Molecular and biological characterization of gamma-interferon-inducible lysosomal thiol reductase in silver carp (Hypophthalmichthys molitrix)
Abstract Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in the processing of major histocompatibility complex (MHC) class II-restricted antigens by catalyzing disulfide bonds reduction. Herein, a GILT homolog (ScGILT) was identified from silver carp. Its open reading frame covers 771 base pairs, encoding a protein of 256 amino acids that possesses GILT signature sequence CQHGX 2 ECX 2 NX 4 C, active-site CXXC motif, and two potential N-linked glycosylation sites. The predicted tertiary structures of ScGILT and other GILTs were quite similar in shape and positional arrangement of the key motifs. ScGILT mRNA was constitutively expressed in all detected tissues, with high-level expression in fish immune organs, spleen and head kidney. After stimulation with lipopolysaccharide, the expression of ScGILT mRNA significantly increased in spleen and head kidney cells, and ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant ScGILT fused with a His 6 tag was expressed and purified, and could reduce the interchain disulfide bonds of IgG at pH 4.5. These results suggested that ScGILT was capable of catalyzing disulfide bonds reduction, and then might play an important role in the processing of MHC class II-restricted antigens in silver carp. Highlights The GILT homolog from silver carp (ScGILT) was identified. The ScGILT had typical characteristic sequences, and its predicted structure was similar to that of other GILTs. The ScGILT mRNA was expressed at the high levels in fish immune organs, spleen and head kidney. After stimulation with LPS, ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant His6-ScGILT could reduce the interchain disulfide bonds of IgG at pH 4.5.
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