The first characterization of gene structure and biological function for echinoderm translationally controlled tumor protein (TCTP)

Highlights

• We first identified a cDNA of TCTP form a tropic sea cucumber.

• This is the first report on the gene structure of TCTP in marine echinoderm species.

• We demonstrated the anti-oxidation and heat shock protein activity of StmTCTP.

• We demonstrated that StmTCTP may serve as an immune-related protein against bacterial and viral infections in sea cucumber.

Abstract

Translationally controlled tumor protein (TCTP) is a multifunctional protein that existed ubiquitously in different eukaryote species and distributed widely in various tissues and cell types. In this study, the gene structure and biological function of TCTP were first characterized in echinoderm. An echinoderm TCTP named StmTCTP was identified from sea cucumber (Stichopus monotuberculatus) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. 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 with a deduced molecular weight of 19.80 kDa and a predicted isolectric point of 4.66. Two deduced signal signatures termed TCTP1 and TCTP2, a microtubule binding domain, a Ca²⁺ binding domain and the conserved residues forming Rab GTPase binding surface were found in the StmTCTP amino acid sequence. For the gene structure, StmTCTP contains four exons separated by three introns. The anti-oxidation and heat shock protein activities of recombinant TCTP protein were also demonstrated in this study. In addition, the expression of StmTCTP was found to be significantly upregulated by polyriboinosinic polyribocytidylic acid [poly (I:C)], lipopolysaccharides (LPS) or inactivated bacteria challenge in in vitro primary culture experiments of coelomocytes, suggested that the sea cucumber TCTP might play critical roles not only in the defense against oxidative and thermal stresses, but also in the innate immune defense against bacterial and viral infections.

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.