α-Gal Epitope Research Articles

Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene: MODULATION OF THE BIOSYNTHESIS OF TERMINAL STRUCTURES BY A BISECTING GlcNAc

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

Expression of xenoantigen transformed human cancer cells to be susceptible to antibody-mediated cell killing

The carbohydrate epitope, αGal epitope, is known as a major xenoantigen. The epitope exists abundantly in non-primate mammals and has recently been found on C-type retroviruses. Humans and Old World monkeys have anti-αGal antibody, a natural antibody. The present study was performed to examine if the αGal epitope could be used as a new target of gene therapy against human cancer. Bovine α1–3 galactosyltransferase (α1–3 GT) cDNA which produces the αGal epitope was electrophoretically transfected into the human pancreatic cancer cell line, MIA PaCa-2 and the human hepatocellular carcinoma cell line, huH7. The expression of the αGal epitope was confirmed by flow cytometry, using specific binding with IB4 lectin conjugated with fluorescein isothiocyanate. Transfected MIA PaCa-2 cells and huH7 cells showed a positive log shift for the αGal epitope. Next, we examined whether human cancer cells expressing the αGal epitope could be lysed by natural antibodies using the complement-dependent cytotoxic cross-match test. The results showed that transfected MIA PaCa-2 cells and huH7 cells were effectively lysed by human natural antibodies, and the killing was observed with any serum irrelevant of blood type. The results indicate that transfection of the functional α1–3 GT gene into human cancer cells can lead to their transformation, making them susceptible to lysis by natural antibodies, and, thus, useful for gene therapy.

Synthesis of α-Gal epitope derivatives with a galactosyltransferase–epimerase fusion enzyme

α-Gal epitopes are carbohydrate structures bearing an α- d-Gal p-(1→3)-β- d-Gal p terminus and are the main cause of antibody-mediated hyperacute rejection in xenotransplantation. Nine monosaccharides and ten disaccharides were evaluated as substrates for a fusion protein, which contains both α-(1→3)-galactosyltransferase and uridine-5′-diphosphogalactose 4-epimerase. Four disaccharide and six trisaccharide α-Gal epitope derivatives were synthesized utilizing this novel fusion enzyme.

Induction of B-cell tolerance by retroviral gene therapy

AbstractThe primary immunologic barrier to overcome before clinical xenotransplantation can be successful is rejection mediated by preformed natural antibodies in the host, directed toward a single carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (αGal) present on porcine tissue, encoded for by the enzyme glucosyltransferase UDP galactose:β-D-galactosyl-1,4-N-acetyl-D-glucosaminide α(1-3)galactosyltransferase (EC 2.4.1.151) or simply αGT. Although we have shown previously that a gene therapy approach could be used to prevent production of natural antibodies specific for αGal, the ability to induce and maintain tolerance after rigorous antigen challenge would be required if similar approaches are to be used clinically. Here, we demonstrate in αGT knockout mice (GT0 mice), which, like humans, contain in their serum antibodies that bind αGal, that the efficient transduction and expression of a retrovirally transduced αGT gene in bone marrow–derived cells induces stable long-term tolerance to the αGal epitope. GT0 mice reconstituted with αGT-transduced bone marrow cells were unable to produce antibodies that bind αGal after extensive immunization with pig cells. Furthermore, using ELISPOT assays, we were unable to detect the presence of B cells that produce αGal reactive antibodies after immunization, suggesting that such B cells were eliminated from the immunologic repertoire after gene therapy. Interestingly, after tolerance to αGal is induced by gene therapy, the antiporcine non-αGal humoral response changes from a predominantly IgM to an IgG response. This suggests that once the natural antibody barrier is eliminated by the induction of tolerance, the antipig response changes to a typical T-cell–dependent response involving isotype switching. Thus, gene therapy approaches may be used to overcome immunologic responses leading to xenograft rejection, and similar gene therapy approaches could be used to overcome autoimmunity.

