis, wound repair angiogenesis,transcriptional regulation of macrophage, etc.In current work, on the basis of molecular biology techniques,we extracted a variety of main tissue, constructed spleen cDNA library, make use of semi-nested to identify a new the salmonoid cathelicidin:CATH_BRALE. It is a kind of strong alkaline polypeptide containing199amino acids among them15alkaline amino acid residues, and the pI is12.43, the molecular weight is5.2006kDa,the static charges is+13.The mature peptide contains53amino acids, the sequence is:RRSKARGGSRGSKMGRKDSKGGSRGRPGSGSRPGGGSS I AGASRGDRGGTRN A. Sequence alignment with other representative salmonid cathelicidins displayed distinctive feature of current lenok cathelicidin, the six-amino-acid tandem repeated sequence of RPGGGS detected in a variable number of copies among fish cathelicidins, suggesting the existence of a genetically unstable region similar to that found in some mammalian cathelicidins. Expression of CATH_BRALE is predominantly found in gill, with lower levels in the gastrointestinal tract and spleen. The homology modeled structure of CATH_BRALE exhibits structural features of antiparallel (3-sheets flanked by a-helices that are representative of small cationic cathelicidin family peptides. Phylogenetic analysis indicated that the bootstrap is99%between CATH_BRALE and CATH-2which comes from Atlantic salmon. CATH_BRALE possesses much stronger antimicrobial activity against gram-negative bacteria than that of the human ortholog, LL-37. The growth of two typical fish bacterial pathogens, gram-negative bacterium of Aeromonas salmonicida and Aeromonas hydrophila was substantially inhibited by synthetic CATH_BRALE, with both MICs as low as9.38μM. What is more, CATH_BRALE showed very modest hemolytic activity, lysing only6.8%of erythrocytes.