Review
Defensins of vertebrate animals

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Abstract

During the past year, novel β-defensins of mice and men have been identified, together with a novel defensin subfamily (the circular or ‘θ’ minidefensins) in primates. Insight into the evolution of defensins has been obtained from structural studies, and several mechanisms related to microbial resistance to defensins have been delineated. There is now convincing evidence that defensins augment adaptive immune responses.

Introduction

The presence of small ‘cationic antimicrobial proteins’ in rabbit and guinea pig granulocytes was first reported in the mid-1960s, 1., 2., but almost 20 years passed before these peptides (α-defensins) were sequenced and shown to be principal constituents of these granulocytes and human neutrophils. α-Defensins are small peptides with 29–35 residues and a ‘hallmark’ six-cysteine motif, whose Cys1–Cys6, Cys2–Cys4 and Cys3–Cys5 pairing forms three intramolecular disulfide bonds. Of the six known human α-defensins, four (human neutrophil peptide [HNP]-1–4) are expressed primarily by granulocytes and certain lymphocytes. The other two, HD-5 and -6, are expressed principally by intestinal Paneth cells [3••].

β-Defensins were discovered in bovine granulocytes and tracheal epithelial cells about 10 years ago. They differ from α-defensins by having up to ∼45 residues; a different cysteine pairing (Cys1–Cys5, Cys2–Cys4, Cys3–Cys6) and spacing; a smaller, less anionic propiece; genes with two, instead of three, exons; and relatively more lysines than arginines. But their almost identical shapes and the juxtaposition of their genes on chromosome 8p22–p23 indicates a common ancestry for α- and β-defensins. The unusual circular (θ) minidefensins, which were identified recently in rhesus monkeys [4], are discussed below.

In addition to the α-, β-, and θ- defensins of vertebrates, several families of cysteine-rich antimicrobial peptides found in invertebrates and plants are called defensins. Interested readers can learn about these peptides from recent reviews 5., 6..

Section snippets

β-Defensins take center stage

Human β-defensin (HBD)-2, a 41-residue peptide, was discovered in scales shed from the skin of psoriasis patients. It is also produced by other epithelial cells and has potent activity against Gram-negative bacteria and Candida, but not against Gram-positive Staphylococcus aureus. Its synthesis by epithelial cells can be induced by microorganisms and by cytokines (e.g. TNF-α and IL-1β) [7].

In solution, the structure of HBD-2 is a triple-stranded β sheet, wherein strands 2 and 3 form a β hairpin

Circular defensins

Although a handful of circular peptides are known in plants [24], RTD-1 —the circular minidefensin described by Selsted and co-workers [4]—is the first such peptide to be identified in an animal. In an elegant study, these workers showed that RTD-1 has two different mRNA precursors, each coded by a mutated α-defensin gene that has acquired a stop codon between its third and fourth cysteine codons. The translated gene products are trimmed and their ends spliced together, as shown schematically

Defensin processing

All α-defensins undergo post-translational proteolytic processing from precursors (preprodefensins) that contain a signal sequence, an anionic propiece with about 45 amino acids, and a mature, carboxy-terminal defensin domain. The processing of α-defensins was first studied in human HL-60 cells, which over a duration of 4–24 hours generated the mature, 29–30-amino-acid HNP-1 and -2 defensin peptides from their 94-amino-acid precursor. Proteolysis proceeded via two main intermediates: a

Defensin expression

HBD-1 is expressed by mammary gland epithelia [30], and is present in human breast milk in concentrations of roughly 1–10μg/ml [31]. Mammary expression of β-defensin peptide(s) may afford protection against mastitis, or may contribute to neonatal host defenses either directly or by priming the adaptive immune system.

In vitro, primary human bronchial epithelial cells cultured without immunostimulants expressed HBD-1 mRNA; however, HBD-2 and -3 were expressed only after stimulation with

Immunostimulatory activities of defensins

The induction of β-defensin synthesis in epithelial cells and the recruitment of α-defensin-rich granulocytes from the blood leads to high local concentrations of defensins within hours of infection. In addition to exerting direct antimicrobial effects, defensins seem to facilitate and amplify subsequent adaptive immune responses. Murine spleen cells stimulated by human α-defensins show increased in vitro proliferation and cytokine production, and defensin administration to mice in vivo leads

Bacteria fight back

Like most other antimicrobial peptides, defensins are cationic (positively charged) molecules that engage negatively charged microbial surface components via electrostatic interactions. Their ‘docking sites’ on bacterial surfaces can include the lipid A moiety of LPS, (lipo)teichoic acids, and anionic membrane phospholipids, such as phosphatidyl glycerol and cardiolipin. Three ways that bacteria increase the odds of surviving a close encounter with defensins (and other antimicrobial peptides)

Defensins and chemokines

Although generally larger (8–10 kDa) than defensins, many chemokines resemble defensins by having clusters of positively charged amino acids and a tertiary structure stabilized by disulfide bonds. Because defensins have chemokine-like chemotactic activity, it seemed pertinent to ask whether chemokines are, in turn, antimicrobial. In a study of 11 representative members of the various chemokine families [44], only the three IFN-γ-inducible chemokines (IP-10/CXCL10, I-TAC/CXCL11 and MIG/CXCL9)

Renal defensins

Rabbit RK-1 is an unusual α-defensin that lacks the many arginines and high net positive charge characteristic of other peptides in this subfamily, Nevertheless, it resembles other α-defensins in its three-dimensional solution structure (which includes turns and a triple-stranded antiparallel β sheet) [46]. Not only was RK-1 active against E. coli, it also activated Ca2+ channels [46]. Two murine Paneth-cell α-defensins were shown early on to stimulate chloride secretion from polarized

Evolution of defensins

Defensins have been around for quite a long time. Their primary structures—aside from the conserved cysteines and a few other residues that also have structural roles— are highly varied. The following papers illuminate the evolutionary history of mammalian defensins and provide insights into the wide-ranging functions of peptides with a β-defensin motif.

Mammals descended from reptiles (therapsids), which diverged from other reptilia as long as 220 million years ago. The modern mammals include

Conclusions

The increased exploration of defensins and other antimicrobial peptides has been propelled by the powerful methods now available to examine peptide expression, structure and function. It also reflects increasing interest in innate immunity, and the desire to identify the corresponding effector mechanisms for the large number of newly discovered pattern-recognition receptors and their transduction pathways.

Evidence that antimicrobial peptides have important roles in antimicrobial defense of

References and recommended reading

Papers of particular interest, published within the annual period of review,have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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