Structural and Functional Consequences Induced by Post-Translational Modifications in α-Defensins

Abstract

HNP-1 is an antimicrobial peptide that undergoes proteolytic cleavage to become a mature peptide. This process represents the mechanism commonly used by the cells to obtain a fully active antimicrobial peptide. In addition, it has been recently described that HNP-1 is recognized as substrate by the arginine-specific ADP-ribosyltransferase-1. Arginine-specific mono-ADP-ribosylation is an enzyme-catalyzed post-translational modification in which NAD(+) serves as donor of the ADP-ribose moiety, which is transferred to the guanidino group of arginines in target proteins. While the arginine carries one positive charge, the ADP-ribose is negatively charged at the phosphate moieties at physiological pH. Therefore, the attachment of one or more ADP-ribose units results in a marked change of cationicity. ADP-ribosylation of HNP-1 drastically reduces its cytotoxic and antibacterial activities. While the chemotactic activity of HNP-1 remains unaltered, its ability to induce interleukin-8 production is enhanced. The arginine 14 of HNP-1 modified by the ADP-ribose is in some cases processed into ornithine, perhaps representing a different modality in the regulation of HNP-1 activities.

Figure 1

Schematic representation of the enzyme-catalyzed reversible mono-ADP-ribosylation reaction. The diagram shows the enzymatic transfer of the ADP-ribose portion from NAD to the guanidine group of an arginine target. Nicotinamide, the remaining portion of NAD, is simultaneously released. The native arginine is restored by the reaction catalyzed by an ADP-ribosylarginine hydrolase that cleaves the α-glycosidic bond with the release of ADP-ribose.

Figure 2

Density electrostatic surface map of HNP-1 peptide. Positive charges conferred by arginine residues are shown in blue; the negative charge conferred by glutamic acid residue is in red. The secondary structure of the peptide was taken from PDB database (3GNY) and the relative density electrostatic surface map was obtained using PyMOLWin software.

Figure 3

Schematic structure of the dimeric form of HNP-1 interacting with the cell membrane. Arg residues that confer a positive net charge to HNP-1 (green) and that are involved to membrane interaction are highlighted in cyan and blue. Negatively charged groups of the cell membrane such as phosphatidyl glycerol or cardiolipin head groups are highlighted in red. Arg14 and Arg24 is the primary and secondary site of ADP-ribosylation, respectively. The schematic representation was made using Visual Molecular Dynamics (VMD) software; HNP-1 secondary structure was taken from PDB database (3GNY) while cell membrane model was created with VMD tool.

Figure 4

Schematic diagram of the HNP-1 ADP-ribosylation. The picture shows that ART1 catalyzes the transfer of an ADP-ribose unit from NAD to HNP-1 on Arg14 with simultaneous release of nicotinamide (NAM) in the extracellular space. ART1 is anchored via a GPI tail to the outer leaflet of the cell membrane.

Figure 5

Different mechanisms of processing and cleavage of ADP-ribosylated proteins. The complete removal of the ADP-ribose, catalyzed by an ADP-ribosylarginine hydrolase is indicated in red. The picture in blue frame shows the phosphodiesterase-catalyzed cleavage of the ADP-ribosylated-protein, giving a phosphoribosylated protein. The picture in the green frame represents the nonenzymatic processing of the ADP-ribosylated protein to the ornithinylated form as described for HNP-1.