A glutamic acid decarboxylase (CgGAD) highly expressed in hemocytes of Pacific oyster Crassostrea gigas

https://doi.org/10.1016/j.dci.2016.05.010Get rights and content

Highlights

  • A GAD homologous gene was identified from Pacific oyster Crassostrea gigas.

  • CgGAD can promote the production of GABA after transfection into HEK293 cells.

  • CgGAD was detected mostly in ganglion and granulocytes of oysters.

  • CgGAD could respond dynamically to LPS stimulation.

Abstract

Glutamic acid decarboxylase (GAD), a rate-limiting enzyme to catalyze the reaction converting the excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA), not only functions in nervous system, but also plays important roles in immunomodulation in vertebrates. However, GAD has rarely been reported in invertebrates, and never in molluscs. In the present study, one GAD homologue (designed as CgGAD) was identified from Pacific oyster Crassostrea gigas. The full length cDNA of CgGAD was 1689 bp encoding a polypeptide of 562 amino acids containing a conserved pyridoxal-dependent decarboxylase domain. CgGAD mRNA and protein could be detected in ganglion and hemocytes of oysters, and their abundance in hemocytes was unexpectedly much higher than those in ganglion. More importantly, CgGAD was mostly located in those granulocytes without phagocytic capacity in oysters, and could dynamically respond to LPS stimulation. Further, after being transfected into HEK293 cells, CgGAD could promote the production of GABA. Collectively, these findings suggested that CgGAD, as a GABA synthase and molecular marker of GABAergic system, was mainly distributed in hemocytes and ganglion and involved in neuroendocrine-immune regulation network in oysters, which also provided a novel insight to the co-evolution between nervous system and immune system.

Introduction

The GABAergic system is a ubiquitous inhibitory neurotransmitter system which cooperates with excitatory glutamatergic system to maintain a balance in the central nervous system (CNS) of vertebrates (Murphy et al., 2005, Shen et al., 2014). This system exists not only in nervous system, but also in immune cells like monocytes and macrophages to exert a profound effect on the immune function (Dionisio et al., 2011). In vertebrates, GABAergic system is related to suppression of immune-mediated pro-inflammatory reactions (Reyes-García and Garcia-Tamayo, 2009), modification of cell proliferation and migration of the dendritic cells (Fuks et al., 2012, Jin et al., 2013). In addition, GABAergic system also participates in immune-related diseases, such as psoriasis, experimental autoimmune encephalomyelitis, rheumatoid arthritis and Alzheimer disease (Limon et al., 2012, Nigam et al., 2010). GABAergic system is also detected in nervous system of some invertebrates, including Caenorhabditis elegan (Mclntlre et al., 1993), Haliotis asinina (Soonklang et al., 2013), Eledone cirrhosa (Cornwell et al., 1993), and it plays effective roles in the regulation of larval swimming (Katow et al., 2013), settlement and metamorphosis during development of sea urchin and bivalve mollusc (García-Lavandeira et al., 2005). However, the information about the distribution and immunomodulatory function of GABAergic system is still very limited in invertebrates.

GABAergic system is composed of four primary parts: i) GABA synthase: glutamic acid decarboxylase (GAD); ii) GABA catabolism enzyme: GABA transaminase (GABA-T); iii) GABA transporters (GAT); iv) GABA receptors (Dionisio et al., 2011). Among them, GAD is the key rate-limiting synthase of GABA and the starter of GABAergic system, which is always used as a molecular marker of GABAergic neurons. It catalyzes the reaction from excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA) with the cofactor pyridoxal 5′-phosphate (PLP) (Taherzadeh et al., 2015, Jin et al., 1999). Two GAD isoforms, GAD65 and GAD67, have been identified in vertebrates. Although both isoforms of GAD can synthesize GABA, they are encoded by different independently regulated genes, and have distinct biochemical properties and intracellular distributions (Erlander and Tobin, 1991). GAD65 appears to be targeted to membranes and nerve endings, and preferentially synthesizes GABA for vesicular release. Whereas GAD67 is widely distributed in cells, and preferentially synthesizes cytoplasmic GABA (Soghomonian and Martin, 1998). So far, only one GAD isoform has been identified in the primitive invertebrates. For example, one GAD gene locus was identified in Caenorhabditis elegans and it was necessary for synaptic transmission in GABAergic nervous system (Mclntlre et al., 1993, Jin et al., 1999). In Dugesia japonica, only one GAD gene was reported and required for GABA biosynthesis and photosensitivity (Nishimura et al., 2008). Therefore, it has been postulated that the two GAD isoforms from phylogenetically distant organisms evolved from a common ancestor (Dunathan and Voet, 1974).

