Toward Understanding the Cationicity of Defensins

Abstract

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in ␣-defensins but not in ␤-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human ␣-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six ␣-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ( diMe Lys), and homo-Arg ( homo Arg). In addition, we prepared human ␤-defensin 1 (hBD1) and Lys3 Arg hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and Lys3 Arg hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the ␣-defensin than in the ␤-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.

Trp-26 Imparts Functional Versatility to Human α-Defensin HNP1

“…Shown in Fig. 1 are survival curves of S. aureus exposed to each defensin at concentrations varying from 0.195 to 50 M. For wild type HNP1, complete killing of S. aureus, a reduction in the number of colonies by at least 6 orders of magnitude, was achieved at 25 M. Replacement of Arg residues (Arg-5, Arg-14, Arg-15, or Arg-24) resulted in attenuated bactericidal activity, affirming the functional importance of cationicity in HNP1-mediated bacterial killing (29,30). Conversely, substituting Ala for Glu-13, the only anionic residue of HNP1, enhanced the killing of S. aureus.…”

“…Hydrophobicity Rather Than Cationicity Dictates HNP1 Strain Selectivity in Bacterial Killing-Many earlier mutational studies also aimed to probe abundant but less conserved Arg residues in ␣-defensins (29,30). Although the importance of cationicity is self-evident in defensin-mediated bacterial killing, cationicity alone does not explain defensin strain selectivity.…”

“…Prior mutational studies of ␣-defensins focused primarily on conserved elements such as disulfide bonding (23,24), an invariant Gly residue (25), a conserved salt bridge (26 -28), and the often abundant but less conserved Arg residues (29,30). However, the loss of many of those conserved structural elements in ␣-defensins is often functionally inconsequential in vitro.…”

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

“…The mature domain is in bold typeface, and the maturation site is denoted by an arrow. The boxed residues, Arg 49 and Glu 57 in proHD5 or Arg 6 and Glu 14 in HD5, form a salt bridge in the structure and are highly conserved in all known mammalian ␣-defensins.…”

“…Detailed biochemical, functional, and structural characterizations of these synthetic defensin/pro defensin molecules help to establish that the conserved Arg 6 -Glu 14 (HD5 numbering) salt bridge is critical not only for correct processing of proHD5 by trypsin but also for proteolytic stability of mature HD5 in the presence of its processing enzyme. 49 and Glu 57 in proHD5. Preparation of correctly folded synthetic HD5 used for this work was essentially as described previously (31).…”