1.C.85 The Pore-Forming Beta-Defensin (β-Defensin) Family

β-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, certain members of the β-defensin superfamily have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. Rohrl et al. (Rohrl et al. 2008) have identified mouse β-defensin 14 (mBD14, Defb14) as an orthologue of human β-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cysteine linkages, characteristic of the β-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Rohrl et al. (Rohrl et al. 2008) also detected mBD14 expression in immature CD11c+ bone marrow-derived dendritic cells. The expression of mBD14 could be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-gamma. Furthermore, expression of mBD14 seemed to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. Both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, based on structural and functional similarities, mBD14 appears to be an orthologue of hBD3 (Rohrl et al., 2008).

β-defensins are important in mammalian immunity, displaying both antimicrobial and chemoattractant activities. These glycopeptides are mainly expressed in epithelial cells and have been shown to form pores in biological membranes (Zanich et al, 2003). Their 3-D structures are known (2NLS_A; Hover et al, 2000, Hover et al, 2001). Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant (Taylor et al., 2008). Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. The chemoattractant activities of HBD3 are restored by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position is inactive.

A range of overlapping linear fragments do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated (Taylor et al., 2008), and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design. 

Shafee et al. 2016 have suggested that defensins and small defensin-like proteins fall into two superfamilies, which they call the cis-defensins (broadly distributed in living organisms) and the trans-defensins (narrowly distrubuted).  They suggest that these two groups of proteins converged to show similar sequences, secondary and tertiary structures, and disulfide connectivities, with overlapping organismal sources and functions, in spite of their independent origins.  The functions of these short proteins vary tremendously including pore formation, bacterial and fungal toxicity, lipid targeting, toxic receptor and channel interactions, fertilization, protease inhibiton and stress adaptation.  However, as noted by the authors, alternative pathways involving divergent evolution from a common evolutionary source could have also occurred although they consider this possibility less likely (Shafee et al. 2017).

The generalized transport reaction catalyzed by β-defensin is:

small molecules (in) small molecules (out)