Comparative N-Linked Glycan Analysis of Wild-Type and α1,3-Galactosyltransferase Gene Knock-Out Pig Fibroblasts Using Mass Spectrometry Approaches

Park, Haemin; Kim, Yoon-Woo; Kim, Kyoung-Jin; Kim, Yong Baek; Yang, Yung‐Hun; Jin, Jang Mi; Kim, Young Hwan; Kim, Byung‐Gee; Shim, Hosup; Kim, Yun‐Gon

doi:10.14348/molcells.2015.2240

Cited by 6 publications

(3 citation statements)

References 37 publications

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“…Because the cornea is an avascular organ, the effect of hyperacute rejection involving alpha Gal is expected to be less than that of a solid organ such as the kidney and heart. In addition, GTKO porcine cornea may increase the expression levels of non-Gal antigens because the level of non-Gal antigens from GTKO porcine fibroblasts is much higher than that from WT porcine fibroblasts ( 64 ). Indeed, elevated levels of non-Gal IgG were reported in rejected GTKO porcine corneas after corneal xenotransplantation.…”

Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation

Yoon

Ryu

et al. 2022

Front. Immunol.

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Although pig-to-non-human primate (NHP) corneal xenotransplantation has shown long-term graft survival, xenogeneic antigen-related immune responses are still stronger than allogeneic antigen-associated responses. Therefore, there is an unmet need to investigate major rejection pathways in corneal xenotransplantation, even with immunosuppression. This study aimed to identify biomarkers in aqueous humor for predicting rejection and to investigate rejection-related pathways in grafts from NHPs transplanted with porcine corneas following the administration of steroids combined with tacrolimus/rituximab. NHPs who had received corneas from wild-type (WT) or α-1,3-galactosyltransferase gene-knockout (GTKO) pigs were divided into groups with or without rejection according to clinical examinations. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the proteomes of corneal tissues or aqueous humor. The biological functions of differentially expressed proteins (DEPs) were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathways and protein–protein interaction network analysis. Among the 66 DEPs in aqueous humor, complement proteins (C3, C5, and C9) and cholesterol metabolic proteins (APOA1 and APOA2) were related to xenogeneic rejection as biomarkers, and alternative pathways of the complement system seemed to be important in xenogeneic graft rejection. Among the 416 DEPs of the cornea, NF-κB1 and proteosomes (PSMD7, PSMA5, and PSMD3) seemed to be related to xenogeneic graft rejection. Additionally, oxidative phosphorylation and leukocyte activation-related pathways are involved in rejection. Overall, our proteomic approach highlights the important role of NF-κB1, proteosomes, oxidative phosphorylation, and leukocyte activation-related inflammation in the cornea and the relevance of complement pathways of the aqueous humor as a predictive biomarker of xenogeneic rejection.

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Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation

Yoon

Ryu

et al. 2022

Front. Immunol.

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“…A higher expression of NeuGc has been demonstrated on GTKO pig tissue (i.e., heart, liver, kidney) and cells (fibroblast cells) compared to NeuGc expression on WT cells, suggesting that deletion of the α1,3-galactosyltransferase gene leads to a ‘compensatory’ increased expression of sialylated glycans, including NeuGc 25,26 . In contrast, in our previous experience we did not document any difference in expression of NeuGc between WT and GTKO pig corneas by immunofluorescence staining 15 .…”

Section: Discussionmentioning

confidence: 99%

Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

Lee

Miyagawa

Long

et al. 2016

Cornea

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Purpose Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (i) to document the lack of NeuGc expression on corneas and aortas, and cultured endothelial cells (aortic [AECs]; corneal [CECs]) of GTKO/NeuGcKO pigs, and (ii) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. Methods Wild-type (WT), GTKO, and GTKO/NeuGcKO pig were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and to determine human IgM and IgG binding to tissues. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. Results Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither human nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared to binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. Conclusions The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.

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“…A study of total N-glycans from membrane glycoproteins, derived from specific pathogen-free miniature pig kidneys, was carried out, and the result showed that α-gal was the most abundant compound among the neutral pig N-glycans. However, the various N-glycan structures of Neu5Gc and Gala1-3Lex epitope as new non-gal carbohydrate antigens were identified by mass spectrometry ( 46 , 63 ). Therefore, non-gal carbohydrate antigens may be the next complication in the transplantation from animals to humans.…”

Section: Clinical Exploitationmentioning

confidence: 99%

Characteristics of α-Gal epitope, anti-Gal antibody, α1,3 galactosyltransferase and its clinical exploitation (Review)

Huai

Pan

Yang

et al. 2015

International Journal of Molecular Medicine

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The α-Gal epitope (Galα1,3Galα1,4GlcNAc-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 α-Gal epitope, humans have a distinct anti-α-gal reactivity, which is responsible for hyperacute rejection of organs transplanted from α-gal donors. In addition, the α1,3 galactosyltransferases (α1,3GT) can catalyze the synthesis of the α-Gal epitope. Therefore, the α1,3GT gene, which encodes the α1,3GT, is developed profoundly. The distributions of the α-Gal epitope and anti-Gal antibody, and the activation of α1,3GT, reveal that the enzyme of α1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human α1,3-GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the α1,3GT 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 α-Gal epitope and anti-Gal antibody, they can be applied to clinical exploitation. Knocking out the α1,3GT gene will eliminate the xenoantigen, Gal(α1,3)Gal, so that the transplantation of α1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the α-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 α-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, α-Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.

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Comparative N-Linked Glycan Analysis of Wild-Type and α1,3-Galactosyltransferase Gene Knock-Out Pig Fibroblasts Using Mass Spectrometry Approaches

Cited by 6 publications

References 37 publications

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation

Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation

Characteristics of α-Gal epitope, anti-Gal antibody, α1,3 galactosyltransferase and its clinical exploitation (Review)

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