Alpha-galactosyl Epitopes Research Articles

Immunologic analyses of bovine bone treated with a novel tissue sterilization process

This study evaluates the efficacy of removal of xeno-antigens from bovine bone using a patented BioCleanse process for decellularization of allograft tissues for clinical implantation. BioCleanse deploys a combination of chemicals and several high pressure rinses to achieve standardized sterility assurance levels. This method produces sterile grafts without reducing allograft bone biomechanical properties and effectively removes cells, lipids, and other sources of antigenic material from human allografts for clinical use. In this investigation, BioCleanse is evaluated for its potential in removing xenograft antigens from bovine bone grafts followed by immunologic evaluation in the subcutaneous pouch of immunocompetent rats. The alpha-galactosyl (alpha-gal) epitope with the structure Galalpha1-3Galbeta1-4GlcNAc-R constitutes a critical component of xenoantigens and its removal using BioCleanse from bovine bone was compared with tissue levels of unprocessed bone. The relative degree of antigen removal was also determined through measuring the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha, and through the use of histologic grading of cellular infiltrates into bone. Compact cortical bone inhibited immune cell migration but cancellous bone demonstrated cellular increase and bone resorption in the untreated control group. The alpha-gal xenoantigen level was significantly lower in both cortical (P < 0.001) and cancellous bone (P < 0.001) compared with controls. TNF-alpha levels were significantly (P < 0.001) reduced compared with untreated controls when human acute monocytic leukemia cells were exposed to cortical or cancellous bone. BioCleanse effectively removed xenoantigens and inflammatory markers justifying a follow up study in primates to determine these benefits in a model that is primed with preformed xeno-antibodies responsible for hyperacute rejection in hard tissues.

The Possible Contribution of Anti-Gal to Graves' Disease

Anti-Gal is a natural antibody specific for the alpha-galactosyl epitope. Previous studies suggested that Graves' disease (GD) patients had elevated anti-Gal titers compared to normal controls, but titers returned to normal after treatment. We developed an anti-Gal enzyme-linked immunosorbent assay (ELISA) using the property of anti-Gal to bind tightly to mouse laminin. We found no significant correlations between anti-Gal and thyroidstimulating immunoglobulin (TSI) or free thyroxine (T(4)) in untreated hyperthyroid GD patients (n = 15) without clinical ophthalmopathy or euthyroid, previously treated GD patients with ophthalmopathy. There was a significant regression between TSI and free T(4) in the hyperthyroid patients (p < 0.01). Addition of total anti- Gal antibody to the regression showed a trend toward improved correlation (p = 0.15 for improved correlation relative to TSI and free T(4) alone), suggesting it may stimulate GD thyroid tissue. However, in contrast to previous studies we found hyperthyroid patients (n = 20) had lower levels of anti-Gal immunoglobulin G (IgG) (18.4 +/- 4.0 vs. 41.8 +/- 8.9) than normals (n = 36 p < 0.05). Interestingly, hyperthyroid patients without clinical ophthalmopathy tended to have lower IgG anti-Gal levels than euthyroid patients with ophthalmopathy (p = 0.1). Hyperthyroidism significantly lowers anti-Gal, but the possible increase of anti-Gal in patients with ophthalmopathy suggests anti-Gal may play a role in ophthalmopathy, or may reflect the euthryoid status of these patients. This trend needs further study.

Isolation of the Feline α1,3‐Galactosyltransferase Gene, Expression in Transfected Human Cells and its Phylogenetic Analysis

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.

Structure-Based Engineering of E. coli Galactokinase as a First Step toward In Vivo Glycorandomization

In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing alpha(1,3)galactosyl epitopes.

The major barrier for xenotransplantation in humans is the presence of alpha(1-3) Galactosyl epitopes (alphaGal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-alphaGal Ab to cells expressing alphaGal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express alphaGal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in alphaGalactosyltransferase knockout (alphaGT KO) mice which, like humans, do not express alphaGal on their cell surfaces and can produce anti-alphaGal Ab. Forty-five percent of mice with preexisting anti-alphaGal Ab rejected alphaGal positive melanoma cells (B16alphaGal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, alphaGal negative cells succumbed to melanoma. The rejection of B16alphaGal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16alphaGal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of alphaGal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in alphaGal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies.

Sensitivity to human serum of gammaretroviruses produced from pig endothelial cells transduced with glycosyltransferase genes.

