Alpha-galactosyl Epitopes Research Articles

Implication of glycolipids in lens fiber development.

Mammalian lens contains Lewis(x), sialyl-Lewis(x) and alpha-galactosyl epitopes in neolactoseries glycosphingolipids. The expression of these three epitopes is not observed in lens epithelial cells, but is immunohistochemically detected in the inner cortical fibers and the lens nucleus. In embryonic chick lens, sialyl-Lewis(x)-containing gangliosides were also detected in the transitional zone and elongating lens fibers. Thus, the Lewis(x), sialyl-Lewis(x) and alpha- galactosyl epitopes may be associated with the differentiation and maturation of lens epithelial cells to lens fibers.

Reduction of the Major Swine Xenoantigen, the α-Galactosyl Epitope by Transfection of the α2,3-Sialyltransferase Gene

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.

Xenoantigens and xenoantibodies.

Major efforts are being directed towards determining and modifying the glycosylated epitopes on pig vascular endothelial cells, against which human natural antibodies are directed. Genetic engineering techniques are being used in an effort to knock out or replace the major alpha galactosyl (alpha Gal) epitopes in mice, but to-date these have been only modestly successful in prolonging functional survival of such modified organs. Competitive glycosylation involving insertion of the gene for alpha 1,2 fucosyltransferase results in reduction of alpha Gal expression but also of presentation of hitherto cryptic antigens against which natural human antibodies are directed or could develop. The introduction of the gene for N-acetylglucosaminyltransferase III has been demonstrated to significantly reduce alpha Gal expression, and the intracellular expression of single chain Fv antibodies against alpha 1,3 galactosyltransferase also represses this enzyme activity. Several other carbohydrate antigens have been identified that could act as targets for human natural antibodies, and these include Gal alpha 1-3Le(x), Hanganutziu-Deicher, Tn, and Forssman antigens. The alternative approach, namely, the depletion of the recipient's natural antibodies, is proving difficult, but techniques for inducing B cell tolerance are being explored. The induction of a state of mixed hematopoietic chimerism in alpha Gal knockout mice has resulted in tolerance to the alpha Gal antigen. Tolerance to the SLA antigens of miniature swine is also being attempted in baboons by the transfer of SLA Class II genes into baboon bone marrow cells. It is hoped that one or a combination of these approaches may overcome the problem created by the presence of pig antigens against which humans have xenoreactive antibodies.

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues.

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.

Alteration of glycosylation renders HIV sensitive to inactivation by normal human serum.

Retroviruses from various mammalian species, excluding humans, are effectively inactivated in normal human serum (NHS). Recent studies have shown that NHS inactivation of retroviruses occurs through natural Ab recognition of a terminal glycosidic moiety on the viral envelope that is acquired during replication in the host cell. This carbohydrate structure (the alpha-galactosyl epitope) is expressed on the cells of most mammals, with the exception of humans and other Old World primates. In this study, NHS sensitivity of HIV was assessed following viral propagation in human cells that were manipulated to express the alpha-galactosyl epitope. HUT-78 cells were transduced with an exogenous alpha1-3-galactosyl transferase gene, which codes for the terminal glycosyl transferase responsible for generation of the alpha-galactosyl epitope. The transduced HUT-78 cells expressed high levels of the alpha-galactosyl epitope on their membrane surface, rendering them sensitive to killing in NHS. Similarly, HIV passaged through these cells acquired the alpha-galactosyl epitope in association with the envelope glycoprotein gp120 and was also effectively inactivated in NHS. Viral inactivation was abolished by the addition of a synthetic disaccharide that contains the alpha-galactosyl epitope, indicating that virolysis is mediated by anti-alpha-galactosyl natural Ab. These results demonstrate that, like other retroviruses bearing the alpha-galactosyl epitope, HIV modified to express this epitope is inactivated in NHS. Furthermore, these data suggest that expression of the alpha-galactosyl epitope on the surface of viruses may have implications in the interspecies transmission of such viruses to humans.

Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells.

