Synthesis and characterization of human alpha-defensins 4-6.

Human alpha-defensins are small, Cys-rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high-quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human alpha-defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N-diisopropylethylamine (DIEA) in situ neutralization/2-(1 H-benzotriazolyl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid-phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10-16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony-counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.

From pro defensins to defensins: synthesis and characterization of human neutrophil pro alpha-defensin-1 and its mature domain.

Human neutrophil alpha-defensins (HNPs) are small, cationic, Cys-rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre-proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro-peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45-residue pro-peptide and the C-terminal functional domain. Here we described, total chemical synthesis of the 75-residue human neutrophil pro alpha-defensin-1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met45-Ala46 peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys1-Cys6, Cys2-Cys4 and Cys3-Cys5, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro-peptide binds to HNP1 intermolecularly with an apparent Kd value of 6.2 microM at pH 7.4, confirming the mode of intramolecular inactivation of human alpha-defensin precursors.