A new cytokine receptor in fish involved in immunity and development
Mudjekeewis D. Santos
Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Konan 4-5-7 Minato-ku, Tokyo 108-8477 Japan
National Fisheries Research and Development Institute of the Philippines, 940 KPI Bldg., Quezon Ave., Quezon City, Philippines
Cytokines are important protein molecules involved in numerous physiological processes in animals including immune regulation, host defense, reproduction, development, blood formation and energy metabolism. In fact, some of these cytokines have been produced in recombinant form in humans; granulocyte colony stimulating factors for the treatment of neutropenia (loss of neutrophils) and Interleukin 11 for the treatment thrombocytopenia (loss of platelets) during cancer therapy. To initiate their action, these cytokines bind to their specific receptors (called cytokine receptors in general) that are found on the surface of target cells. Upon the formation of the cytokine-cytokine receptor complex, the receptor will trigger numerous intracellular signal pathways such as the Janus kinase/Signal transducers and activators of transcription (Jak/STAT) pathway that lead to expression of other genes and their corresponding proteins to catalyze designated functions. In mammals, cytokine receptors are well studied but not their counterparts in fish, even though fish is considered one of the most important food sources and evolutionarily, one of the most successful vertebrate taxa in the animal kingdom with more than 2,000 member species.
Using data from Expressed Sequence Tags (ESTs) derived from Japanese flounder, and employing molecular cloning through Polymerase Chain Reaction (PCR) walking combined with Random Amplifiation of cDNA Ends (RACE-PCR), bioinformatic tools, phylogenetic analysis, and Reverse Transcription – PCR (RT-PCR) techniques, we discovered a novel cytokine receptor involved in development and immunity, and named it as Japanese flounder glycoprotein 130 homologue or JfGPH.
|Figure 1. Japanese flounder gp130 homologue (JfGPH) structure and transcript (Accession no. AB281273). A) Partial JfGPH open reading frame (ORF) transcript. Primers F65 and R1295 (Table 1.), which starts from bp 148 and bp 2401, respectively were used to produce the ~2.2 kb amplicon that spans 93% of the ORF. B) Gene organization showing 11 exons (boxes), 10 introns (line) and untranslated regions (black shade). C) Amino acid sequence exhibiting the extracellular region (uppercase) signal peptide (uppercase bold), glycosylation sites (uppercase italics), the cytokine binding domain with the four conserved cysteine (C) residues (arial font bold with connecting line), WSXWS motif (uppercase bold boxed), transmembrane region (uppercase boxed), intracellular region (lowercase), Box 1 (lowercase bold boxed), Box 3 (lowercase italics boxed) and tyrosine (Y) residues (lowercase bold italics). D) Schematic drawing of the JfGPH protein structure including position of regions in item B plus the 3 fibronectin III (FnIII) domains.|
The JfGPH molecule exhibited the unique type I cytokine receptor motifs i.e. having a cytokine binding domain (CBD) containing two pairs of conserved cysteine (C) residues, a WSXWS motif, 3 fibronectin domains all in the extracellular region. It is also composed of the Jak binding domains Box 1 and Box 2, and a STAT 3 binding motif (Box 3) in the cytoplasmic region suggesting its mediatory role for Jak/STAT signal pathway. The JfGPH complementay DNA (cDNA) is about 3 kb encoding 801 amino acid residues with a predicted molecular weight of 90 kDa and its gene has an 11-exon/10-intron architecture. Structural and phylogenetic analysis of JfGPH amino acid sequence revealed that it is indeed novel and an ancestral receptor to the type-1 cytokine receptor family probably found only in teleost. JfGPH gene is ubiquitously expressed in Japanese flounder tissues and in its natural embryo (HINAE) cell line showing its critical role in teleost physiological functions similar to gp130 in higher vertebrates. High expression of JfGPH transcripts in immune-related tissues and, in ovary and embryo-derived cell line suggest its role in immune responses, and reproduction/development, respectively. In vitro stimulation of spleen, kidney, peripheral blood leukocytes (PBLs) and HINAE revealed that JfGPH is down-regulated by polyinosinic:polycytidylic acid (poly I:C), an interferon (IFN) inducer, showing an apparent control of the JfGPH’s expression during IFN-induced Jak/STAT signaling.
|Figure 2. Expression of Japanese flounder gp130 homologue (JfGPH) gene in response to polyinosinic:polycytidylic acid (poly I:C). JfGPH, Mx and β-actin transcript expression in vitro in Japanese flounder spleen, kidney, peripheral blood leukocytes (PBLs) and Hiame Natural Embryo (HINAE) cell line at 1, 3 and 6 hrs post-stimulated with the double stranded NA, polyI:C.|
The discovery of this cytokine receptor has deepened the understanding of fish immune and development system in particular and of the molecular evolution of vertebrate physiology in general. It also adds to the increasing evidence of the “more genes in fish” concept and has implications to the tremendous evolutionary success of the fish taxon.
 Mudjekeewis D. Santos, Motoshige Yasuike, Hidehiro Kond, Ikuo Hirono and Takashi Aoki. 2007. A novel type-1 cytokine receptor from fish involved in the Janus kinase/Signal transduces and activators of transcription (Jak/STAT) signal pathway. Molecular Immunology. 44: 3355-3363.
 Mudjekeewis D. Santos, Motoshige Yasuike, Ikuo Hirono and Takashi Aoki. 2006. The granulocyte colony stimulating factors (CSF3s) of fish and chicken. Immunogenetics. 56: 422-432.
Mudjekeewis D. Santos is a 3rd year Ph.D. student at the Tokyo University of Marine Science and Technology taking up Applied Marine Biosciences. He is also the current president of the Science and Technology Advisory Council Japan Chapter (STAC-J) (www.stacj.org). Back in the Philippines, he is the Chief of the Pelagic Vertebrate Resources Section of the National Fisheries Research and Development Institute (http://nfrdi.da.gov.ph/).