This is a competing renewal application to continue studies to identify endogenous antimicrobial peptides from chicken and porcine leukocytes.
Three specific aims are described. 1) Preparative and analytical protein chemistry methods will be used to purify the non-gallinacin antimicrobial peptides of chicken leukocytes and to ascertain the molecular characteristics of these peptides including primary protein sequence and unique structural characteristics (e.g., circular dichroism, disulfide pairing, etc.). Several highly sensitive assays will be applied to measure antibiotic activity against bacterial and fungal pathogens. In particular, extracts of chicken peritoneal exudate cells will be separated by chromatography. Fractions exhibiting antimicrobial activity will be further purified using preparative continuous acid urea gel electrophoresis and/or reversed-phase HPLC. Peptides of interest will be microsequenced. In some instances it may be necessary to derive the entire sequence from peptides produced from the holopeptide by limited proteolysis. 2) Another aspect of this collaboration will be to analyze the structure of prophenin-l, an unusually proline-rich peptide, to examine its post-translational processing and determine the contributions of its reiterated decamer motif to the peptide's tight binding of LPS and to its selective antimicrobial activity. To accomplish this specific aim, well-characterized synthetic peptide fragments will be tested for their ability to protect against LPS-mediated endotoxin shock in mice. 3) Finally, molecular biologic techniques will be used to clone and sequence the genes of avian antimicrobial peptides. Many of the prophenin-related peptides that have been discovered in pigs and cows exhibit highly conserved sequences within the propeptide region. Thus, screening for genes that exhibit homology to the propeptide region may allow the discovery of similar antimicrobial peptides in chickens. Total RNA from chicken bone marrow cells will be used to construct a cDNA library using standard techniques. The library will be screened with synthetic oligonucleotide """"""""guessmers"""""""" based on reverse translation of the least degenerate part of the N-terminal sequence of each precursor protein. Hybridizing cDNA clones will then be subcloned, sequenced, and used to screen a commercially obtained chicken genomic library. The cloning of peptide cDNAs and genes will also permit future studies of the regulation of antimicrobial peptide synthesis by microbial substances and cytokines.
