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Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation:Implications for Natural Killer T-Cell Biology

Significance Statement

Long time hypothesized pig iGb3 enzyme involved in xeno-transplantation found to be functionally inactive in vivo.

Galα3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation and acute vascular rejection of xenografts. The enzyme α1,3-galactosyltransferase enzyme 1 (α1,3GalT1, EC 2.4.1.51) synthesizes Galα3Gal epitopes (Galα3Gal{Beta}4GlcNAc-R) on the cell surface in almost all mammals with exceptions of humans, apes, and Old World monkeys. In addition, isoglobotriaosylceramide (iGb3) synthase (iGb3S, EC 2.4.1.87), another member of the α1,3-glycosyltransferase family, initiates the synthesis of isoglobo-series glycosphingolipids (GSLs). Starting from iGb3, a series of α3Gal-terminated GSLs, B4, B5, and B6, can be synthesized. This raises the possibility that iGb3S may synthesize the Galα3Gal structure in α3GalT1−/− animals.

Galα3Gal epitopes are found to be recognized by both humoral and cellular arms of immunity in human that α3GalT1 gene is inactivated as pseudogene.  Antibodies against Galα3Gal epitopes exist in human, and mediate HAR through complement mediated lysis and antibody dependent cellular cytotoxity to grafted organs and vascular structures during xenotransplatation. Galα3Gal{Beta}4Glc{Beta}1-Cer (iGb3), a glycospingolipid, is recognized by invariant natural killer T (NKT) cells, a critical type of innate immune cells functionally similar to NK cells. The semi-invariant NKT-cell receptor recognizes glycolipid antigens presented by the monomorphic, evolutionally conserved CD1d molecule. Pig iGb3 enzyme, serves as a potential candidate which synthesizes stimulatory ligand in pig organs that causes rejection of α1,3-galactosyltransferase-1-deficient, xenotransplanted organs through activating NKT cells.

Although the in vitro activity of pig iGb3 enzyme was reported a decade ago, it was never studied whether this enzyme functions in living mammals. In 2009, two groups in Switzerland and USA independently published negative findings of iGb3 in pig organs and pig endothelial cells derived from α3GalT1-/- animals (1-2). Extensive studies using NMR and mass spectrometry were subsequently published after and a final conclusion was reached by Journal of Carbohydrate Chemistry (3-5). To the great surprise of all fields of clinical immunology, glycobiology, and carbohydrate chemistry, iGb3 enzyme is clearly inactive in all major organs examined, and does not synthesize any iGb3 or related structure.

This surprising finding may be explained by the fact that NKT cells, like the other innate lymphocyte type natural killer cells, were originally created to battle pathogens such as  parasites, viruses and bacteria. The loss of iGb3 in mouse, pig, and human (except extremely low abundance in thymus) suggest that the immune system has evolved to eliminate the expression of such self antigenic ligands in main organs during evolution. However, a minimum amount of iGb3 and potentially other structurally related glycolipids, are preserved in thymus and immune organs to mediate the development and function of NKT cells.

The functional inactivation of iGb3 synthase in major organs might be similar to the inactivation of the other member of α3GT family (α3GalT1), that is believed to be caused by immune selection pressure when human and Old World monkeys produce anti-carbohydrate antibodies toward gut bacteria which share structural similarities with Galα3Gal epitope. However, the molecular mechanisms responsible for iGb3 enzyme inactivation remain unclear. Unlike αGalT1 which is a clear pseudogene in human and Old World monkeys with multiple frameshift mutations and nonsense codons, the mouse, pig, and human iGb3 enzymes are active in vitro by cell transfection assays. The exact in vivo mechanisms that they are suppressed and functionally become pseudogene in organs might be caused by complex regulation at transcriptional level, or posttranscriptional level in Golgi apparatus with potential involvement of other chaperon proteins.

Reference

1. Yung, GLP, Li YS, Borsig L, Karpova M, Zhou DP. Absence of isoglobotrihexosylceramide 3 and the alpha gal xeno-antigen in endothelial cells derived from alpha-1,3galactosyltransferase knock out (galtko) pigs. XENOTRANSPLANTATION. 2009 Sept. 16(5): 366-367

2. Zhou DP, Hawke, D, Li YS, Levery SB. Lack of Igb3 and Isoglobo-Series of Glycosphingolipids in Pig Organs Related to Xenotransplantation: Implications for NKT Cell Biology. 2009 Nov. GLYCOBIOLOGY 19(11) :1304-1304

3. Diswall M, Gustafsson A, Holgersson J, Sandrin MS, Breimer ME. Antigen-binding specificity of anti-{Alpha}Gal reagents determined by solid-phase glycolipid-binding assays. A complete lack of {Alpha}Gal glycolipid reactivity in {Alpha}1,3GalT-KO pig small intestine. Xenotransplantation. 2011 Jan-Feb;18(1):28-39.

4. Puga Yung GL, Li Y, Borsig L, Millard AL, Karpova MB, Zhou D, Seebach JD. Complete absence of the {Alpha}Gal xenoantigen and isoglobotrihexosylceramide in {Alpha}1,3galactosyltransferase knock-out pigs. Xenotransplantation. 2012 May-Jun;19(3):196-206.

5. Tahiri F, Li Y, Hawke D, Ganiko L, Almeida I, Levery S, Zhou D. Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation: Implications for Natural Killer T-Cell Biology. J Carbohydr Chem. 2013 Jan 1;32(1):44-67.

Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation- global medical discovery

 

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

Tahiri F, Li Y, Hawke D, Ganiko L, Almeida I, Levery S, Zhou D.

J Carbohydr Chem. 2013 Jan 1;32(1):44-67.

MD Anderson Cancer Center, University of Texas, Houston, Texas, USA.

Abstract

{Alpha}-1,3-Terminated galactose residues on glycoproteins and glycosphingolipids are recognized by natural anti-α-1,3-galactose antibodies in human serum and cause hyperacute rejection in pig-to-human xenotransplantation. Genetic depletion of α-1,3-galactosyltransferase-1 in pigs abolishes the hyperacute rejection reaction. However, the isoglobotriosylceramide (iGb3) synthase in pigs may produce additional α-1,3-terminated galactose residues on glycosphingolipids. In both α-1,3-galactosyltranserase-1 knockout mice and pigs, cytotoxic anti-α-1,3-galactose antibodies could be induced; thus, a paradox exists that anti-α-1,3-galactose antibodies are present in animals with functional iGb3 synthases. Furthermore, iGb3 has been found to be an endogenous antigen for natural killer T (NKT) cells, an innate type of lymphocyte that may initiate the adaptive immune responses. It has been reasoned that iGb3 may trigger the activation of NKT cells and cause the rejection of α-1,3-galactosyltransferase-1-deficient organs through the potent stimulatory effects of NKT cells on adaptive immune cells (see ref.([20])). In this study, we examined the expression of iGb3and the isoglobo-series glycosphingolipids in pig organs, including the heart, liver, pancreas, and kidney, by ion-trap mass spectrometry, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 ug/mL of the total iGb3/Gb3 mixture is present (see ref.([35])). We did not detect iGb3 or other isoglobo-series glycosphingolipids in any of these organs, although they were readily detected in mouse and human thymus and dendritic cells. The lack of iGb3 and isoglobo-series glycosphingolipids in pig organs indicates that iGb3 is unlikely to be a relevant immune epitope in xenotransplantation.

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