Effects of a single nucleotide polymorphism in the chicken NK-lysin gene on antimicrobial activity and cytotoxicity of cancer cells

Abstract

NK-lysin is an effector protein of the innate immune system and an important component of host protection. We isolated a SNP in the NK-lysin coding sequence among different chicken breeds. This A to G substitution at the position 271 nucleotide in the ORF results in an Asn (N) to Asp (D) amino acid alteration. We synthesized two 30-aa peptides (N29N and N29D) to compare the biological activity of the helix 2-loop-helix 3 region of NK-lysin resulting from the polymorphic gene. Both peptides were found to be cytotoxic in bacteria and tumor cell cultures at micromolar concentrations. The N29N peptide, however, exhibited greater antibacterial and anticancer activity than the N29D peptide. Circular dichroism spectroscopy of the two peptides in negatively charged single unilamellar vesicles showed spectra typical of α-helical peptides. The helical profile of N29D was reduced substantially compared with that of N29N. However, no structural change was observed in neutral vesicles. ζ-Potential measurements of liposomes incubated with increasing peptide concentrations allowed surface charge neutralization with a negatively charged lipid, but not with a zwitterionic lipid. This result suggests that a difference in electrostatic interaction between lipid membranes and the helical peptides results from the polymorphic gene and is subsequently an important factor in cell lytic activity of variant NK-lysin peptides.

Fig. 1.

SNPs of the NK-lysin gene in chicken. (A) The chromatograms of nucleic acid sequence revealed an A-to-G transition. (B) PCR-SSCP showed three different patterns (lanes 1 and 3, AA genotype; lanes 2 and 4, GG genotype; lane 5, AG genotype). (C) Asn (N) to Asp (D) amino acid alteration produced by nonsynonymous SNP.

Fig. 2.

Antibacterial properties of N29N and N29D peptides against Gram-negative bacteria, E. coli and P. aeruginosa, and Gram-positive bacteria, S. aureus and L. monocytogenes in black and gray color, respectively. Different concentrations of N29N and N29D peptides were incubated with each bacterium and its viability compared with control.

Fig. 3.

Cytotoxicity of N29N and N29D determined by cell viability assay against SW480, U937, SNU-1 and normal human lymphocytes. Cells were incubated with peptide concentrations of 1, 5, 10, 20, 30, and 50 μM. N29N and N29D treatments are represented in black and gray, respectively. The average was calculated from four independent experiments.

Fig. 4.

NK-lysin induced apoptosis in SNU-1 cells—FACS flow cytometry. After cell staining with annexin V-FITC and PI, the apoptotic cells (annexin V⁺/PI⁻ and annexin V⁺/PI⁺) were analyzed by a dot-plot using a flow cytometer. The numbers in the quadrants of each plot indicate the percentage of annexin-positive (apoptotic) cells. The figure is representative of three replicates.

Fig. 5.

(A) CD spectra of N29N and N29D peptides in 10 mM phosphate buffer (solid line), DPPC liposomes (dotted line), and DPPG liposomes (dashed line) in black and gray color, respectively. (B) α-Helical contents of N29N and N29D using CDNN deconvolution software.

Fig. 6.

Effects of N29N and N29D on the ζ-potential properties of phospholipid liposomes. Peptide concentrations of 0 to 500 μM were tested for negatively charged DPPG and neutral DPPC liposomes to determine ζ-potential. Data shown for ζ-potential represent mean values from at least two independent liposome preparations.