Induction of B-cell tolerance by retroviral gene therapy

The primary immunologic barrier to overcome before clinical xenotransplantation can be successful is rejection mediated by preformed natural antibodies in the host, directed toward a single carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (αGal) present on porcine tissue, encoded for by the enzyme glucosyltransferase UDP galactose:β-D-galactosyl-1,4-N-acetyl-D-glucosaminide α(1-3)galactosyltransferase (EC 2.4.1.151) or simply αGT. Although we have shown previously that a gene therapy approach could be used to prevent production of natural antibodies specific for αGal, the ability to induce and maintain tolerance after rigorous antigen challenge would be required if similar approaches are to be used clinically. Here, we demonstrate in αGT knockout mice (GT0 mice), which, like humans, contain in their serum antibodies that bind αGal, that the efficient transduction and expression of a retrovirally transduced αGT gene in bone marrow–derived cells induces stable long-term tolerance to the αGal epitope. GT0 mice reconstituted with αGT-transduced bone marrow cells were unable to produce antibodies that bind αGal after extensive immunization with pig cells. Furthermore, using ELISPOT assays, we were unable to detect the presence of B cells that produce αGal reactive antibodies after immunization, suggesting that such B cells were eliminated from the immunologic repertoire after gene therapy. Interestingly, after tolerance to αGal is induced by gene therapy, the antiporcine non-αGal humoral response changes from a predominantly IgM to an IgG response. This suggests that once the natural antibody barrier is eliminated by the induction of tolerance, the antipig response changes to a typical T-cell–dependent response involving isotype switching. Thus, gene therapy approaches may be used to overcome immunologic responses leading to xenograft rejection, and similar gene therapy approaches could be used to overcome autoimmunity.

Changing the Donor Cofactor of Bovine α1,3-Galactosyltransferase by Fusion with UDP-galactose 4-Epimerase: MORE EFFICIENT BIOCATALYSIS FOR SYNTHESIS OF α-Gal EPITOPES

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.

Genes coding for anti-Gal in knock-out mice for the α1,3Galactosyltransferase gene: analysis by hybridomas

THE interaction between the natural anti-Gal antibody and the a-gal epitope (Gala1-3Galb1-4GlcNAc-R) on xenograft cells results in rejection of the xenograft. Moreover, activity of anti-Gal increases by manyfold in xenograft recipients as a result of immune response to a-gal epitopes on the graft. This elicited anti-Gal greatly exacerbates xenograft rejection. A potential method for prevention of anti-Gal production in xenograft recipients is the treatment of such patients with anti-idiotype antibodies for anti-Gal. Such antibodies may neutralize anti-Gal and destroy cells capable of producing this antibody. To determine the possible diversity of such anti-idiotypes, the anti-Gal Ig genes have to be analyzed. As a first step, we studied the diversity of the immunoglobulin genes coding for this antibody in a1,3galactosyltransferase (a1,3GT) knock-out (KO) mice. These mice lack a-gal epitopes and are capable of producing large amounts of anti-Gal, upon immunization with xenograft cell membranes that express a-gal epitopes. Anti-Gal hybridomas were produced by fusion of splenocytes from KO mice, immunized with rabbit red cell membranes, with the mouse myeloma fusion partner X63.Ag.653. Hybridomas from several mice were screened for secretion of anti-Gal in ELISA with synthetic a-gal epitopes linked to BSA, as solid-phase antigen, as previously described. The specificity of these monoclonal antibodies was confirmed by demonstrating their specific binding to glycoproteins that express the a-gal epitope, such as mouse laminin and bovine thyroglobulin. Three hybridomas producing anti-Gal IgM were isolated, each from a different mouse. The Ig genes in these hybridomas were cloned and sequenced. All three anti-Gal clones contained heavy chain (VH) genes derived from the germline Ig gene VH22.1 and light chain (VL) genes derived from the germline Ig gene K5.1 (Table 1). These anti-Gal coding genes contained various somatic mutations in CDR1 and CDR2 regions. Some mutations were also found in frame work (FR) 2 and 3 regions. In addition, the CDR 3 regions differed significantly from one clone to the other. Interestingly, the sequences of the VL genes were much more conserved than those of the VH genes. Analysis of replacement (R) versus silent (S) mutations demonstrated a much higher proportion of R mutations in the CDR regions than in the FR regions (Table 1). Overall, these findings indicated that the Ig gene repertoire for anti-Gal is highly restricted in KO mice, and it consists primarily of one light and one heavy chain Ig genes. This limited repertoire is likely to reflect the structural

T cells interacting with the α-Gal epitope: studies in α1,3Galactosyltransferase knock-out mice

