BovineNK-lysin: Copy number variation and functional diversification

Abstract

NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30–35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer’s patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

“…As previously described [9,10], four 30 amino acids long linear peptides corresponding to the functional regions of bovine NK-lysin (helices 2 and 3) of NK1 [VIIHVTSKVCSKMGLWSI LCNQMMKKYLNR], NK2A [TVIEVASKMCSKMRLLKGLCKSITKRFLRR], NK2B [TVIEA ASKVCGKMGPLKGLCKSITKRFLRR] and NK2C [TVIEEASKVCSKMRLLKGLCKSIMK KFLRT]) were synthesized by Peptide 2.0 Inc (Chantilly, VA), with over 95% purity. Lyophilized peptides (1 mg aliquot) were stored at -20˚C, dissolved in phosphate-buffered saline (PBS, pH 7.4) and stored in aliquots again at -20˚C until used.…”

“…Human granulysin as well as porcine and bovine NK-lysin, have also been described as AMPs [7][8][9]. Granulysin and NK-lysin are found in the cytosolic granules of cytotoxic T-lymphocytes and NK cells and have been found to exhibit activity against a variety of Gram positive and Gram negative bacteria, viruses, fungi, parasites and tumor cells [8][9][10][11][12]. Both granulysin and NK-lysin are structurally related to the saposin-like protein (SAPLIP) family of lipid binding proteins [8,12,13].…”

“…Both granulysin and NK-lysin are structurally related to the saposin-like protein (SAPLIP) family of lipid binding proteins [8,12,13]. Although most mammalian species, including humans and swine, have only a single antimicrobial protein gene (granulysin or NK-lysin), the cattle genome is known to contain four functional NK-lysin genes, NK1, NK2A, NK2B and NK2C [10]. Gene expression analysis has revealed that while NK1 and NK2A were highly expressed in the intestinal Peyer's patches, while NK2C was highly expressed in the lungs [10,14].…”

“…Although most mammalian species, including humans and swine, have only a single antimicrobial protein gene (granulysin or NK-lysin), the cattle genome is known to contain four functional NK-lysin genes, NK1, NK2A, NK2B and NK2C [10]. Gene expression analysis has revealed that while NK1 and NK2A were highly expressed in the intestinal Peyer's patches, while NK2C was highly expressed in the lungs [10,14]. It has also been previously reported that the potent antimicrobial activity of bovine NK-lysin is associated with helix 2 through helix 3 regions [9,10,14].…”

The Immunology of Bovine Respiratory Disease

“…Although humans and pigs have only a single NK-lysin gene, cattle have 4 functional NK-lysin genes, NK1, NK2A, NK2B, and NK2C. 80,81 NK-lysin gene expression is up-regulated in the lungs of animals infected with Mycoplasma bovis, M haemolytica, P multocida, BVDV, BRSV, and BHV-1. 80 In vitro, NK-lysin has antimicrobial activities against M haemolytica, P multocida, and H somni, although susceptibility to the 4 individual NK-lysin peptides differs between species.…”