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Gal epitope expression and immunological properties in iGb3S deficient mice.

ABSTRACT: The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.

SUBMITTER: Shao A

PROVIDER: S-EPMC6194060 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Gal epitope expression and immunological properties in iGb3S deficient mice.

Shao Anliang A Xu Liming L Wu Xi X Liu Susu S Lu Yan Y Fan Changfa C

Scientific reports 20181018 1

The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using ...[more]

PMID: 30337628

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Project description:IntroductionAlpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS.MethodsEight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs.ResultsCompared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature.ConclusionTSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.

Project description:The ?-Gal epitope (Gal?1,3Gal?1,4GlcNAc?R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and Old World monkeys. However, the anti-Gal antibody (~1% of immunoglobulins) is naturally generated in human, and is found as the immunoglobulin G (IgG), IgM and IgA isotypes. Owing to the specific binding of the anti?Gal antibody with the ??Gal epitope, humans have a distinct anti???gal reactivity, which is responsible for hyperacute rejection of organs transplanted from ??gal donors. In addition, the ?1,3 galactosyltransferases (?1,3GT) can catalyze the synthesis of the ??Gal epitope. Therefore, the ?1,3GT gene, which encodes the ?1,3GT, is developed profoundly. The distributions of the ??Gal epitope and anti?Gal antibody, and the activation of ?1,3GT, reveal that the enzyme of ?1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human ?1,3?GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the ?1,3GT gene in ancestral primates attribute to the mutations under a stronger selective pressure. However, on the basis of the structure, the mechanism and the specificity of the ??Gal epitope and anti?Gal antibody, they can be applied to clinical exploitation. Knocking out the ?1,3GT gene will eliminate the xenoantigen, Gal(?1,3)Gal, so that the transplantation of ?1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the ??Gal epitope expressed through the application of chemical, biochemical and genetic engineering can be exploited for the clinical use. Targeting anti?Gal?mediated autologous tumor vaccines, which express ??Gal epitope to antigen?presenting cells, would increase their immunogenicity and elicit an immune response, which will be potent enough to eradicate the residual tumor cells. For tumor vaccines, the way of increasing immunogenicity of certain viral vaccines, including flu vaccines and human immunodeficiency virus vaccines, can also be used in the elderly. Recently, ??Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.

Project description:The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Gal?(1,3)Gal?(1,4)GlcNAc?, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-?-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Gal?(1,3)Gal?(1,4)GlcNAc?-BSA is recognized by purified anti-?-Gal Abs from chronic ChD patients ?230-fold more strongly than by anti-?-Gal Abs from sera of healthy individuals (NHS anti-?-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Gal?(1,3)Gal?(1,4)GlcNAc? ?20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Gal?(1,4)GlcNAc? and the monosaccharide GlcNAc? or GlcNAc? conjugated to BSA are poorly or not recognized by purified anti-?-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Gal? moiety for recognition by Ch anti-?-Gal Abs and the lack of Abs against nonself Gal?(1,4)GlcNAc? and GlcNAc? glycotopes. The substantial difference in binding of Ch vs. NHS anti-?-Gal Abs to Gal?(1,3)Gal?(1,4)GlcNAc?-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Gal?(1,3)Gal?(1,4)GlcNAc?-BSA NGP was then used to immunize ?1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Gal?(1,3)Gal?(1,4)GlcNAc? glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

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Gal epitope expression and immunological properties in iGb3S deficient mice.

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Gal epitope expression and immunological properties in iGb3S deficient mice.

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