Abstract
Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as CgGLS-1) was identified from Pacific oyster Crassostrea gigas, whose open reading frame was of 1836 bp. CgGLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica. The enzyme activity of recombinant CgGLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. CgGLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. CgGLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p < 0.01) after lipopolysaccharide (LPS) stimulation. The CgGLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of CgGLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of CgmGluR6, CgAP-1, cytokines CgIL17-5 and CgTNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of CgCaspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that CgGLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines CgIL17-5 and CgTNF-1, as well as hemocyte apoptosis.
Highlights
Phosphate-activated glutaminase hereinafter, referred to as GLS, is an amidohydrolase enzyme catalyzes the reaction of glutamine (GLN) to glutamate (Glu) and a mmonia[1]
A homologue of GLS was identified from C. gigas with the main purposes to (1) examine its mRNA expression level in different tissues, and in the response against bacterial stimulation, (2) determine its enzyme activity to catalyze the hydrolytic deamidation of glutamine to Glu, (3) examine the apoptotic rate, Glu concentration and mRNA expression of CgIL17-5, CgTNF-1 and CgCaspase[3] in oyster hemocytes after the inhibition of CgGLS-1, hope to explore the function of GLS in bacteria-induced immune response in oyster
CgGLS-1was clustered with the kidney-type GLS of C. virginica and Mizuhopecten yessoensis (Fig. 1c), indicating that it exhibited higher homology to kidney-type GLS. Another phylogenic tree based on the amino acid sequences of CgGLS-1 and kidney-type GLS from other species was constructed to investigate the evolutionary status of CgGLS-1 (Fig. 1d)
Summary
Phosphate-activated glutaminase hereinafter, referred to as GLS, is an amidohydrolase enzyme catalyzes the reaction of glutamine (GLN) to glutamate (Glu) and a mmonia[1]. Glu at ~ 1,000-fold lower content of 1 × 10–6 M could regulate mGluRs to modulate IL-6 production and enhance the secretion of tumor necrosis factor (TNF)-α, IFNγ, IL-2, and IL-1018 These findings indicate that Glu is involved in regulating the function and survive of the immune cells, and contribute to the protection of the host from invading pathogens. A homologue of GLS was identified from C. gigas (designated as CgGLS-1) with the main purposes to (1) examine its mRNA expression level in different tissues, and in the response against bacterial stimulation, (2) determine its enzyme activity to catalyze the hydrolytic deamidation of glutamine to Glu, (3) examine the apoptotic rate, Glu concentration and mRNA expression of CgIL17-5, CgTNF-1 and CgCaspase[3] in oyster hemocytes after the inhibition of CgGLS-1, hope to explore the function of GLS in bacteria-induced immune response in oyster
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