Woodchuck lymphotoxin-α, -β and tumor necrosis factor genes: structure, characterization and biological activity

Abstract

We cloned and characterized the woodchuck tumor necrosis factor (TNF) and lymphotoxin-α, -β (LT-α, -β) cDNAs, genes and proteins to facilitate study of the functions of these cytokines during the course of woodchuck hepatitis virus (WHV) infection. Woodchuck cDNA and genomic DNA libraries were screened with woodchuck-specific DNA probes to isolate the cDNA and gene clones for TNF, LT-α and LT-β. The cDNAs for woodchuck TNF, LT-α and LT-β code for proteins of 233, 205 and 310 amino acids respectively. The polypeptide encoded by each gene among woodchucks, humans and mice can differ: the human TNF, LT-α and LT-β genes encode polypeptides of 233, 205 and 244 amino acids respectively, whereas the mouse TNF, LT-α and LT-β genes encode polypeptides of 235, 202 and 306 amino acids respectively. In the woodchuck, there are four exons for TNF, four exons for LT-α and three exons for LT-β. The RNA splicing patterns for TNF, LT-α and LT-β genes are identical among woodchucks, humans and mice, except that the human LT-β gene contains four exons. The woodchuck TNF gene promoter contains consensus sequences for binding of AP-1, AP-2, C/EBPβ, CRE, Egr-1, Ets, NF-AT, NF-κB and SP-1 transcription factors. LT-α has AP-2, Ets, NF-κB, SP-1 and STAT binding sites, and LT-β has Egr-1/SP-1, Ets and NF-κB binding sites. The bacterially expressed woodchuck TNF and LT-α proteins exhibited cytotoxic activities on both mouse L929B and woodchuck A 2 cells in the presence of actinomycin D. The specific activities of TNF and LT-α were 2.62×10 8 units/mg and 2.22×10 3 units/mg respectively for L929B cells, and 1.05×10 9 units/mg and 3.56×10 4 units/mg respectively for A 2 cells. However, only woodchuck TNF showed cytotoxic activity on human HepG 2 cells, with a specific activity of 6.55×10 7 units/mg in the presence of actinomycin D. The data obtained from this study will be useful to future investigations of the TNF and LT anti-tumor and anti-viral activities, and their therapeutic potential in the woodchuck model for human hepatitis B virus (HBV).