Impact of Pro Segments on the Folding and Function of Human Neutrophil α-Defensins

Human neutrophil α-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 α-defensins and/or pro α-defensins, we chemically attached the proHNP1 pro peptide or wtpro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg10 (polyR), Ser10 (polyS), and crpro 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, crpro peptide and wtpro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as α-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 α-defensins fold in vivo.