Structural and functional characterization of a novel molluskan ortholog of TRAF and TNF receptor-associated protein from disk abalone (Haliotis discus discus).

Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology.

[1]  Genhong Cheng,et al.  The signaling adaptors and pathways activated by TNF superfamily. , 2003, Cytokine & growth factor reviews.

[2]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[3]  C. Goggin,et al.  Perkinsus, a protistan parasite of abalone in Australia: A review , 1995 .

[4]  P. Vandenabeele,et al.  TTRAP, a Novel Protein That Associates with CD40, Tumor Necrosis Factor (TNF) Receptor-75 and TNF Receptor-associated Factors (TRAFs), and That Inhibits Nuclear Factor-κB Activation* , 2000, The Journal of Biological Chemistry.

[5]  J. Whisstock,et al.  The Inositol Polyphosphate 5-Phosphatases and the Apurinic/Apyrimidinic Base Excision Repair Endonucleases Share a Common Mechanism for Catalysis* , 2000, The Journal of Biological Chemistry.

[6]  Jehee Lee,et al.  First molluscan TNF-alpha homologue of the TNF superfamily in disk abalone: molecular characterization and expression analysis. , 2009, Fish & shellfish immunology.

[7]  C. Scheidereit,et al.  NF-kappaB and the innate immune response. , 2000, Current opinion in immunology.

[8]  Yang Zhang,et al.  I-TASSER: a unified platform for automated protein structure and function prediction , 2010, Nature Protocols.

[9]  W. Stoffel,et al.  Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis , 1998 .

[10]  Lingling Wang,et al.  cDNA cloning and characterization of a new member of the tumor necrosis factor receptor family gene from scallop, Chlamys farreri , 2011, Molecular Biology Reports.

[11]  Ju-Young Kim,et al.  FOXO3a Turns the Tumor Necrosis Factor Receptor Signaling Towards Apoptosis Through Reciprocal Regulation of c-Jun N-Terminal Kinase and NF-&kgr;B , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[12]  J. Jarosz,et al.  Molluscan immune defenses. , 1997, Archivum immunologiae et therapiae experimentalis.

[13]  W. Stoffel,et al.  Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Y.C. Chen,et al.  Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome , 2000, Letters in applied microbiology.

[15]  Huan Zhang,et al.  A TRAF and TNF receptor-associated protein (TTRAP) in mollusk with endonuclease activity. , 2011, Developmental and comparative immunology.

[16]  R. Beyaert,et al.  ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling. , 2009, Biochemical pharmacology.

[17]  Jehee Lee,et al.  A novel Fas ligand in mollusk abalone: molecular characterization, immune responses and biological activity of the recombinant protein. , 2009, Fish & shellfish immunology.

[18]  Yang Zhang,et al.  COFACTOR: an accurate comparative algorithm for structure-based protein function annotation , 2012, Nucleic Acids Res..

[19]  Xiaoyan Song,et al.  First molluscan TNFR homologue in Zhikong scallop: molecular characterization and expression analysis. , 2009, Fish & shellfish immunology.

[20]  K. Caldecott,et al.  A human 5′-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage , 2009, Nature.

[21]  Limei Qiu,et al.  Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri. , 2007, Fish & shellfish immunology.

[22]  K. Terahara,et al.  Mechanisms and immunological roles of apoptosis in molluscs. , 2008, Current pharmaceutical design.

[23]  D. MacEwan.  TNF receptor subtype signalling: differences and cellular consequences. , 2002, Cellular signalling.

[24]  M. Katan,et al.  Sequence analysis identifies TTRAP, a protein that associates with CD40 and TNF receptor-associated factors, as a member of a superfamily of divalent cation-dependent phosphodiesterases. , 2001, Biochemical and biophysical research communications.

[25]  P. Scheurich,et al.  The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. , 2001, Cellular signalling.

[26]  B. Beutler,et al.  Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. , 1985, Science.

[27]  Y. Matsuo,et al.  A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I , 1996, Protein science : a publication of the Protein Society.

[28]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[29]  C.-Y. Huang,et al.  Withering Syndrome of the Small Abalone, Haliotis diversicolor supertexta, Is Caused by Vibrio parahaemolyticus and Associated with Thermal Induction , 2001, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[30]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[31]  Jehee Lee,et al.  Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): molecular characterization and expression analysis. , 2011, Fish & shellfish immunology.

[32]  T. Nishizawa,et al.  A virus isolated from juvenile Japanese black abalone Nordotis discus discus affected with amyotrophia , 1999 .

[33]  Jehee Lee,et al.  Molecular evidence for the existence of lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus). , 2010, Fish & shellfish immunology.

[34]  Runzhao Li,et al.  EAPII interacts with ETS1 and modulates its transcriptional function , 2003, Oncogene.

[35]  Samuel H. Wilson,et al.  A role for p53 in base excision repair , 2001, The EMBO journal.

[36]  Narmada Thanki,et al.  CDD: a Conserved Domain Database for the functional annotation of proteins , 2010, Nucleic Acids Res..