Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex

• Published in: Immunogenetics (New York) Vol. 56; no. 7; pp. 518 - 530
• Main Authors: Long, Scott, Wilson, Melanie, Bengtén, Eva, Clem, L William, Miller, Norman W, Chinchar, V Gregory
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01-10-2004
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Blotting, Western; Calcium - metabolism; Carcinogens - pharmacology; Caspase 8; Caspases - genetics; Caspases - metabolism; Cytotoxicity; Death Domain Receptor Signaling Adaptor Proteins; DNA, Complementary - analysis; Fas Ligand Protein; fas Receptor - genetics; fas Receptor - metabolism; Fas-Associated Death Domain Protein; Fibroblasts - drug effects; Fibroblasts - metabolism; Ictaluridae - genetics; Ictalurus punctatus; Ionophores - pharmacology; Irradiation; Lymphocytes; Lymphocytes - drug effects; Lymphocytes - metabolism; Macrophages - drug effects; Macrophages - metabolism; Mammals; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Molecular Sequence Data; Proteins; Receptors, Tumor Necrosis Factor - genetics; Receptors, Tumor Necrosis Factor - metabolism; Sequence Homology, Amino Acid; Tetradecanoylphorbol Acetate - pharmacology; Ultraviolet radiation; Ultraviolet Rays
• ISSN: 0093-7711; 1432-1211
• DOI: 10.1007/s00251-004-0701-2

To elucidate cytolytic mechanisms in the channel catfish, lysates from catfish lymphoid and fibroblast cell lines were screened by Western blot analysis using a panel of antibodies reactive with components of the mammalian apoptotic pathway. Strong reactivity with three proteins (approximate Mr 70,000, 37,000, and 15,000) was seen using an antibody targeted to mammalian Fas ligand (FasL). The sizes of the two smaller proteins are consistent with their tentative designation as membrane-bound (37,000 Mr) and soluble (15,000 Mr) FasL. Treatments known to induce FasL in mammalian systems (e.g., PMA/calcium ionophore, UV-irradiation) induced expression of the 37,000- Mr protein in catfish T-cell lines. Moreover, expression of the 37,000- Mr protein in clonal T cells was up-regulated by increasing cell density. At the nucleotide level, homologues of Fas receptor (FasR), FADD, and caspase 8 were identified and characterized. These gene products likely constitute the teleost equivalent of the death-inducing signaling complex (DISC). FADD was constitutively expressed in all (T, B, macrophage, and fibroblast) cell lines examined as well as in peripheral blood lymphocytes (PBL), whereas FasR and caspase 8 were expressed in all cell lines except CCO, a FasL-positive fibroblast line. In contrast to FasL, expression of FasR and caspase 8 was inversely proportional to cell density. Collectively these studies identified four membrane-proximal proteins involved in the initiation of apoptosis in channel catfish and suggest that mechanisms of cell-mediated cytotoxicity in teleosts are similar to those used by mammals.