Anti-Gal and human red cell aging.
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Animals↗
Biomedical subjects
S B Shohet
Publications and source records attributed to S B Shohet.
Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells.
The study of the expression of alpha-galactosyl epitopes on various mammalian cells is of particular interest, since as much as 1% of circulating IgG antibodies in humans interact with this carbohydrate residue. This natural antibody, designated "anti-Gal," was previously found to bind to terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R on biochemically defined glycolipids (Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med. 162, 573-582; Galili, U., Buehler, J., Shohet, S. B., and Macher, B. A. (1987) J. Exp. Med. 165, 693-704). The expression of anti-Gal binding epitopes on nucleated cells from various mammalian species was studied by immunostaining with this antibody. The binding of anti-Gal to various cells was correlated with the binding of the lectin Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin). The BS lectin also interacts with alpha-galactosyl residues and particularly with high affinity with Gal alpha 1----3Gal beta 1----4GlcNAc residues. We observed a striking evolutionary pattern in the expression of these epitopes on mammalian nucleated cells. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and lymphoid cells of nonprimate mammals, prosimians, and New World monkeys readily bound both anti-Gal and BS lectin. However, no such binding was detectable on cells of Old World monkeys, apes, and humans. Measurment of the binding of radiolabeled BS lectin to the various nucleated cells suggests that cells binding anti-Gal express 10(6) to 3.5 x 10(7) alpha-galactosyl epitopes, most of which, based on the anti-Gal specificity, seem to have the structure of Gal alpha 1----3Gal beta 1----4GlcNAc-R. The absence of these epitopes from human cells results from diminished activity of the enzyme alpha 1----3 galactosyltransferase, which catalyzes the following reaction. Gal beta 1----4GlcNAc-R + UDP-Gal(alpha 1----3-galactosyltransferase)----Gal alpha 1----3Gal beta 1----4GlcNAc-R + UDP This enzyme, which participates in the glycosylation of cell membrane glycoconjugates in nonprimate mammals, prosimians, and New World monkeys, appears to have been suppressed in Old World primates as a result of evolutionary events which occurred 20-30 million years ago. It is argued that an anomalous activity of this enzyme in man may result in initiation of autoimmune diseases because of the de novo expression of Gal alpha 1----3Gal beta 1----4GlcNAc-R epitopes recognized by anti-Gal.
ABO Blood-Group System↗
Interaction of erythrocyte protein 4.1 with phospholipids. A monolayer and liposome study.
We have studied the interaction of purified human erythrocyte protein 4.1 with phospholipid membranes by monitoring both the increase in surface pressure of monolayers at the air/water interface and the change in permeability in liposomes to fluorescent molecules, in the presence of protein 4.1. Protein 4.1 penetrated into monolayers of brain phosphatidylserine (PS) and egg phosphatidylcholine (PC), even above surface pressures of 30 mN/m. Protein 4.1 increased the permeability of negatively charged PS, but not PC, liposomes, measured as the increase in fluorescence when encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylenebispyridinium bromide (DPX) or carboxyfluorescein were released into the medium. The interaction of protein 4.1 with PS large unilamellar vesicles (LUV) was increased as the pH and the ionic strength were lowered, and decreased as the Ca2+ or Mg2+ concentrations and ionic strength were raised. In order to study the relevance of these measurements to the erythrocyte, we prepared LUV of synthetic lipid mixtures characteristic of both the inner and the outer membrane leaflets. Protein 4.1 increased the permeability of inner, but not outer, leaflet LUV at both pH 6.0 and 7.4. These observations suggest that negatively charged phospholipid domains around the protein 4.1 high-affinity protein-binding site(s) may contribute to the anchoring of protein 4.1 to the cytoplasmic surface of the red cell membrane.
Blood Proteins↗
Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora.
