A novel horse alpha-defensin: gene transcription, recombinant expression and characterization of the structure and function

Abstract

Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: theta-, beta-, and alpha-defensins. Synthesis of alpha-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine alpha-defensin DEFA (defensin alpha) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known alpha-defensins from primates and glires displayed a homology with Paneth-cell-specific alpha-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analysed structurally by CD and molecular modelling. To examine the antimicrobial properties, a radial diffusion assay was performed with 12 different micro-organisms and the LD90 (lethal dose killing > or =90% of target organism) and MBC (minimal bactericidal concentration) values were examined. DEFA1 showed an antimicrobial activity against different Gram-positive and Gram-negative bacteria and against the yeast Candida albicans. Using viable bacteria in combination with a membrane-impermeable fluorescent dye, as well as depolarization of liposomes as a minimalistic system, it became evident that membrane permeabilization is at least an essential part of the peptide's mode of action.

Figure 1. Transcriptional analysis of DEFA1 in various equine tissues

DEFA1 is solely transcribed in the small intestine (lane 9, lower panel). The product size is approx. 340 bp in length. β-Actin was used as a positive control (upper panel), water was used instead of cDNA template as a negative control (lane 15).

Figure 2. DEFA1 nucleotide sequence and the amino acid sequence and alignment of the DEFA1 primary structure with those of other α-defensins

(A) The DEFA1 cDNA sequence is depicted in lower case letters, with the amino acid sequence in upper case letters. The sequence (nucleotides and amino acids) of the mature peptide is depicted in bold, and the start codon is underlined. (B) Comparison of the DEFA1 amino acid sequence with the amino acid sequences of equine [ECA (Equus caballus)] DEFA5L, human [HSA (Homo sapiens)] α-defensins DEFA5, DEFA6 and HNP-3, chimpanzee [PTR (Pan troglodytes)] DEFA5, rat [RNO (Rattus norwegicus)] DEFA5, NP-3 and defensin 6, and rhesus macaque [MMU (Macaca mulatta)] α-defensin 5. The boxes indicate highly conserved residues among the primary translation products of equine defensin and the other α-defensin genes. The connectivity of the cysteines typically present in α-defensins is indicated by lines. The length of the peptides is denoted at the C-terminus.

Figure 3. Recombinant expression of DEFA1

(A) SDS/PAGE of the cell pellet before induction (lane 2) and after induction (lane 3) and the supernatant from unstimulated bacterial cells (lane 4) and stimulated bacterial cells (lane 5). Lane 1, molecular-mass markers. The His₆-tagged fusion peptide was only detected in the pellet of stimulated cells, and has a molecular mass of approx. 10 kDa. SN, supernatant. (B) Time-dependent analysis of peptide expression after induction in the supernatant (upper panel) and the pellet (lower panel) fractions after 0.5 h (lane 2), 1 h (lane 3), 1.5 h (lane 4), 2 h (lane 5), 2.5 h (lane 6), 3 h (lane 7), 3.5 h (lane 8) and 4 h (lane 9). The molecular-mass markers are shown in lane 1. (C) Digestion of the recombinant peptide with Factor Xa. Lane 1, molecular-mass markers; lane 2, the uncleaved peptide with a mass of approx. 10 kDa; lane 3, cleaved product. The mature peptide reveals a molecular mass of approx. 5 kDa. SN, supernatant.

Figure 4. RP-HPLC purification of DEFA1 and MALDI–TOF MS of the eluted fractions

(A) The chromatogram on the left-hand side shows three conspicuous peaks appearing at elution times of 13.4, 14.0 and 15.7 min. Eluted fractions were termed F1 to F3 and analysed by MS. An RP-HPLC analysis of the undigested peptide is shown on the right-hand side, the eluted fraction was termed F0. (B) MALDI–TOF MS of the fractions F0–F3. Fraction F0 shows a mass peak of 9046.1 Da for the uncleaved product. Fraction F1 shows an unknown peptide mixture with peptide masses of approx. 4090, 5003 and 5035 Da. Fractions F2 and F3 contain peptides of the calculated average mass of cleaved DEFA1 (4071.8 Da), whereas in fraction F2, the His₆-tag (4987.5 Da) was eluted in addition to the mature peptide. In fraction F3, a homogenous peptide without any contaminants was detected.

Figure 5. CD spectrum of DEFA1

The spectrum was recorded between 190 nm and 250 nm in 0.01% (v/v) TFA, with a minimum at 203 nm.

Figure 6. Antimicrobial activities of DEFA1 in the radial diffusion assay

Peptide concentration (x-axis) plotted against the diameter of the microbial growth inhibition zone (y-axis) after incubation for 12 h. (A) Growth inhibition of various E. coli strains. (B) Growth inhibition of Staph. aureus, Staph. epidermidis, P. aeruginosa, Bac. megaterium, and C. albicans. The arrangement of measuring points are a result of adjusted peptide concentrations of 4.7, 9.3, 18.7, and 37.3 μM.

Figure 7. Membrane permeabilization of Bac. megaterium induced by DEFA1

Membrane damage of the bacteria was measured fluorometrically using the SYTOX Green dye. The binding of the dye to the DNA in membrane-compromised target cells resulted in an increase in fluorescence. Antibacterial activity of the peptides is expressed as a percentage of permeabilized bacteria. (A) Time kinetics of membrane permeabilization induced by DEFA1 measured for different concentrations of peptide at various incubation periods. (B) Comparison of the membrane-permeabilizing effects of DEFA1 (◆,◇) and cecropin P1 (■,□) after incubation of Bac. megaterium for 30 min with each peptide at various concentrations and either at pH 5.2 (◆,■) or at pH 7.4 (◇,□).

Figure 8. Time course of pore formation induced by DEFA1

The dissipation of a valinomycin-induced diffusion potential in vesicles of soy bean phospholipids after addition of DEFA1 (1.4 nmol; arrow) (1), the control peptide alamethicin (0.1 nmol) (2) and the peptide solvent (3) was recorded. Pore-forming activity was reflected by the increase in fluorescence as a function of time.

Figure 9. Three-dimensional model of DEFA1

β-Sheet structures are displayed as arrows, the N-terminus is marked with N, the C-terminus with C. Disulfide bonds are depicted as ball-and-stick representations.