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.
Wu
Z
Z
Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA.
Prahl
A
A
Powell
R
R
Ericksen
B
B
Lubkowski
J
J
Lu
W
W
D000595:Amino Acid Sequence; D023082:Defensins / Q000096:biosynthesis; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D023084:alpha-Defensins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
D023082:Defensins; D004220:Disulfides; D010446:Peptide Fragments; D011498:Protein Precursors; D023084:alpha-Defensins; C406413:human neutrophil peptide 1