Anti-alpha-galactosyl immunoglobulin G (anti-Gal) is a natural antibody present in unusually high amounts in human sera. It constitutes as much as 1% of circulating immunoglobulin G in humans and displays a distinct specificity for the carbohydrate epitope galactosyl alpha(1----3) galactosyl (Gal alpha 1----3Gal). Recently, it has been suggested by various investigators that anti-Gal may be related to some autoimmune phenomena, since marked elevation of its titer was found in sera of patients with autoimmune thyroid disorders, rheumatoid arthritis, glomerulonephritis, and Chagas' disease. In view of the ubiquitous presence of anti-Gal in high titers in humans, throughout life, we hypothesized that, analogous with synthesis of anti-blood group antibodies against bacterial antigens, bacteria within normal intestinal flora may provide constant antigenic stimulation for the synthesis of anti-Gal. This hypothesis would imply that anti-Gal may bind to a variety of bacterial strains of human flora. In the present study, the interaction between affinity chromatography-purified anti-Gal and various bacterial strains was studied. By the use of a direct immunostaining assay and an enzyme-linked immunosorbent assay, anti-Gal was found to interact with a variety of Escherichia coli, Klebsiella, and Salmonella strains, some of which were isolates from normal stool. Furthermore, the anti-Gal-binding sites in some strains were found to be present on the carbohydrate portion of bacterial lipopolysaccharides. It is thus suggested that Gal alpha 1----3Gal epitopes in the outer membranes of normal flora enterobacteria may provide a continuous source for antigenic stimulation. Since there is no immune tolerance to the Gal alpha 1----3Gal carbohydrate structure in humans, anti-Gal seems to be constantly produced in response to these enterobacteria. In addition, bacteria which express Gal alpha----3Gal epitopes and which may adhere to various cells mediated binding of anti-Gal to human cell lines. These findings raise the possibility that anti-Gal may damage normal human tissues via inflammatory processes facilitated by bacterial Gal alpha 1----3Gal epitopes.
Antigen-Antibody Reactions↗
Cross-linked polymers in the red cell membranes of a patient with Hb-Koln disease.
Membranes isolated from the red cells of a Hb-Koln patient were shown to contain appreciable amounts of N epsilon-(gamma-glutamyl)lysine, the characteristic product of the post-translational cross-linking of proteins by transglutaminase. Such an apparently irreversible fusion of membrane proteins may contribute to the membrane defect in Hb-Koln disease.
Adult↗
Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13.
The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The human natural anti-Gal IgG. III. The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies.
A well-defined antigen/antibody system was used to evaluate the effect of immune tolerance on the spectrum of specificities of natural antibodies. The antibody used in this study, anti-Gal, is a naturally occurring, polyclonal IgG that constitutes 1% of the circulating IgG in humans. We have previously shown that anti-Gal, purified from AB sera, specifically interacts with glycosphingolipids bearing a Gal alpha 1----3Gal epitope, but not with the closely related B antigen in which the penultimate galactose of the Gal alpha 1----3Gal epitope is fucosylated Gal alpha 1----3(Fuc alpha 1----2)Gal. This narrow specificity was assumed to be the result of an effective immune tolerance mechanism that prevents the expression of antibody clones that can recognize both the Gal alpha 1----3Gal and the self B epitopes. If the assumption that immune tolerance determines the range of anti-Gal specificity is correct, then anti-Gal from individuals lacking the B antigen (A and O blood types) would be expected to interact with both Gal alpha 1----3Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal epitopes. In this study, anti-Gal from the serum of individuals of various blood types was purified by affinity chromatography on Gal alpha 1----3Gal adsorbent and tested for its reaction with the B antigen. Whereas anti-Gal from AB and B individuals only reacted with Gal alpha 1----3Gal epitopes, anti-Gal from A and O individuals reacted with both Gal alpha 1----3Gal and B epitopes. Furthermore, it was determined that the majority of anti-B reactivity in A and O individuals is in fact anti-Gal antibodies capable of recognizing both Gal alpha 1----3Gal and B epitopes. It can be concluded from these results that immune tolerance accurately controls the spectrum of natural antibody specificities by preventing the production of antibody clones that can interact with self antigens.
ABO Blood-Group System↗
Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1----3Gal epitope in primates.
Anti-Gal is a natural antibody, which constitutes as much as 1% of circulating IgG in humans and displays a distinct specificity for the structure Gal alpha 1----3Gal. This glycosidic structure has been found on various tissues of many nonprimate mammals. A comparative study of the occurrence of anti-Gal versus the expression of the Gal alpha 1----3Gal epitope was performed in primates, and a distinct evolutionary pattern was observed. Whereas anti-Gal was found to be present in Old World monkeys and apes in titers comparable to those in humans, its corresponding antigenic epitope is abundantly expressed on erythrocytes of New World monkeys. Immunostaining with anti-Gal of glycolipids from New World monkey erythrocytes indicated that the molecules to which anti-Gal binds are similar to those found in rabbit and bovine erythrocytes. These findings indicate that there is an evolutionary reciprocity between New World and Old World primates in the production of the Gal alpha 1----3Gal structure and the antibody that recognizes it. The expression of the Gal alpha 1----3Gal epitope was evolutionarily conserved in New World monkeys, but it was suppressed in ancestral lineages of Old World primates. The suppression of this epitope was accompanied by the production of anti-Gal. The observed in vivo binding of anti-Gal to human normal senescent and some pathologic erythrocytes implies that the Gal alpha 1----3Gal epitope is present in man in a cryptic form.