T cells interacting with the α-Gal epitope: studies in α1,3Galactosyltransferase knock-out mice

Differential expression of α-Gal epitopes on pig and mouse organs

Differential expression of α-Gal epitopes on pig and mouse organs

Identification of B cells with receptors for α-Gal epitopes (Galα 1-3Galβ1-4GlcNac-R) in xenograft recipients

Identification of B cells with receptors for α-Gal epitopes (Galα 1-3Galβ1-4GlcNac-R) in xenograft recipients

Genes coding evolutionary novel anti-carbohydrate antibodies: studies on anti-Gal production in α1,3galactosyltransferase knock out mice

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The α-gal epitope (Galα1–3Galβ1–4GlcNAc-R) is an autoantigen, abundantly expressed in wild type mice, but absent in α1,3galactosyltransferase knock-out (KO) mice, where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover, one ‘forbidden’ anti-Gal clone, produced in a wild type mouse, was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species.

Anti-Gal antibodies in humans and 1, 3alpha-galactosyltransferase knock-out mice.

Due to the absence of alphaGAL epitopes, humans and galactosyltransferase knock-out (GALT/ KO) mice express high levels of anti-Gal antibodies. We describe the properties of mouse anti-GAL antibodies. Anti-GAL IgG antibodies were quantified by affinity purification. Antibody affinities and avidities were determined in direct binding and competition assays. Antibody-mediated rejection was investigated using hyperimmunized GALT/KO mice as recipients of GAL+ heart allografts. In young GALT/KO mice the levels of anti-GAL antibodies were low. Immunization of GALT/KO mice resulted in increased anti-GAL antibody expression. In mouse serum 0.6% of IgG was specific for alphaGAL compared to 0.5% in human serum. The avidity of purified mouse and human anti-GAL IgG was 30 and 6 nM, the affinity 15 and 50 microM, respectively. The isotype distribution in mouse and human anti-GAL IgG appeared to be similar to the isotype distribution in normal sera. The affinity of mouse and human anti-GAL IgM was 150 and 750 microM, respectively. Immunized GALT/KO recipients of GAL+ heart transplants rejected their grafts within 2 hr although nonimmunized GALT/KO mice retained their grafts for up to 6 days. Immunohistological examination of the rejected GAL+ hearts revealed massive deposition of IgM and IgG on endothelial cells of the graft with a concomitant deposition of complement. Our studies demonstrate that anti-GAL antibodies from immunized GALT/KO mice bind alphaGAL with an avidity/affinity similar to human anti-GAL antibodies and are able to induce hyperacute rejection of GAL+ heart allografts.

Introduction to porcine red blood cells: Implications for xenotransfusion

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells ( pRBCs) as an alternative source for blood transfusion. The domestic pig is considered the most likely donor species for xenotransplantation. However, identification of xenoantigens on porcine erythrocytes and elucidation of their possible roles in antibody-mediated RBC destruction are necessary for developing clinical strategies to circumvent immunological incompatibility between humans and pigs. Although the αGal epitope (Galα1,3Galβ1,4GlcNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-αGal xenoantigens have been identified. The importance of these non-αGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated. Our data suggest that non-αGal xenoantigen(s) identified on the porcine erythrocyte membrane are not only recognized by xenoreactive human natural antibodies but are also involved in complement-mediated hemolysis.

Differential immune responses to alpha-gal epitopes on xenografts and allografts: implications for accommodation in xenotransplantation.

Xenograft recipients produce large amounts of high-affinity anti-Gal IgG in response to Galalpha1-3Galbeta1- 4GlcNAc-R (alpha-gal) epitopes on the graft. In contrast, ABO-mismatched allograft recipients undergo "accommodation," a state of very weak immune response to ABO antigens. These differences in anti-carbohydrate immune response were studied in alpha1,3galactosyltransferase knock-out mice. Pig kidney membranes administered to these mice elicited extensive production of anti-Gal IgG, whereas allogeneic kidney membranes expressing alpha-gal epitopes elicited only a weak anti-Gal IgM response. Anti-Gal IgG response to xenograft membranes depended on helper T cell activation and was inhibited by anti-CD40L antibody. These T cells were activated by xenopeptides and not by alpha-gal epitopes. Moreover, allogeneic cell membranes manipulated to express xenoproteins also induced anti-Gal IgG response. Xenoglycoproteins with alpha-gal epitopes are processed by anti-Gal B cells. Xenopeptides presented by these cells activate a large repertoire of helper T cells required for the differentiation of anti-Gal B cells into cells secreting anti-Gal IgG. Alloglycoproteins with alpha- gal epitopes have very few immunogenic peptides and fail to activate helper T cells. Similarly, ineffective helper T-cell activation prevents a strong immune response to blood group antigens in ABO-mismatched allograft recipients, thus enabling the development of accommodation.

