title: Impact of pro segments on the folding and function of human neutrophil alpha-defensins.
abstract: Human neutrophil alpha-defensins (HNPs) are synthesized in vivo as inactive precursor proteins, i.e. preproHNPs. A series of sequential proteolytic events excise the N-terminal inhibitory pro peptide, leading to defensin maturation and storage in azurophilic granules. The anionic pro peptide, required for correct sub-cellular trafficking and sorting of proHNPs, inhibits the antimicrobial activity of cationic defensins, either inter or intra-molecularly, presumably through charge neutralization. To better understand the role of the pro peptide in the folding and functioning of alpha-defensins and/or pro alpha-defensins, we chemically attached the proHNP1 pro peptide or (wt)pro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg(10) (polyR), Ser(10) (polyS), and (cr)pro peptide, a charge-reversing mutant of the pro peptide where Arg/Lys residues were changed to Asp, and Asp/Glu residues to Lys. Comparative in vitro folding suggested that while all artificial pro segments chaperoned defensin folding, with PEG being the most efficient, the pro peptide catalyzed the folding of proHNPs likely through two independent mechanisms: solubilization of and interaction with the C-terminal defensin domain. Further, the N-terminal artificial pro segments dramatically altered the bactericidal activity of HNP1 against both Escherichia coli and Staphylococcus aureus. Surprisingly, (cr)pro peptide and (wt)pro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as alpha-defensin binding, although their binding modes appeared different. Our findings identify a dual chaperone activity of the pro peptide and may shed light on the molecular mechanisms by which pro alpha-defensins fold in vivo.
authors.affiliation: Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Wu
authors.forename: Xiangqun
authors.initials: X
authors.lastname: Li
authors.forename: Bryan
authors.initials: B
authors.lastname: Ericksen
authors.forename: Erik
authors.initials: E
authors.lastname: de Leeuw
authors.forename: Guozhang
authors.initials: G
authors.lastname: Zou
authors.forename: Pengyun
authors.initials: P
authors.lastname: Zeng
authors.forename: Cao
authors.initials: C
authors.lastname: Xie
authors.forename: Chong
authors.initials: C
authors.lastname: Li
authors.forename: Jacek
authors.initials: J
authors.lastname: Lubkowski
authors.forename: Wei-Yue
authors.initials: WY
authors.lastname: Lu
authors.forename: Wuyuan
authors.initials: W
authors.lastname: Lu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D008567:Membranes, Artificial; D050296:Microbial Viability / Q000187:drug effects; D018832:Molecular Chaperones / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000187:drug effects; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013997:Time Factors; D023084:alpha-Defensins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D008567:Membranes, Artificial; D018832:Molecular Chaperones; D050505:Mutant Proteins; D010455:Peptides; D011498:Protein Precursors; D023084:alpha-Defensins; D012694:Serine