Accumulating evidences have demonstrated that GAD not only functions in the nervous system, but also participates in immunomodulation through synthesizing GABA in vertebrates. It has been reproted that GAD can be detected in dendritic cells and macrophages, and its expression can be induced by LPS stimulation (Bhat et al., 2010). In invertebrates, GAD is always regarded as a useful molecular marker for GABAergic neurons (Nishimura et al., 2008), while its role in immunomodulation is still far from well understood. Recently, it was proposed that immune and neuroendocrine system evolved from a common origin (Ottaviani et al., 2007), and crayfish adult-born neurons were found to be derived from hemocytes, indicating that neuronal precursors may be derived from cells in the innate immune system (Benton et al., 2014). Therefore, the information of the distribution and immune function of GAD in invertebrates may help us to further understand the possible co-evolution of immune system and nervous system.

The Pacific oyster Crassostrea gigas is one of most important marine mollusc, which contributes greatly to the aquaculture industry worldwide. With the convenience of oyster genome information, investigations of the distribution and immune function of GABAergic system in oyster C. gigas will further understand the neuro-endocrine-immune modulation and the relationships between the nervous system and immune system. The purposes of this study were to (1) identify the homologue of GABA synthase GAD in oyster C. gigas, (2) survey the enzyme function and distribution of CgGAD in different tissues and hemocytes, (3) investigate the response of CgGAD after LPS stimulation.

Section snippets

Oysters and LPS stimulation

Adult oysters C. gigas with an average shell length of 12.0–15.0 cm were collected from a local farm in Qingdao, Shandong Province, China, and maintained in the aerated seawater at 15–18 °C for a week before experiments.

For LPS stimulation experiment, a narrowed notch was sawed in the closed side of the oyster shell adjacent to the adductor muscle, and the oysters were acclimated for another one week for experiments. The oysters were randomly divided into 2 groups and each group contained 80

The molecular characters of CgGAD

According to the genome annotation of Pacific oyster C. gigas, three candidate genes of GAD (Genebank Accession NO. JH821014.1, JH819059.1 and XM_011420388.1) were identified from NCBI. The protein encoded by the first gene (JH821014.1) lacked two conserved active site residues (Leu158, Asn179) of GAD. The remaining two candidates were found to be located on the same gene locus, and only one gene product of 1689 bp (XM_011420388.1) was amplified from cDNA library of Pacific oyster. Its open

Discussion

The CNS and the immune system play important roles in the process of homeostasis, adaptation and immune defense in both vertebrate and invertebrate animals (Demas et al., 2011, Mašek et al., 2003). The CNS can regulate the function of immune system through secreting neurotransmitters. However, some neurotransmitters, such as NO and biogenic amines have also been reported in the immunocytes of some protostome species without complex nervous system (Ottaviani and Franceschi, 1996, Jiang et al.,

Acknowledgements

We are grateful to all the laboratory members for their technical advice and helpful discussions. This research was supported by the High Technology Project (863 Program, No. 2014AA093501) from the Chinese Ministry of Science and Technology, grants (Nos. 31530069, 41276169) from National Science Foundation of China, earmarked fund (CARS-48) from Modern Agro-industry Technology Research System, and funds from the Taishan Scholar Program of Shandong.

References (48)

  • Q. Jiang et al.

    The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

    Fish Shellfish Immunol.

    (2013)
  • M. Li et al.

    The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas

    Fish Shellfish Immunol.

    (2016)
  • K. Mašek et al.

    Neuroendocrine immune interactions in health and disease

    Int. Immunopharmacol.

    (2003)
  • K. Nishimura et al.

    Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica

    Neuroscience

    (2008)
  • E. Ottaviani et al.

    The neuroimmunology of stress from invertebrates to man

    Prog. Neurobiol.

    (1996)
  • E. Ottaviani et al.

    Common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidence to in silico approaches

    Trends Immunol.

    (2007)
  • M.G. Reyes-García et al.

    A neurotransmitter system that regulates macrophage pro-inflammatory functions

    J. Neuroimmunol.

    (2009)
  • X. Shen et al.

    Menin regulates spinal glutamate-GABA balance through GAD65 contributing to neuropathic pain

    Pharmacol. Rep.

    (2014)
  • J.J. Soghomonian et al.

    Two isoforms of glutamate decarboxylase: why?

    Trends Pharmacol. Sci.

    (1998)
  • W. Wang et al.

    A novel phagocytic receptor (CgNimC) from Pacific oyster Crassostrea gigas with lipopolysaccharide and gram-negative bacteria binding activity

    Fish Shellfish Immunol.

    (2015)
  • L. Xin et al.

    CgIL17-5, an ancient inflammatory cytokine in Crassostrea gigas exhibiting the heterogeneity functions compared with vertebrate interleukin17 molecules

    Dev. Comp. Immunol.

    (2015)
  • Q.G. Xue et al.

    Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph

    Fish Shellfish Immunol.

    (2001)
  • Y. Ben-Ari

    Excitatory actions of GABA during development: the nature of the nurture

    Nat. Rev. Neurosci.

    (2002)
  • R. Bhat et al.

    Inhibitory role for GABA in autoimmune inflammation

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • Cited by (0)

    View full text