A rat-to-primate cardiac xenograft model has been proposed as an alternative to the clinically relevant but more cumbersome pig-to-primate model for assessing the efficacy of strategies aimed at preventing xenograft hyperacute rejection. As in pig xenografts, the rejection of rat hearts was mediated by the binding of xenoreactive natural antibodies (XNA) and complement activation. The present study was conducted to identify target antigens recognized by cynomolgus and rhesus monkey IgM XNA on rat tissues and cells in comparison with pig cells. The reactivity of rhesus or cynomolgus serum on pig and rat endothelial cells (ECs) was studied by flow cytometry, ELISA, and complement-dependent cytotoxicity, after removal of primate XNA by perfusion of pig livers, immunoadsorption on a Gal alpha(1,3)Gal affinity column, and enzymatic removal of alpha-galactosyl epitopes from the cell surface. Rat and pig EC extracts were also immunoprecipitated with primate serum and resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the Gal alpha(1,3)Gal epitope was analyzed on rat tissues and ECs by immunohistochemistry, flow cytometry, and Western blot, using the isolectin B4 from Griffonia simplicifolia. Removal of primate XNA or of alphaGal epitopes resulted in a decrease in XNA binding to pig and rat cells, leaving a similar degree of residual reactivity in the two species. At least five proteins of 260, 210, 110, 56, and 50 kDa were immunoprecipitated on rat ECs, with molecular weight similar to several proteins identified on pig ECs. These results suggest that primate XNA recognize similar antigens on rat and pig ECs. Rat cells expressed lower levels of the Gal alpha(1,3)Gal epitope than pig cells. A large proportion, but not all, of primate XNA react with this epitope on pig and rat ECs. This study suggests that the rat is a valuable species for the evaluation of genetic engineering strategies on the vascular endothelium aimed at preventing hyperacute xenograft rejection.

Porcine and bovine cartilage transplants in cynomolgus monkey: II. Changes in anti-Gal response during chronic rejection.

The recent advances in avoiding hyperacute rejection by producing transgenic pigs with complement regulatory proteins call for the analysis of posttransplantation changes in anti-Gal activity in the absence of hyperacute rejection. Transplantation of cynomolgus monkeys with porcine or bovine meniscus and articular cartilage enabled the study of anti-Gal IgG response to xenografts that are not subjected to hyperacute rejection. The cartilage implants were kept in suprapatellar pouches of the recipients for 1 or 2 months and anti-Gal activity was measured in the serum at various time intervals after transplantation. Within 2 weeks after transplantation, titer of anti-Gal IgG, in all transplanted monkeys, increased by 20- to 100-fold, as measured in ELISA with synthetic alpha-galactosyl epitopes linked to bovine serum albumin or with mouse laminin. Furthermore, binding of serum anti-Gal to porcine endothelial cells increased by 10-fold or more after transplantation. Complement-mediated cytotoxicity also increased by two- to eightfold after transplantation. The elevated activity of anti-Gal was maintained for the 2-month period during which the grafts were kept in the monkeys, and returned to the pretransplantation level 6 months after graft removal. All these data suggest that the primate immune system responds vigorously to alpha-galactosyl epitopes on xenografts by activating many B lymphocytes that produce increased amounts of anti-Gal IgG, which may also be of high affinity. These antibodies are likely to bind to the xenograft cells, even if these cells express low numbers of alpha-galactosyl epitopes. Such antibody binding may play an important role in chronic rejection of xenografts.

Porcine and bovine cartilage transplants in cynomolgus monkey: I. A model for chronic xenograft rejection.