Glycosyltransferase Genes: Applications to Medical Science.

Glycosylation represents a major posttranslational modification reaction of proteins and plays a pivotal role in a variety of cellular events. N-Acetylglucosaminyltransferases III (GnT-III) and V (GnT-V), and α 1-6 fucosyltransferase are key enzymes in the processing of N-linked glycoproteins, and are heavily involved in cancer progression and metastasis. Very recently we also found that GnT-III is capable of reducing or masking the expression of the αGal epitope, which is a major xenoantigen in swine to human xenotransplantation. In this minireview we focus on the role of the above glycosyltransferases in cancer metastasis as well as in xenotransplantation.

Serum levels of xenoantibodies in different groups of patients awaiting transplantation

THE BINDING of anti-aGal human xenoantibodies (xenoAb) to aGal epitopes expressed on pig endothelial tissues leads to cell activation and complement-mediated hyperacute xenograft rejection. Although all immunocompetent individuals have xenoAb, there is scarcely any information about the occurrence of serum xenoAb in normal people and the influence of immune or metabolic condition of each group of transplantation recipients on serum levels of xenoAb. Additionally, it has been published that human immunodeficiency virus (HIV) seems unable to infect nonhuman T cells, especially nonprimate T cells, so bone marrow xenotransplantation could be a successful therapy in selected cases to restore a competent immune system. Cognate interactions between T and B cells play a critical role for an adequate synthesis of immunoglobulins. For this reason, a severe depletion of CD41 T cells might be responsible for a decrease in xenoAb and for an easier xenograft acceptance. The aim of this study was to investigate the serum concentrations of xenoAb (IgM and IgG) in several human groups: First, a group of normal population (controls) with different ages and blood groups; second, groups of patients waiting for different types of transplantation; and third, a group of HIV-infected patients.

Enhanced Inhibition of Human Anti-Gal Antibody Binding to Mammalian Cells by Synthetic α-Gal Epitope Polymers

Neoglycopolymers of polyacrylamide backbone conjugated with varying densities of Galα1−3Galβ1−4Glcβ trisaccharide epitopes (α-Gal epitopes) were designed and synthesized to study the inhibition of the binding of human natural anti-Gal antibodies to either α-Gal-containing glycoproteins or α-Gal antigens on the surface of mammalian cells. An inhibition ELISA using mouse laminin and a flow cytometry assay using pig kidney cells (PK15) were established to determine the binding affinity of the synthesized polymers. In comparison to the α-Gal monomer (Galα1−3Galβ1−4GlcNHAcβ), the α-Gal polymers dramatically enhanced the inhibition of human anti-Gal antibodies (IgG, IgM, and IgA) binding to mouse laminin or mammalian cells. Increases of 7.8 × 103- and 5.0 × 104 -fold in inhibitory potential of polymer 7C to IgA and IgM (with IC50s of 7.0 and 5.6 nM respectively) were observed over the monomer in inhibition ELISA. The results also indicated that binding enhancement of α-Gal polymers is greater for anti-Gal IgA a...

Recent Progress in Glycochemistry and Green Chemistry

Abstract: This two-part article will focus on the most recent research efforts from the Wang's group in areas of glycochemistry and green chemistry. In the first part, we wish to address the progress in synthesis, conformational analysis, and transformations of some biologically active a­ galactosyl oligosaccharides. Carbohydrate structures containing Gala1-3Galterminus (a-Gal epitopes) are xenoactive antigens and are considered to be the major cause for hyperacute rejection in xenotransplantation. The latest chemo-enzymatic synthesis of a-galactosyl epitopes using a recombinant a1,3-galactosyltransferase is described. Along with an efficient chemical synthesis of an a-Gal trisaccharide, these synthetic approaches provide an easy access to this important class of oligosaccharides. Conformational analysis of an a-Gal epitope using various NMR techniques and molecular modeling is discussed. These studies provide important information in structure-function relationship and binding conformation of a-Gal epitopes and their interaction with anti-Gal antibody. In another aspect, novel glycoconjugates containing receptor-specific ligands and a-Gal epitopes have been synthesized as potential immunotherapeutic agents.

