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

ABSTRACT: 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.

SUBMITTER: Long S

PROVIDER: S-EPMC1364530 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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

Long Scott S Wilson Melanie M Bengtén Eva E Clem L William LW Miller Norman W NW Chinchar V Gregory VG

Immunogenetics 20040916 7

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, ...[more]

PMID: 15375637

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Project description:Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3' and 5' untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.

Dataset Information

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

Publications

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

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