Transplantation of discordant xenograft tissues usually results in antibody-mediated hyperacute rejection response. It has been speculated that because cartilage has a limited vascular, neural, and lymphatic supply, it might be immunologically privileged and may not undergo hyperacute or chronic rejection. Moreover, porcine and bovine cartilage were found to express very low amounts of alpha-galactosyl epitopes (Gal alpha1-3Gal beta1-4GlcNAc-R). To evaluate animal cartilage for possible human transplantation, xenograft meniscal cartilage was transplanted from pigs and cows into the suprapatellar pouches of six cynomolgus monkeys (group 1). In a second group of six monkeys (group 2), porcine meniscal cartilage and porcine articular cartilage plugs were evaluated. During the 2-month evaluation period in group 1, all monkeys displayed an extensive humoral response to the xenograft, as indicated by the increase in production of antibodies against bovine and porcine cartilage. Upon explant, all meniscal cartilage samples in this group demonstrated histological evidence of chronic rejection, including fibroplasia, encapsulation, mononuclear infiltrates, foreign body giant cells, and eosinophilic infiltrates. There was no difference between the response seen in untreated tissues and that seen in tissues treated with UV irradiation or ozone oxidation. In group 2, the menisci explanted after 1 month displayed extensive infiltration of eosinophils alone or eosinophils mixed with mononuclear cells. The mononuclear infiltrates consisted primarily of CD4+ and CD8+ T cells and of macrophages. The articular cartilage plugs demonstrated only a small area of fibrous encapsulation and leukocyte infiltration at the periphery. This study suggests that xenograft cartilage tissue does not appear to be immunoprivileged and is unsuitable for human implantation due to a chronic rejection mechanism, which is evident already within 1 month after transplantation. In addition, this study may serve as a general model for the primate immune response against xenografts in the absence of hyperacute rejection.

Developmental changes in carbohydrate antigens in embryonic rat lens.

The functions of glycosphingolipids, especially those containing the alpha-galactosyl epitope, were investigated during the development and differentiation of rat lens. Glycosphingolipids in embryonic lens tissue were mainly composed of neolacto-series glycosphingolipids and sialic acid-containing ganglio-series gangliosides GM3 and GD3. These glycosphingolipids and gangliosides were widely expressed on cell membranes in the lens vesicle and the elongating lens fibers. In particular, the expression of neolacto-series glycosphingolipids with the alpha-galactosyl epitope was found to be associated with the differentiation and interaction of lens fibers. Glycoproteins with the alpha-galactosyl epitope was also involved in the elongation of lens fibers. The expression of the glycoproteins was highly specific in elongating lens fibers when these were examined in head sections obtained at various embryonic stages. Thus, the alpha-galactosyl epitope on glycosphingolipids and glycoproteins appears to be associated with the differentiation and elongation of lens fibers in the rat. Evolution-related changes in the expression of carbohydrate antigens are also discussed in relation to the development and cell-to-cell interaction of lens fibers in mammals.

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes.

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2Kb or H-2Kd gene-transfected melanoma cells was associated with reduced adherence to endothelial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demonstrated reduced ability to invade Matrigel that paralleled up-regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2Dd/Ld genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2Kb transfected cells was associated with alterations in cell surface carbohydrates with appearance of alpha-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes.

Abnormal expression of galactosyl(alpha 1-->3) galactose epitopes in squamous cells of the uterine cervix infected by human papillomavirus.

This study describes the presence of alpha-galactosyl epitopes on 12 cervical biopsy samples with features of human papillomavirus infection (HPV) and different stages of cervical intraepithelial neoplasia. An avidin-biotin-peroxidase assay with a monoclonal antibody recognizing gal(alpha 1-->3)gal residues was strongly positive in 5 of 12 cases. None of the controls stained (p = 0.02). Immunostaining was intense in the areas with the highest viral load (koilocytes and keratinocytes) and absent in malignant foci. Immunostaining was also absent in normal exo- and endocervical epithelium of 12 controls with no features of HPV infection. A faint background staining in cases and controls was evident, but similar. These initial findings suggest that alpha-galactosyl epitopes are expressed in cervical squamous cells infected with HPV, turning them vulnerable to lysis by natural anti-alpha-galactosyl antibodies.

A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody.

Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence of anti-alpha-galactosyl natural Ab. Blockade or depletion of anti-alpha-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti-alpha-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the alpha-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-alpha-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the alpha-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.

ENZYMATIC REMOVAL OF ALPHA-GALACTOSYL EPITOPES FROM PORCINE ENDOTHELIAL CELLS DIMINISHES THE CYTOTOXIC EFFECT OF NATURAL ANTIBODIES

Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.