ANTI-GAL ANTIBODIES FROM HUMANS AND 1,3??-GALACTOSYLTRANSFERASE KNOCKOUT MICE.

54 All mammals, except Old World monkeys including humans, express the enzyme 1,3α-galactosyltransferase that couples terminal αGAL epitopes to glycoproteins and glycolipids. As a result of the lack of αGAL epitopes human serum contains a high level of natural antibodies recognizing αGAL. These anti-GAL antibodies have been identified as a major obstacle for the successful transplantation of GAL+ organs into primates. Mice have been generated in which the 1,3α-galactosyltransferase gene is knocked-out by homologous recombination (GAL/TKO mice). In young GAL/TKO mice however, the levels of anti-GAL antibodies were below detection limit. In order to increase IgG and IgM anti-GAL antibody titers in GAL/TKO mice, an immunization protocol was developed in which rabbit red blood cells were used as a source of GAL epitopes. Repeated immunizations were necessary to increase anti-GAL antibody titers. Following immunization. the observed titers varied from 1:100 to 1:1000. Anti-GAL antibodies from mice and humans were compared in binding studies. Direct binding assays of human or mouse anti-GAL IgG or IgM to BSA/GAL showed that antibody titers in immunized mice were higher than those in humans (1:1000 vs 1:40). The affinity of anti-GAL antibodies was measured in competition assays using soluble α1-3,β1-4-Dgalactotriose. Antibody binding to BSA/GAL was measured in the presence of increasing concentrations of galactotriose. The concentration of galactotriose inhibiting antibody binding by 50% was 20 uM for human and mouse anti-GAL IgG antibodies. In contrast, IgM anti-GAL antibodies had a substantially lower affinity (800 uM for human IgM Vs 100 uM for mouse IgM). When hyperimmunized mice were used as recipients of GAL+ heart transplants, hyperacute rejection of these grafts (<2 hrs) was observed. In non-immunized GAL/TKO mice GAL+ hearts remained functional for up to 6 days. Immunohistological examination of the rejected GAL+ hears revealed massive deposition of IgM on endothelial cells of the graft with a concomitant deposition of complement. Therefore, our studies demonstrate that anti-GAL antibodies expressed in immunized GAL/TKO mice bind αGAL with similar affinity as human anti-GAL antibodies and cause hyperacute rejection of GAL+ heart transplants.

INDUCTION OF IN VITRO ACCOMODATION OF PORCINE ENDOTHELIAL CELLS HUMAN ANTI-??GAL XENOANTIBODIES

283 Pre-incubation of porcine aortic endothelial cells (PAECs) with heat-inactivated human serum (HS) or HS immunoglobulin fractions has been shown to induce resistance to subsequent (1,2) complement-mediated lysis (CML). The purpose of this study was to evaluate the ability of purified human anti-αGal antibodies to induce resistance of PAECs to lysis by human complement, a phenotype characteristic of in vitro accomodation. Human anti-αGal antibody was absorbed from human serum obtain from 3 healthy donors using an αGal-coupled affinity colum. PAECs were incubated with the antibody for 3, 5, and 6 days and were compared with a control group of PAECs incubated with serum free media (SFM) for similar time periods. Following incubation, antibody-treated PAECs were assessed for their resistance to complement-mediated lysis (CML) using a Cr-release assay. (Table)TableThe results indicate that purified human anti-αGal antibodies induce in vitro accommodation of PAECs, and that the extent to which the PAECs become "accommodated" depends on both the concentration of antibody and the duration of incubation. In addition, incubation of PAECs with unfractionated HS from the same donors, for the same time periods did not induce resistance to CML. Similarly, the αGal-depleted HS did not induce resistance to CML and in some instances appeared to be cytotoxic to PAECs after less than 48 hour incubation. Overall, the results of this study suggest that manipulation of the interaction between human anti-αGal antibodies and the αGal epitope could yield strategies that induce graft accomodation which could help make xenotransplantation clinically feasible.