Effects of a single nucleotide polymorphism in the chicken NK-lysin gene on antimicrobial activity and cytotoxicity of cancer cells.
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.
World Class University Biomodulation Major, Department of Agricultural Biotechnology,  Seoul National University, Seoul 151-742,  Korea.
Mi Ok
MO
Lee
Eun-Hee
EH
Kim
Hyun-Jun
HJ
Jang
Mi Na
MN
Park
Hee-Jong
HJ
Woo
Jae Yong
JY
Han
James E
JE
Womack
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D060326:Chemistry Techniques, Synthetic; D002645:Chickens / Q000235:genetics* / Q000276:immunology; D002942:Circular Dichroism; D003601:Cytotoxicity Tests, Immunologic; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D007113:Immunity, Innate / Q000235:genetics*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D011487:Protein Conformation; D011510:Proteolipids / Q000235:genetics*; D055672:Static Electricity
D017931:DNA Primers; C093238:NK-lysin; D010455:Peptides; D011510:Proteolipids