ENZYMATIC REMOVAL OF ALPHA-GALACTOSYL EPITOPES FROM PORCINE ENDOTHELIAL CELLS DIMINISHES THE CYTOTOXIC EFFECT OF NATURAL ANTIBODIES

Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric

Anti‐Gal activity In diabetic patients transplanted with fetal porcine Islet cell clusters

The natural anti-Gal antibody seems to create a major obstacle for discordant xenotransplantation in humans. Anti-Gal, which is produced in large amounts in humans (1% of circulating IgG), interacts specifically with the carbohydrate structure Gal alpha 1-3Gal beta 1-4Glc-NAc-R (termed the alpha-galactosyl epitope). This epitope is present in large amounts on porcine cells, as well as on cells of other nonprimate mammals (1 x 10(6) to 35 x 10(6) epitopes/cell). The interaction of anti-Gal with alpha-galactosyl epitopes on the xenograft was found to mediate the immune destruction of discordant xenografts. In the present study, the human immune response to alpha-galactosyl epitopes on xenografts was assessed by measuring changes in anti-Gal titers and affinity in sera of diabetic patients transplanted with fetal porcine islet cell clusters. The activity of this antibody was assessed by a hemagglutination assay with RBC, by ELISA with mouse laminin as a solid-phase antigen, and by equilibrium dialysis with the radiolabeled free haptenic form of the alpha-galactosyl epitope, i.e. [3H]Gal alpha 1-3Gal beta 1-4GlcNAc. All assays revealed a marked increase in anti-Gal activity after transplantation. The increase in anti-Gal titers ranged between 8- and 64-fold. A similar increase was observed in the binding of free alpha-galactosyl epitopes to anti-Gal, as assayed in equilibrium dialysis. Immunoglobulin concentration did not increase after transplantation, suggesting that the observed increase in anti-Gal activity is the result of a specific immune response against alpha-galactosyl epitopes on the xenograft. The elevation in anti-Gal activity was observed in all three immunoglobulin classes and the highest activity was found within the IgG class. Analysis of IgG binding to fixed porcine endothelial cells suggested that most of the observed increased activity against these cells in transplanted patients may be attributed to the elevation in anti-Gal activity.

Human anti-Gal heavy chain genes. Preferential use of VH3 and the presence of somatic mutations.

Anti-Gal is the most abundant natural Ab known in humans. It interacts specifically with the carbohydrate structure Gal alpha 1-3 Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope), constitutes approximately 1% of circulating Ig, and is found in all three isotypes in the serum. Anti-Gal is produced in Old World monkeys, apes, and humans, and in no other mammalian species. The objective of this study was to determine the VH genes involved in the synthesis of anti-Gal. B lymphocytes from various individuals were transformed by EBV, the clones producing anti-Gal were isolated, the specificity and affinity of the Abs were determined, and the VH genes were sequenced. The affinity of anti-Gal clones for the free radiolabeled alpha-galactosyl epitope ranged between 1.1 x 10(6) M-1 and 5 x 10(8) M-1. Eight of the nine clones studied used VH3 family genes and one clone used a VH1 family gene. Four of the five VH3 genes used were found to form a cluster of related sequences, suggesting that functional constraints may lead to the use of VH3 genes with structural motifs that are suited for specific interactions with the alpha-galactosyl epitope. Comparison with known germline sequences for all of the clones studied and analysis of autologous germ-line genes in two of the clones indicate that anti-Gal VH genes undergo somatic mutations. These somatic mutations may provide a pool of variants that are available for affinity maturation.

Variations in Activity of the Human Natural Anti-Gal Antibody in Young and Elderly Populations

Anti-Gal is a natural antibody which constitutes approximately 1% of circulating IgG in humans and which interacts specifically with the mammalian carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). The purpose of this study was to determine whether there are age-associated changes in the affinity of anti-Gal in elderly individuals. The age-associated variations in affinity of anti-Gal were determined by the extent of this antibody binding to solid-phase alpha-galactosyl epitopes (mouse laminin) in ELISA, performed at various serum dilutions with serum samples from 140 elderly and 127 young individuals. The data were expressed as the mean of optical density (O.D.) values measured at serum dilutions of 1:40, 1:80, and 1:160, and correlated with the affinity of anti-Gal as measured in equilibrium dialysis. The proportion of individuals with low-affinity anti-Gal is sixfold higher in the elderly than in the young population. Whereas low-affinity binding of anti-Gal (mean O.D. value < or = 1.0) was observed in only 6% of the young population, as many as 35% of the elderly population displayed such low-affinity anti-Gal. Anti-Gal affinity in ELISA assay correlated with the affinity of this antibody as measured in equilibrium dialysis. The proportion of individuals with low affinity anti-Gal increases in the elderly population in comparison with the young population. The possible molecular mechanisms which may result in the age-associated decrease in the antibody affinity are discussed.

