Full length articleThe first characterization of gene structure and biological function for echinoderm translationally controlled tumor protein (TCTP)
Introduction
The translationally controlled tumor protein (TCTP), also referred to Q23 [1], P21 [2], P23 [3], histamine releasing factor (HRF) [4] and fortilin [5], was first given the name more than 20 years ago by Gross et al., who obtained a cDNA sequence from a human mammary carcinoma cDNA library with probes derived from the translationally controlled, growth-related mouse tumor protein p23 [6]. After that, TCTP has been demonstrated to be a highly conserved protein that existed ubiquitously in different eukaryote species and distributed widely in various tissues and cell types by plenty of data [7].
The amino acid sequences of TCTP share not any similarity with other known protein families [7]. Only the solution structure of TCTP from yeast, Schizosaccharomyces pombe has been determined by NMR spectroscopy which indicated that this protein is structurally similar to two small guanine nucleotide-free chaperones, namely Mss4 and Dss4 [8]. TCTP and Mss4/Dss4 are now therefore structurally grouped into one protein superfamily [7].
TCTP displays many biological functions and implicates in a wide variety of important cellular processes, including calcium-binding [9], tubulin-binding [10], release of histamine [4], anti-apoptosis [5], embryo development [11], cell division [10], growth regulation[10], cytoskeleton regulation [12], tumor reversion [13], and gene regulation [14]. Additionally, TCTP also exhibits extracellular cytokine-like function, as it can induce the production of interleukins from basophils and eosinophils [15], [16], implicating that this protein may be involved in a variety of inflammatory processes [7].
The synthesis of TCTP is regulated at both transcriptional and translational levels [17], [18]. The mRNA levels of TCTP may response to various stresses, such as starvation [19], heat stress [20], oxidative stress [21], calcium stress [22] and challenges of heavy metals [23], toxins and drugs [24], [25]. In addition, intracellular and extracellular signals, such as growth signals [26] and cytokines [27], could result in induction or reduction of TCTP levels. In the translational regulation of TCTP, it has been confirmed that the protein synthesis of TCTP may be positively or negatively regulated by the eukaryotic initiation factor 4E (eIF4E) [28] and the dsRNA-dependent protein kinase PKR [26].
Compared to vertebrates, research on the biological roles of invertebrate TCTP is very limited. Currently, the cDNA sequences of TCTP have been cloned from arthropod [29], [30], [31], [32], [33], [34], mollusk [35] and annelida [23], and some biological functions of TCTP in these species have also been explored. In four shrimp species (Penaeus monodon, Penaeus japonicus, Fenneropenaeus chinensis, and Penaeus indicus), TCTP has been shown to be an important innate immune factor in defense of viruses or bacterial pathogen infection [29], [30], [31]. However, in echinoderm, only three cDNA sequences of Strongylocentrotus purpuratus (NCBI Reference Sequence: XM_790526.2), Holothuria glaberrima (GenBank: GU191016.1), and Apostichopus japonicus (GenBank: DQ359952.1) have been obtained. To date, study on the gene structure and biological function of TCTP in Phylum Echinodermata, the highest group of invertebrates, is blank. In order to fill this gap and make clear the roles of TCTP in echinoderm, we cloned the full-length cDNA and gene sequence of TCTP from the tropical sea cucumber, Stichopus monotuberculatus, one of the start-up aquaculture species in South China. Furthermore, the temporal expression profile of TCTP transcript after challenge with lipopolysaccharide (LPS), Vibrio alginolyticus and polyriboinosinic-polyribocytidylic Acid [poly (I:C)], as well as the anti-stress function of TCTP recombinant protein were also investigated.
Section snippets
Animals and tissue collection
Healthy sea cucumbers (S. monotuberculatus) weighted about 200 g were collected from the Coast of Weizhou Island in Guangxi, China, and kept in aerated and filtrated seawater (35 g/L salinity) at 32 °C for a week before experiments. After anesthesia on ice for 30 min, the sea cucumbers were dissected by cutting through the calcareous ring with a razor blade. The coelomic fluids were immediately gathered and centrifuged at 1000× g for 10 min at 4 °C to harvest the coelomocytes. Then, the
Molecular cloning and sequence analysis of StmTCTP
The complete cDNA sequence of StmTCTP was obtained using 3′/5′ RACE technique and deposited in GenBank under accession no. KJ710436.1. The StmTCTP cDNA is 1219 bp in length, containing a 5′-untranslated region (UTR) of 77 bp, a 3′-UTR of 623 bp and an open reading frame (ORF) of 519 bp that encoding a protein of 172 amino acids (Fig. 1). A polyadenylation signal (AATAAA) is located at 31-bp upstream of the poly-A tail. The putative protein has a deduced molecular weight of 19.80 kDa and a
Discussion
In this report, a TCTP gene named StmTCTP was identified from the sea cucumber, S. monotuberculatus. Coupled with the data from cDNA sequence analysis, protein structure and function domain prediction, and phylogenetic analysis, it could be referred that our cloned StmTCTP is one new member of TCTP family. To our knowledge, this is the first research on TCTP in echinoderm.
Compared with vertebrates, the gene structure of TCTP in invertebrates was less studied. In this study, the TCTP gene from
Acknowledgments
This study was funded by the Key Projects in the National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012BAD18B03), Guangdong Province and CAS Cooperation Program (2012B091100272), and the Science & Technology Promoting Projects for Oceanic & Fishery in Guangdong Province (A201100D01, A201101D02).
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2015, Fish and Shellfish ImmunologyCitation Excerpt :For measurement of StmCaM mRNA expression, StmCaM protein content and cumulative NO production, the durations of bacterial challenge and inhibitor treatment were fixed to 3 h, 12 h and 24 h, respectively. For StmCaM mRNA measurement, total RNA was extracted from sea cucumber tissue samples or primary coelomocytes and reversely transcribed into first-strand cDNA as described before [29]. The real-time PCR was carried out in RotorGene RG-3000 (Roche) by using an SYBR Premix Ex Taq™ II (TaKaRa) with specific primers and PCR conditions (Suppl. 1).