Reduction of pig cell-surface alpha-galactosyl (Gal) epitope, Galalpha1, 3Galbeta1, 4GlcNAc-R, by the introduction of glycosyltransferase genes is effective in suppressing hyperacute rejection (HAR) in pig-to-human xenotransplantation. The transmission of porcine endogenous retroviruses (PERVs) has been recognized as a potential risk factor associated with xenotransplantation. In this study, effects of the introduction of glycosyltransferase genes to pig cells on the sensitivity of gammaretroviruses to human serum were investigated. Pig endothelial cells (PEC), PEC transduced with alpha1,2 fucosyltransferase (FT), alpha2,3 sialyltransferase (ST), or N-acetylglucosaminyltransferase III (GnT-III), and human embryonic kidney (HEK) 293 cells were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection. Then, the cells were further infected with PERV subtype B (PERV-B) or feline leukemia virus subgroup B (FeLV-B). Culture supernatants of the infected cells were mixed with human serum (HS) and then inoculated to HEK293 cells. The inoculated cells were histochemically stained and lacZ-positive blue foci were counted. Glycosyltransferase activity, xenoantigenicity, and alpha-Gal epitope density in the cells were measured at the time of the infection experiments. PERV-B or FeLV-B particles from the parental PEC were efficiently neutralized by HS, while those from PEC transduced with alpha1,2FT, alpha2,3ST or GnT-III were less sensitive to HS. The transduced PEC exhibited high levels of activity of the introduced glycotransferases, and expressed fewer xenoantigens and cell-surface alpha-Gal epitopes. Our results suggest that gammaretroviruses including PERVs produced by transgenic pigs, that are generally modified to reduce the cell-surface alpha-Gal epitope to overcome the HAR in xenotransplantation, are less sensitive to HS.

Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels.

We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

Structure of bovine alpha-1,3-galactosyltransferase and its complexes with UDP and DPGal inferred from molecular modeling.

A homology model of alpha-1,3-galactosyltransferase (alpha-1,3-GalT), the retaining enzyme responsible for the formation of alpha-galactosyl epitopes, has been developed by means of molecular modeling using the SpsA glycosyltransferase structure. A protein-ligand docking approach was used to model alpha-1,3-GalT complexed with UDP and UDP-Gal. The comparison of structural features found in the alpha-1,3-GalT homology model with available structural data on this class of enzymes revealed similarities in the UDP-binding pocket. In the predicted structure of the complexes, the pyrophosphate interacts with the DVD motif (Asp-225, Val-226, and Asp-227) of alpha-1,3-GalT through the Mn(2+) cation. The uridine part of the UDP binds into the well-defined cavity that consists of Phe-134, Tyr-139, Ile-140, Val-136, Arg-194, Arg-202, Lys-209, Asp-173, His-218, and Thr-137 in a conformation that is generally observed in the crystal structures of other glycosyltransferase complexes.

Xenotransplantation for brain repair: reduction of porcine donor tissue immunogenicity by treatment with anti-Gal antibodies and complement.

Transplantation of embryonic neural tissue is a potential treatment for Parkinson's disease. Because human donor material is in short supply, porcine xenografts are considered a useful alternative. Current immunosuppressive therapies fail, however, to protect intracerebral neural xenografts from host CD4 T lymphocytes. To reduce the immunogenicity of porcine donor tissue, we attempted to remove microglial cells with antibodies against the alpha-galactosyl epitope (Galalpha1,3Galbeta1,4GlcNAc-R), or anti-Gal, and complement, and studied whether this pretreatment can reduce direct and indirect T-cell responses to the tissue. Brain tissue from 27-day-old pig embryos was dissociated and treated with human anti-Gal and rabbit complement. The microglial content was analyzed by flow cytometry. [3H]thymidine incorporation in cocultures of the brain cells and purified human CD4 T cells was used to determine direct T-cell responses. Indirect T-cell responses were studied by grafting pretreated and control-pretreated (no anti-Gal) nigral tissue into the lesioned striatum of immunocompetent rats with 6-hydroxydopamine-induced hemiparkinsonism. Amphetamine-induced circling behavior was used to measure graft function. Anti-Gal and complement reduced the microglial content to 11-24% of control and abolished the ability of the brain cells to induce human CD4 T-cell proliferation. Pretreated nigral tissue reduced hemiparkinsonism by more than 50% in five of eight rats at some point during the 10-week follow-up. Rats receiving control-pretreated nigral tissue did not display this degree of improvement. Pretreatment with anti-Gal and complement can reduce the immunogenicity of porcine neural tissue, and might, therefore, be a valuable alternative or supplement to immunosuppression in neural xenotransplantation.

Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alpha-galactose antigens.

Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.

α-Galactosyl epitopes on glycoproteins of porcine renal extracellular matrix

The pig is the donor animal of choice for human xenotransplantation. In the most relevant pig-to-baboon model, pig organs transplanted into baboons are hyperacutely rejected by natural xenoantibodies, which mainly bind to alpha-galactosyl (alphaGal) epitopes expressed at the surface of endothelial cells. Recent advances in controlling hyperacute rejection have led to improved survival of these xenografts, and it is now important to identify alphaGal binding sites in other cells and tissues that may be subject to immunologic attack. To this end, we have studied whether alphaGal antibodies bind to glycated proteins of the extracellular matrix in the kidney and other organs most likely to be used for human xenotransplantation. High-titer anti-alphaGal antibodies, similar to human natural xenoantibodies, were prepared in baboons, and their reactivity with components of pig extracellular matrix was tested by serology and immunohistology. The antibodies recognized epitopes of immobilized murine, bovine or porcine thyroglobulin, laminin, heparan sulfate proteoglycans, and fibronectin. In sections of pig tissue, the antibodies bound to endothelial and certain epithelial cells, as shown in previous studies, and also to mesenchymal cells, basement membranes, and extracellular matrices, in which they colocalized with matrix glycoproteins, especially laminin and heparan sulfate proteoglycans. These results suggest that when pig xenografts can be made to survive for prolonged periods, the reactivity of alphaGal antibody with matrix molecules can induce basement membrane and matrix lesions similar to those induced in laboratory animals by antilaminin and antiheparan sulfate proteoglycans antibodies.

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases.

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.

Molecular Cloning of Endo-β-galactosidase C and Its Application in Removing α-Galactosyl Xenoantigen from Blood Vessels in the Pig Kidney

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.

Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-alpha-galactosyl antibodies.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.

Alpha-Galactosyl epitopes on glycoproteins of porcine renal extracellular matrix

alpha-Galactosyl epitopes on glycoproteins of porcine renal extracellular matrix

Production of alpha-galactosyl epitopes via combined use of two recombinant whole cells harboring UDP-galactose 4-epimerase and alpha-1,3-galactosyltransferase.

alpha-Galactosyl epitopes (or alpha-Gal, oligosaccharides with a terminal Galalpha1,3Gal sequence) are a class of biologically important oligosaccharides in great demand in bulk quantities for basic and clinical studies on preventing hyperacute rejection in pig-to-primate organ xenotransplantation. A truncated bovine alpha-1, 3-galactosyltransferase, the key enzyme responsible for the biosynthesis of the terminal structure of alpha-Gal, was cloned and overexpressed previously. The acceptor specificity was further studied in the present paper, and lactose and galactose derivatives were found to be good acceptors. To develop a more proficient reaction process, we report herein an example of an efficient enzymatic synthesis of alpha-Gal oligosaccharides catalyzed by the combination of two recombinant Escherichia coli whole cells harboring the genes of a UDP-galactose 4-epimerase and the alpha-1, 3-galactosyltransferase, respectively. Using lactosyl azide (LacN(3)) as the acceptor for the glycosyltransferase, the combined use of the two recombinant cells efficiently produced alpha-Gal epitope Gal alpha1,3LacN(3) in 60-68% yield.

Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling.

Ligation of alpha-galactosyl epitopes on endothelial cells by naturally occurring human antibodies causes hyperacute rejection in porcine-to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeiraea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelial "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa protein. We have studied two 130-kDa junctional adhesion molecules, CD31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4-mediated activation. The cellular distribution of these molecules, their susceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined. Porcine CD31 and VE-cadherin were cloned. Recombinant proteins and monoclonal antibodies were prepared. The distribution and phosphorylation of CD31 and VE-cadherin in confluent PAECs activated with IB4 or human serum were studied by confocal microscopy and Western blotting, respectively. IB4 caused rapid redistribution of CD31 and VE-cadherin away from cell junctions and tyrosine-phosphorylation of CD31 but not VE-cadherin. A monoclonal antibody to CD31 also triggered tyrosine phosphorylation of this molecule, but brief exposure of PAECs to normal human serum did not. Tyrosine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphoproteins. Both IB4 and natural human antibodies bound to porcine CD31 but not to VE-cadherin. Cell adhesion tests showed that porcine and human CD31 are functionally incompatible. Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junctions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signaling complex, which may have a significant role in the response of the xenograft vascular endothelium.

