Woodchuck lymphotoxin-alpha, -beta and tumor necrosis factor genes: structure, characterization and biological activity.
We cloned and characterized the woodchuck tumor necrosis factor (TNF) and lymphotoxin-alpha, -beta (LT-alpha, -beta) 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-alpha and LT-beta. The cDNAs for woodchuck TNF, LT-alpha and LT-beta 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-alpha and LT-beta genes encode polypeptides of 233, 205 and 244 amino acids respectively, whereas the mouse TNF, LT-alpha and LT-beta genes encode polypeptides of 235, 202 and 306 amino acids respectively. In the woodchuck, there are four exons for TNF, four exons for LT-alpha and three exons for LT-beta. The RNA splicing patterns for TNF, LT-alpha and LT-beta genes are identical among woodchucks, humans and mice, except that the human LT-beta gene contains four exons. The woodchuck TNF gene promoter contains consensus sequences for binding of AP-1, AP-2, C/EBPbeta, CRE, Egr-1, Ets, NF-AT, NF-kappaB and SP-1 transcription factors. LT-alpha has AP-2, Ets, NF-kappaB, SP-1 and STAT binding sites, and LT-beta has Egr-1/SP-1, Ets and NF-kappaB binding sites. The bacterially expressed woodchuck TNF and LT-alpha proteins exhibited cytotoxic activities on both mouse L929B and woodchuck A2 cells in the presence of actinomycin D. The specific activities of TNF and LT-alpha were 2.62x10(8) units/mg and 2.22x10(3) units/mg respectively for L929B cells, and 1.05x10(9) units/mg and 3.56x10(4) units/mg respectively for A2 cells. However, only woodchuck TNF showed cytotoxic activity on human HepG2 cells, with a specific activity of 6.55x10(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 antitumor and anti-viral activities, and their therapeutic potential in the woodchuck model for human hepatitis B virus (HBV).
Li
D H
DH
Department of Microbiology, Pathology and Parasitology, North Carolina State University College of Veterinary Medicine, Raleigh 27606, USA. dhli@leland.stanford.edu
Havell
E A
EA
Brown
C L
CL
Cullen
J M
JM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D015870:Gene Expression; D005796:Genes / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D008233:Lymphotoxin-alpha / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D053308:Lymphotoxin-beta; D008392:Marmota / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D018076:DNA, Complementary; C505886:LTB protein, human; C505887:Ltb protein, mouse; D008233:Lymphotoxin-alpha; D053308:Lymphotoxin-beta; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D005982:Glutathione Transferase