Molecular cloning, expression and functional characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene from mandarin fish (Siniperca chuatsi)

Abstract

Interferon-γ-inducible lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. For this important function in the immune system, we cloned a GILT gene homologue from mandarin fish (designated mGILT), a kind of precious freshwater fish with high market value. Through reverse transcription PCR and rapid amplification of cDNA ends (RACE) strategies, we obtained the full-length cDNA of mGILT, which consists of 1008 bp with a 771 bp open reading frame, encoding a protein of 256 amino acids, with a putative molecular weight of 28.47 kDa. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 6 conserved cysteines. The result of real-time quantitative PCR showed that mGILT mRNA was expressed in a tissue-specific manner. In addition, the expression of mGILT mRNA was obviously up-regulated in splenocytes and kidney after induction with lipopolysaccharide (LPS). Recombinant mGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Further study revealed that mGILT exhibit thiol reductase activity on IgG substrate. These results suggest mGILT is highly likely to play a role in the immune responses in mandarin fish.

Keywords: Interferon-γ-inducible lysosomal thiol reductase; Mandarin fish; Real-time quantitative PCR; Thiol reductase activity; cDNA cloning.