Anti-alpha-galactosyl immunoglobulin A (IgA), IgG, and IgM in human secretions

Anti-alpha-galactosyl (anti-Gal) is a natural human serum antibody that binds to the carbohydrate Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha-galactosyl epitope) and is synthesized by 1% of circulating B lymphocytes in response to immune stimulation by enteric bacteria. We were able to purify secretory anti-Gal from human colostrum and bile by affinity chromatography on silica-linked Gal alpha 1,3Gal beta 1,4GlcNAc. We found similar secretory anti-Gal antibodies in human milk, saliva, and vaginal washings. Secretory anti-Gal from milk and saliva was exclusively immunoglobulin A (IgA); that from colostrum and bile also contained IgG and IgM isotypes. Serum was also found to contain anti-Gal IgM and IgA in addition to the previously reported IgG. Anti-Gal IgA purified from colostrum and bile had both IgA1 and IgA2. Secretory anti-Gal from saliva, milk, colostrum, and bile agglutinated rabbit erythrocytes (RRBC) and bound to bovine thyroglobulin, both of which have abundant alpha-galactosyl epitopes. The RRBC-hemagglutinating capacity of human saliva, milk, bile, and serum was specifically adsorbed by immobilized Gal alpha 1,3Gal beta 1,4GlcNAc but not by Gal alpha 1,4Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, Gal beta 1,4GlcNAc, Gal beta 1,4GlcNAc alpha 1,2Man, or Fuc alpha 1,2Gal beta 1,4GlcNAc. No RRBC-hemagglutinating activity could be detected in rat milk, rat bile, cow milk, or rabbit bile, suggesting a restricted species distribution for secretory anti-Gal similar to that found for serum anti-Gal. Colostral anti-GaI IgA bound strongly to a sample of gram-negative bacteria isolated from the throats and stools of well children as well as to an Escherichia coli K-1 blood isolate. Colostral anti-GaI IgA inhibited the binding of a Neisseria meningitidis strain to human buccal epithelial cells, suggesting that this antibody may play a protective role at the mucosal surface.

Characterization of neutral glycosphingolipids in rat lens

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Alpha-galactosyl (Galalpha1-3Galbeta1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate alpha1,3galactosyltransferase expressed in E.coli.

Developing methods for in vitro synthesis of the carbohydrate structure Galalpha1-3Galbeta1-4GlcNAc-R (termed the alpha-galactosyl epitope) on human tumour cells may be of potential clinical significance in cancer immunotherapy. Tumour vaccines with this epitope would be opsonized in vivo by the natural anti-Gal antibody, which is present in large amounts in humans, and which interacts specifically with alpha-galactosyl epitopes. Binding of anti-Gal to alpha-galactosyl epitopes on tumour cell membranes is likely to increase uptake of the cell membranes by antigen-presenting cells, such as macrophages, via the adhesion of the Fc portion of anti-Gal to Fc receptors on these cells. This, in turn, may increase processing and presentation of tumour-associated antigens by antigen-presenting cells, and induce an effective immune response against tumour cells with these antigens. The present study describes a method for the synthesis of alpha-galactosyl epitopes on human cells (red cells used as a model) by recombinant alpha1,3galactosyltransferase (rec. alpha1,3GT) expressed in bacteria. Escherichia coli was transformed with cDNA of the luminal portion of New World monkey rec. alpha1,3GT linked to six histidines (His)6 at the N-terminus. The enzyme produced by the bacteria was isolated from bacterial lysates on a nickel-Sepharose column and eluted with imidazole. This recombinant enzyme displayed acceptor specificity similar to that of rec. alpha1,3GT produced in COS cells. Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine acceptors, then subjected to rec. alpha1,3GT activity. This enzyme synthesized at least 4 x 10(4) alpha-galactosyl epitopes/red cell. These epitopes were found to be accessible for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4 lectin. It is argued that the method presented can be used for the synthesis of alpha-galactosyl epitopes on membranes of autologous tumour vaccines in humans.