title: Identification and characterization of a gamma-interferon-inducible lysosomal thiol reductase homolog from guinea pig (Cavia porcellus) that exhibits thiol reductase activity in vitro.
abstract: Gamma-interferon-inducible lysosomal thiol reductase (GILT) is a key enzyme in the antigen processing and presentation pathway whereby it reduces disulfide bonds at an acidic pH. In this study, a homolog of GILT from guinea pigs (designated gpGILT) was identified and characterized using bioinformatic methods and bioactivity assays. The open reading frame of gpGILT is 705bp in length and encodes 234 amino acids, with a putative molecular weight of about 25.85kDa. The structure of gpGILT is similar to those of humans and zebrafish, containing six introns and seven exons. The deduced primary structure of the gpGILT protein includes all of the typical features of other known GILT proteins, including an active-site motif, CXXC, a GILT signature sequence, CQHGX2ECX2NX4C, three potential Asn-linked glycosylation sites, and six other conserved cysteines. The predicted tertiary structures of gpGILT, human GILT, and mouse GILT are quite similar in shape and positional arrangement of the key motifs modeled on the same template. Amino acid sequence-based alignment and phylogenetic analysis showed that gpGILT is most closely related to that from the rat, with an identity of 68.40%. Additionally, the constitutive expression and immune response to lipopolysaccharide (LPS) challenge of gpGILT were tested using real-time quantitative polymerase chain reaction. A tissue-specific expression pattern in selected tissues and remarkable up-regulation of gpGILT mRNA in spleen and blood within 12h of LPS stimulation were observed, suggesting that GILT functions as an immunological surveillance-related factor in both innate and adaptive immunity. Soluble recombinant gpGILT produced in E. coli could reduce the interchain disulfide bonds of IgG in an acidic reaction system in vitro, suggesting thiol reductase activity in antigen processing. The results of this study provide a better understanding of the molecular characteristics of gpGILT and are a useful reference for further investigation of its involvement in antigen processing and immunological surveillance using the laboratory guinea pig.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Qilu Institute of Pharmaceutical Research, Qilu Pharmaceutical Co. Ltd, Jinan 250100, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Ma
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Cao
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Runze
authors.initials: R
authors.lastname: Tang
authors.affiliation: Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China. Electronic address: zhangjiaxin60633@163.com.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China. Electronic address: zhangshuangquan@njnu.edu.cn.
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs / Q000235:genetics* / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D007371:Interferon-gamma; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: Gamma-interferon-inducible lysosomal thiol reductase; Guinea pig; Immune response; Thiol reductase activity