Characterization of two thymosins as immune-related genes in common carp (Cyprinus carpio L.)

https://doi.org/10.1016/j.dci.2015.01.003Get rights and content

Highlights

  • Full cDNA sequences of two common carp thymosins have been identified.

  • The expression patterns of common carp thymosin genes in different tissues and during embryogenesis have been determined.

  • The transcriptional changes of carp thymosin genes after spring viraemia of carp virus (SVCV) infection have been detected.

  • The expressions of T lymphocyte-related genes increased after transient over-expression of carp thymosins in zebrafish.

Abstract

Prothymosin alpha (ProTα) and thymosin beta () belong to thymosin family, which consists of a series of highly conserved peptides involved in stimulating immune responses. ProTα b and Tβ are still poorly studied in teleost. Here, the full-length cDNAs of ProTα b and Tβ-like (Tβ-l) were cloned and identified in common carp (Cyprinus carpio L.). The expressions of carp ProTα b and Tβ-l exhibited rise-fall pattern and then trended to be stable during early development. After spring viraemia of carp virus (SVCV) infection, the carp ProTα b and Tβ-l transcripts were significantly up-regulated in some immune-related organs. When transiently over-expressed carp ProTα b and Tβ-l in zebrafish, these two proteins up-regulated the expressions of T lymphocytes-related genes (Rag 1, TCR-γ, CD4 and CD8α). These results suggest that carp ProTα b and may ultimately enhance the immune response during viral infection and modulate the development of T lymphocytes in teleost.

Introduction

Since 1965, many research groups have dedicated to the study of thymosins, a mixture of small polypeptides ranging from 1 to 15 kDa in mammals (Goodall et al., 1986). According to the different isoelectric points, thymosins are classified into three categories: α-thymosins (pH < 5.0), β-thymosins (5.0 < pH < 7.0) and γ-thymosins (pH > 7.0) (Hannappel and Huff, 2003). Prothymosin alpha (ProTα), the precursor of α-thymosins, is a small acidic nuclear protein, containing 109–113 amino acids depending on the species, consisting mainly of aspartic and glutamic acid residues (more than 50%) with few hydrophobic amino acids and without any aromatic or sulfur amino acids (Gast et al., 1995). Beta thymosins (s) are a family of highly conserved polar 5-kDa polypeptides consisting of 40–44 amino acid residues. More than 15 kinds of s have been reported (Huff et al., 2001).

ProTα is widely distributed and highly conserved in mammalian tissues and cells (Pineiro et al., 2000). More than 10,000 copies per cell of ProTα are found in kidney, liver, spleen, normal lymphocytes (predominantly T cells), human T-cell leukemia virus-infected T cells and myeloma cells (B-cell lineage) (Eschenfeldt and Berger, 1986). Several studies have demonstrated ProTα's function in immune regulatory activity and potentiation of the immune system (Cordero et al, 1991, Oates et al, 1988). Recent studies documented that ProTα also played a regulatory role in apoptosis because of the significance of this process in development, cell turnover, and tumorigenesis (Emmanouilidou et al, 2013, Jiang et al, 2003, Qi et al, 2010, Tripathi et al, 2011). First identified from a cytokine-like activity, s play significant roles in various physiological processes. (Low et al., 1981). Later studies showed that s' mRNA and protein levels were changed rapidly during differentiation or by different stimuli (Hall, 1991, Huff et al, 2001). Meanwhile, the archetypical Tβ, Tβ4 was found to play a fundamental role in the host defense mechanism (Gondo et al., 1987). Li et al. (2007) reported that human recombinant Tβ4 can promote lymphocyte proliferation and differentiation. Lee et al. (2009) suggested that Tβ4 was a key activator of NK cell cytotoxicity. Two types of isolated from Chinese mitten crab were also proved to induce human cell proliferation (Gai et al., 2009). A recent study indicated that homolog may be involved in the immune response in abalone (Kasthuri et al., 2013). All these studies indicated that ProTα and play important roles in physiological regulation of immunity.

To date, ProTα and genes have been well studied in mammals. However, in teleost, the ProTα gene has only been reported in zebrafish (Danio rerio) and spotted ray (Torpedo marmorata) (Donizetti et al, 2008, Prisco et al, 2009). Interestingly, Donizetti et al. showed that ProTα was duplicated in zebrafish (ProTα a and ProTα a b). isoforms have also been identified from several fishes including trout (Salmo gairdneri) (thymosin β11 and thymosin β12), perch (Perca fluviatilis ) (thymosin β12) and paradise fish (Macropodus opercularis) (β-thymosin) (Anathy et al, 2003, Erickson-Viitanen, Horecker, 1984, Low et al, 1992). ProTα was reported to inhibit the replication of virus, and it increased the expression of MHC I which presented endogenous peptides to cytotoxic T cells to kill the virally infected cells and some cancer cells (Baxevanis et al, 1994, Baxevanis et al, 1995, Haritos et al, 1985, Romani et al, 2004). A study reported one of the Tβs, Tβ4, was a key activator of NK cell cytotoxicity (Lee et al., 2009). These studies indicated that carp ProTα b and Tβ-l genes may also play important roles in NK/Tc-mediated cytolysis in viral clearance. Here, we reported the structure and organization of carp (Cyprinus carpio L.) ProTα b and Tβ-l genes and determined their dynamic expression during early development and expression changes after the SVCV infection. We also examined the expression of several immune-related genes in zebrafish model in which carp ProTα b or Tβ-l proteins were over-expressed. Our study will provide insights into the molecular structure and organization of ProTα b and Tβ-l genes and their biological functions in teleost.

Section snippets

Experimental carp

Common carp (Cyprinus carpio L.) at the age of 9 months were supplied by the Henan Institute of Aquaculture, Henan, China. These experimental carps were transported to the laboratory, and transferred to large indoor tanks (1000 L) equipped with a recirculating water system and maintained under water temperature (15–18 °C) conditions, then acclimated to laboratory conditions for at least 3 days (3 d). The gill, spleen, thymus, head kidney, kidney, intestine, liver, peripheral blood, muscle and

Characteristics of carp ProTα b and Tβ-l genes

The full-length cDNA of carp ProTα b was 1059 base pair (bp) (GenBank accession no. KJ586576) containing an open reading frame that encoded for 107 amino acids. Four RNA instability motifs (ATTA, ATTTA, AT**TA) were presented in the 3′-UTR. The polyadenylation signal AATAA was 50 bp upstream of poly (A) (Fig. 1A). Amino acid sequence analysis indicated that 53% amino acid residues were acidic and no aromatic amino acid was found in the polypeptide. The theoretical isoelectric point of carp

Discussion

The discovery of thymosins in the mid 1960s emerged from investigations of the role of the thymus in the development of the vertebrate immune system (Goldstein et al., 1966). These small proteins have been well studied as particularly attractive molecules for stimulating immune responses in mammals thereafter, and some of which have already progressed from the laboratory to the clinic (Goldstein, 2007, Goldstein, Badamchian, 2004). Until now, the roles and functions of thymosins in teleost are

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 30671599, 30871912), and the National Basic Research Program of China (Grant No. 2009CB118704). We would like to thank Prof. Pin Nie and Xudong Xu (Institute of Hydrobiology, Chinese Academy of Sciences) for technical supports and valuable advices.

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