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Fractionation of Fab glycosylated immunoglobulin G with concanavalin A chromatography unveils new structural properties of the molecule.

ABSTRACT: Concanavalin A (ConA) chromatography has been extensively used to separate asymmetric Immunoglobulin G (IgG), which possesses oligosaccharide attached to one of the two F(ab')2 arms, from symmetric IgG with no glycan attached to Fab fragments. In this study, applying affinity chromatography, silver stain, Western blot and lectin stain techniques, N- linked oligosaccharide attached to Fab fragment was demonstrated to be exposed on the surface of the protein and be accessible by ConA. In contrast, N- linked oligosaccharide attached to asparagine (Asn) 297 of IgG Fc was located in the inside of the natural protein and was inaccessible by ConA. In addition to asymmetric IgG, there are also detectable level of IgG with both F(ab')2 arms glycosylated that has not been reported previously. The discoveries of new basic molecular structure of IgG would have implications in understanding the function and properties of this important immune molecule with clinical applications.

SUBMITTER: Huang T

PROVIDER: S-EPMC5058747 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Fractionation of Fab glycosylated immunoglobulin G with concanavalin A chromatography unveils new structural properties of the molecule.

Huang Tao T Chen Xueling X Gu Huan H Zhao Conghui C Liu Xingmu X Yan Meiling M Deng Xiaodong X Zhang Zaiping Z Gu Jiang J

Oncotarget 20160501 21

Concanavalin A (ConA) chromatography has been extensively used to separate asymmetric Immunoglobulin G (IgG), which possesses oligosaccharide attached to one of the two F(ab')2 arms, from symmetric IgG with no glycan attached to Fab fragments. In this study, applying affinity chromatography, silver stain, Western blot and lectin stain techniques, N- linked oligosaccharide attached to Fab fragment was demonstrated to be exposed on the surface of the protein and be accessible by ConA. In contrast, ...[more]

PMID: 27145274

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Project description:Study questionHow does the placenta protect the fetus from immune rejection by the mother?Summary answerThe placenta can produce IgG that is glycosylated at one of its Fab arms (asymmetric IgG; aIgG) which can interact with other antibodies and certain leukocytes to affect local immune reactions at the junction between the two genetically distinct entities.What is known alreadyThe placenta can protect the semi-allogenic fetus from immune rejection by the immune potent mother. aIgG in serum is increased during pregnancy and returns to the normal range after giving birth. aIgG can react to antigens to form immune complexes which do not cause a subsequent immune effector reaction, including fixing complements, inducing cytotoxicity and phagocytosis, and therefore has been called 'blocking antibody'.Study design, size, durationEighty-eight human placentas, four trophoblast cell lines (TEV-1, JAR, JEG and BeWo), primary culture of human placental trophoblasts and a gene knock-out mouse model were investigated in this study.Participants/materials, setting, methodsThe general approach included the techniques of cell culture, immunohistochemistry, in situ hybridization, immuno-electron microscopy, western blot, quantitative PCR, protein isolation, glycosylation analysis, enzyme digestion, gene sequencing, mass spectrophotometry, laser-guided microdissection, enzyme-linked immunosorbent assay, pulse chase assay, double and multiple staining to analyze protein and DNA and RNA analysis at the cellular and molecular levels.Main results and the role of chanceThree major discoveries were made: (i) placental trophoblasts and endothelial cells are capable of producing IgG, a significant portion of which is aberrantly glycosylated at one of its Fab arms to form aIgG; (ii) the asymmetrically glycosylated IgG produced by trophoblasts and endothelial cells can react to immunoglobulin molecules of human, rat, mouse, goat and rabbit at the Fc portion; (iii) asymmetrically glycosylated IgG can react to certain leukocytes in the membrane and cytoplasm, while symmetric IgG from the placenta does not have this property.Limitations, reasons for cautionMost of the experiments were performed in vitro. The proposed mechanism calls for verification in normal and abnormal pregnancy.Wider implications of the findingsThis study identified a number of new phenomena suggesting that aIgG produced by the placenta would be able to react to detrimental antibodies and leukocytes and interfere with their immune reactions against the placenta and the fetus. This opens a new dimension for further studies on pregnancy physiology and immunology. Should the mechanism proposed here be confirmed, it will have a direct impact on our understanding of the physiology and pathology of human reproduction and offer new possibilities for the treatment of many diseases including spontaneous abortion, infertility and pre-eclampsia. It also sheds light on the mechanism of immune evasion in general including that of cancer.

Dataset Information

Fractionation of Fab glycosylated immunoglobulin G with concanavalin A chromatography unveils new structural properties of the molecule.

Publications

Fractionation of Fab glycosylated immunoglobulin G with concanavalin A chromatography unveils new structural properties of the molecule.

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