Calculated with the residue masses and pKa set used on this site; defensin masses include −2.016 Da per disulfide.
The peptide arm of innate immunity
Vertebrate immunity is usually described in terms of antibodies and lymphocytes, which take days to respond to a new pathogen. Acting long before them is the innate system, and a large part of its chemical armoury consists of small antimicrobial peptides made constantly by epithelial cells and stored in the granules of white blood cells.
Humans produce two main groups: the defensins, a large family of disulfide-rich β-sheet peptides, and a single cathelicidin, whose active peptide is called LL-37. Together they cover the skin, the airways, the gut and the surface of the eye, and they are released in large amounts when neutrophils encounter bacteria. The general principles of how such peptides work are covered in the guide to antimicrobial peptides; this page looks at the human ones specifically.
Defensins
Defensins are 18 to 45 residues long, cationic, and stabilised by three disulfide bonds. Their fold is a small, twisted β-sheet. Human defensins fall into two classes, distinguished by the spacing and pairing of their cysteines:
| Class | Disulfide pairing | Examples | Made by |
|---|---|---|---|
| α-Defensins | C1–C6, C2–C4, C3–C5 | HNP-1 to HNP-4; HD-5, HD-6 | Neutrophils; Paneth cells of the small intestine |
| β-Defensins | C1–C5, C2–C4, C3–C6 | hBD-1 to hBD-4 and many others | Epithelial cells of skin, airway, urogenital tract |
| θ-Defensins | Three disulfides in a cyclic backbone | Rhesus θ-defensin (RTD-1) | Old World monkeys; the human gene is inactive |
The two pairings are a good illustration of why connectivity matters. Both classes have six cysteines, so there are 15 possible ways to pair them, as calculated in the guide to disulfide bonds. α- and β-defensins use two different ones, and the result is two related but distinct folds from otherwise similar sequences.
θ-Defensins are the only known cyclic peptides made by animals: two nine-residue fragments are spliced head-to-tail into an 18-residue ring, then closed further by three disulfides. Humans carry the gene but a premature stop codon prevents its expression. Synthetic θ-defensins, sometimes called retrocyclins, have been studied as antimicrobials; the chemistry of such rings is covered in the guide to cyclic peptides.
Defensins are made as inactive precursors with an acidic pro-region that neutralises their positive charge, protecting the cell that makes them. The pro-region is removed as the peptide is packaged or released, a strategy similar to the one melittin's precursor uses, described in the melittin profile.
LL-37
Humans have exactly one cathelicidin gene, CAMP. Its product, hCAP18, consists of a conserved N-terminal cathelin domain and a variable C-terminal peptide. When the protein is released, the protease proteinase 3 cuts off that C-terminal peptide, which begins with two leucines and has 37 residues: hence the name LL-37.
Unlike the defensins, LL-37 contains no cysteines and forms no disulfide bonds. It is unstructured in dilute solution and folds into an amphipathic α-helix on contact with membranes or at higher ionic strength, the conditional folding described in the guide to peptide secondary structure. With six lysines and five arginines against six acidic residues, it carries a net charge of about +6 at physiological pH.
LL-37 appears throughout the site's guides as a calculated example: its isotope pattern in the molecular weight guide, where at 4.5 kDa its monoisotopic peak is no longer the tallest, its charge curve in the isoelectric point guide, and its unusually high hydrophobic moment in the antimicrobial peptides guide, where a strongly negative GRAVY coexists with strong membrane activity.
Expression of the cathelicidin gene is regulated in an unusual way: its promoter contains a vitamin D response element, so the active form of vitamin D increases LL-37 production in several cell types. This link between a vitamin and an antimicrobial peptide has been much studied since its discovery in 2004.
Why salt matters
Both defensins and LL-37 depend on electrostatic attraction to negatively charged bacterial surfaces. Increasing the salt concentration screens those charges and reduces activity, sometimes dramatically. This has practical and biological consequences.