T-antigen (Gal β3 GaINAc α-) containing glycoproteins of human reace

Desialation of cell surface glycoconjugates due to bacterial or viral infection can expose epitopes like T-antigenic structure which can also occur during oncological transformations. Human platelet plasma membrane glycoproteins were isolated by jacalin affinity chromatography. Potential T-antigen containing glycoproteins which were not reported before could be identified on the Western blot using peanut agglutinin-horse radish peroxidase (PNA-HRP) after neuraminidase treatment. Alpha-galactosyl epitopes recognized by anti-gal were found to be absent in human platelet plasma membrane glycoproteins. Under the experimental conditions employed, the Gp IIbα was identified most rich in T-antigenic structures. Probable role of exposed T-antigenic structures and α-galactosyl epitopes in pathological conditions is discussed. The identity of major glycoprotein bands was confirmed by differential lectin-binding studies with Concanavalin A on the Western blot. The higher binding affinity of jacalin for T-antigenic structures when compared to PNA enabled the isolation and detection of the antigen containing platelet surface glycoproteins which were not reported before.

Species-specific distribution of alpha-galactosyl epitopes on the gastric H/K ATPase beta-subunit: relevance to the binding of human anti-parietal cell autoantibodies.

The gastric H/K ATPase beta-subunit, an abundant glycoprotein of the secretory membranes of gastric parietal cells, is the major autoantigen recognized by human parietal cell autoantibodies in gastric autoimmunity. Our previous studies demonstrated that the human autoantibodies recognize the H/K ATPase beta-subunit from a number of species and that glycosylation of the beta-subunit with complex N-glycans is required for autoantibody binding. The N-glycans of the beta-subunit contain polylactosamine chains. The lactosamine chains of the rabbit beta-subunit are terminated with alpha-linked galactosyl residues (alpha-galactosyl epitope) (Tyagarajan et al., Biochemistry, 1996, 35, 3238-3246). Here we have investigated the expression of alpha-galactosyl epitopes on the H/K ATPase beta-subunit from a number of species. Using the alpha-galactosyl binding lectin, BS1-IB4, and naturally occurring anti-alpha-galactosyl antibodies, we have demonstrated that the rat H/K ATPase beta-subunit also contains terminal alpha-galactosyl residues, but not the beta-subunit from pig, dog, and mouse, indicating species-specific differences in the terminal saccharide sequences of the beta-subunit. We also investigated the potential contribution of the alpha-galactosyl epitopes to the binding by human sera. The reactivity of human pernicious anemia serum with gastric parietal cells could not be inhibited with saccharide inhibitors and, in addition, no binding was observed with normal human sera. We conclude that the H/K ATPase beta-subunit oligosaccharides from rabbit and rat are terminated with alpha-galactosyl epitopes, and although the presence of this epitope does not contribute to binding by human parietal cell autoantibodies at the concentrations routinely used, it is recommended that neither rat or rabbit stomachs be used for screening human sera.

Elevated anti-galactosyl antibody titers in endemic goiter.

Anti-Gal is a human polyclonal antibody that constitutes approximately 1% of the circulating immunoglobulin G (IgG), interacts specifically with the mammalian carbohydrate alpha-galactosyl epitope. Furthermore, it was found to mimic in vitro thyrotropin (TSH) effects regarding stimulation for cyclic adenosine monophosphate (cAMP) synthesis, 125I uptake, and cellular proliferation on cultured porcine thyrocytes and on Graves' disease thyrocytes, but not on normal human thyrocytes. As immune activation in sporadic and endemic goiters might play a secondary role in regulating thyrocyte proliferation and function, we evaluated anti-Gal titers in endemic goiter. Serum was obtained from 109 Chagas'-negative patients living in an endemic goiter area of Brazil (Grao Mogol, MG) and 160 controls. The patients were divided into 3 groups, according to their goiter size (World Health Organization [WHO] classification): grade 0 (group 1, n = 24), grade I-II (group 2, n = 41), and grade III-IV (group 3, n = 44). Anti-Gal was assessed by a radioimmunological procedure (results expressed as the percentage of bound radioactivity/total activity [%B/T]). The antibody titer was significantly more elevated in group 1 (mean +/- SEM: 9.27%+/-0.80%), in group 2 (mean +/- SEM: 16.17%+/-0.97%), and in group 3 (20.97%+/-1.30%) than in normal controls (6.46%+/-0.33%). Analysis of the male and female data separately for anti-Gal titer did not substantially alter these results. We concluded that the anti-Gal titer is higher in patients with endemic goiter and presented a possible relationship with the size of goiter. Whether these antibodies contribute to the pathogenesis of the disease needs further clarification.