A γ-aminobutyrate type A receptor-associated protein involved in the immune response of Eriocheir sinensis

Abstract

The γ-aminobutyrate type A receptor-associated protein (GABARAP) is a ubiquitin-like modifier, which is implicated in membrane trafficking and fusion events of γ-aminobutyrate type A receptor, autophagy and apoptosis. In the present study, the gene encoding GABARAP (designated EsGABARAP) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends approach and expression sequence tag (EST) analysis. The full-length cDNA of EsGABARAP was of 457 bp, containing a 5' untranslated region (UTR) of 77 bp, a 3' UTR of 32 bp with a poly(A) tail and an open reading frame (ORF) of 348 bp encoding a polypeptide of 116 amino acids with the predicted molecular weight of 13.81 kDa and theoretical isoelectric point of 8.73. The deduced amino acid sequence of EsGABARAP shared higher similarity (91.8-97.4%) with those of other GABARAPs, and it contained a conserved MAP1_LC3 domain. In the phylogenetic tree, EsGABARAP was firstly clustered with GABARAPs from other animals and then gathered together with the same family proteins of GABARAP. The mRNA expression level of EsGABARAP in six tissues and its temporal expression level in haemocytes of crabs challenged with Listonella anguillarum were determined by quantitative real-time RT-PCR. The mRNA transcripts of EsGABARAP could be detected ubiquitously in the examined tissues, including haemocytes, hepatopancreas, muscle, gill, heart and gonad, with the highest expression level in hepatopancreas. The expression level of EsGABARAP mRNA in haemocytes was up-regulated after L. anguillarum challenge and reached 6.58-fold of that in blank group at 24 h (P < 0.05) and 7.52-fold at 48 h (P < 0.05). The increasing transcripts in haemocytes after L. anguillarum challenge gave the preliminary evidence for the involvement of EsGABARAP as a part of immune response against bacteria challenge in crabs.