In the laboratory, antimicrobial assays run in low-salt buffer can overstate potency compared with physiological conditions, so results are only comparable when the ionic strength is stated. Biologically, the salt sensitivity of human β-defensin 1 and LL-37 was central to an influential hypothesis about cystic fibrosis: that a higher salt concentration in airway surface liquid inactivates these peptides and contributes to chronic infection. The hypothesis has been much debated and other mechanisms are now emphasised, but it shaped a generation of research on airway defence.
More than antibiotics
Direct killing of bacteria is only part of what these peptides do, and possibly not the main part at the concentrations found in tissue. Both families act as signalling molecules:
- Chemotaxis. Defensins and LL-37 attract neutrophils, monocytes and T cells to sites of infection through specific receptors.
- Neutralising endotoxin. LL-37 binds bacterial lipopolysaccharide and can dampen the inflammatory response it would otherwise trigger.
- Wound healing. LL-37 promotes migration of epithelial cells and formation of new blood vessels.
- Antiviral activity. Several defensins interfere with enveloped viruses, including by binding viral glycoproteins.
Because of this breadth, many researchers prefer the term host defence peptides to "antimicrobial peptides" for the mammalian members of these families.
Working with them
Defensins are demanding to prepare. Three disulfides in a short chain mean 15 possible connectivities, and oxidative folding does not always favour the native one, so directed disulfide formation with orthogonal cysteine protection is often needed, as described in the guide to solid-phase synthesis. Confirming the correct pairing requires proteolytic mapping under acidic conditions that prevent disulfide scrambling.
LL-37 is simpler to synthesise, having no cysteines, but it is long and prone to aggregation, and its strong positive charge makes it stick to glass and plastic surfaces at low concentrations. Low-binding tubes and a carrier are advisable, as discussed in the stability guide.
Calculated properties
| Peptide | Length | Disulfides | Average MW | Calculated pI | Charge, pH 7.4 | GRAVY |
|---|---|---|---|---|---|---|
| LL-37 | 37 | 0 | 4493.29 | 11.1 | +5.8 | −0.72 |
| HNP-1 (α-defensin) | 30 | 3 | 3442.05 | 10.4 | +2.8 | +0.30 |
| hBD-2 (β-defensin) | 41 | 3 | 4328.19 | 11.1 | +5.8 | −0.10 |
Masses are for the native peptides, with 2.016 Da subtracted for each disulfide. All three are strongly basic, as expected. Note that the charge calculation treats cysteines as free thiols; in the defensins they are all paired, which removes six weakly acidic groups and makes the real pI slightly higher than the calculated value.
Frequently asked questions
Why does the name LL-37 refer to its sequence?
Because the mature peptide begins with two leucine residues and is 37 residues long. It is a descriptive name given when the cleavage product was characterised.
Do humans have θ-defensins?
The gene is present but carries a premature stop codon, so no peptide is made. Old World monkeys produce them. Synthetic versions have been studied in the laboratory.
Why are defensins made as precursors?
The acidic pro-region neutralises the peptide's positive charge, which prevents it from damaging the membranes of the cell that produces it. It is removed during packaging or release.
Can these peptides be used as antibiotics?
Development has proved difficult. They are degraded by proteases, lose activity at physiological salt concentrations, and can be toxic to host cells at the concentrations needed systemically. Most clinical work has focused on topical use and on engineered analogues, as described in the antimicrobial peptides guide.
References
- Ganz T, Selsted ME, Szklarek D, et al. (1985) Defensins. Natural peptide antibiotics of human neutrophils. Journal of Clinical Investigation 76:1427–1435.
- Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. Journal of Leukocyte Biology 75:39–48.
- Tang YQ, Yuan J, Ösapay G, et al. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated α-defensins. Science 286:498–502.
- Wang TT, Nestel FP, Bourdeau V, et al. (2004) Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. Journal of Immunology 173:2909–2912.
- Hancock REW, Sahl H-G (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature Biotechnology 24:1551–1557.