Animals↗
Red cell biochemical anatomy and membrane properties.
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Erythrocyte Membrane↗
Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton.
Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of non-erythroid tissues. In mammalian erythrocytes, it plays a key role in regulating membrane physical properties of mechanical stability and deformability by stabilizing spectrin-actin interaction. We report here the molecular cloning and characterization of human erythrocyte protein 4.1 cDNA and the complete amino acid sequence of the protein derived from the nucleotide sequence. Probes prepared from the cloned erythrocyte protein 4.1 cDNA hybridized with distinct mRNA species from a wide variety of non-erythroid tissues, including brain, liver, placenta, pancreas, and intestine, implying substantial homology between erythroid and non-erythroid protein 4.1. The availability of cloned erythrocyte protein 4.1 cDNA should facilitate the study of the functional characteristics of this protein in erythroid as well as non-erythroid cells.
Amino Acid Sequence↗
Excessive binding of natural anti-alpha-galactosyl immunoglobin G to sickle erythrocytes may contribute to extravascular cell destruction.
A large proportion of sickle erythrocytes is removed from the circulation by the macrophages of the reticuloendothelial system. In view of the proposed role for natural antibodies in the destruction of normal senescent erythrocytes, we looked for a possible similarity in the antibodies that bind in situ to senescent and sickle cells. Bound IgG molecules were detected by a highly sensitive rosetting antiglobulin test, using K562 myeloid cells. After separation on Stractan density gradients, the 0.6% most dense (senescent) normal cells and the most dense 40% sickle cells displayed membrane-bound IgG as reflected by the high proportion of rosettes formed. No antibody was found on low-density cells of either type. The bound antibodies were readily eluted from both sickle and normal senescent cells by carbohydrates containing alpha-galactosyl residues. These antibodies appear identical to the recently discovered human natural anti-alpha-galactosyl IgG (anti-Gal), an IgG antibody present in high titers in normal sera. Moreover, affinity-purified anti-Gal interacted specifically with sickle and normal cells depleted of the autologous antibodies. A similar pattern of binding to the various erythrocyte subpopulations was observed when the radiolabeled lectin with anti-alpha-galactosyl specificity, Bandeiraea simplicifolia, was used. In vitro phagocytosis of normal and sickle erythrocyte subpopulations correlated with the presence of anti-Gal on these cells. The in situ binding of anti-Gal to a large proportion of sickle erythrocytes may reflect an accelerated physiologic aging process by which immune recognition of prematurely exposed alpha-galactosyl-bearing antigenic sites contributes to shortened cell survival.
Anemia, Sickle Cell↗
Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1----3)-linked galactose residues.
A natural IgG antibody (anti-Gal) with alpha-galactosyl binding specificity has been found in large amounts (0.5 - 1.0% of serum IgG) in all individuals tested. It has been purified by affinity chromatography on a column of melibiose-Sepharose. In addition to its affinity for normal and pathological senescent human red cells, the antibody readily interacts with rabbit red blood cell (RRBC) glycolipids with alpha-galactosyl terminal residues. Two types (glycosidic linkages of 1----3 vs. 1----4) of rabbit red cells glycolipids with terminal alpha-galactosyl residues were tested for antibody binding. The antibody specifically bound to glycolipids with Gal alpha 1----3 terminal residues, and treatment of these glycolipids with alpha-galactosidase abolished binding. Hemagglutination inhibition studies with oligosaccharides of known structure also showed that the antibody binds specifically to glycoconjugates with an alpha 1----3 terminal galactose residue. Anti-Gal did not bind to a human B-active glycolipid, indicating that fucose-linked alpha 1----2 to the penultimate galactose prevents anti-Gal binding. The anti-Gal specificity for RRBC glycolipids also paralleled that of the alpha-galactosyl-specific Bandeiraea simplicifolia lectin. The possible reasons for the occurrence of this unique antibody in human serum are discussed.
ABO Blood-Group System↗
Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes.
In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.
Acyltransferases↗
Erythrocyte membrane rigidity induced by glycophorin A-ligand interaction. Evidence for a ligand-induced association between glycophorin A and skeletal proteins.
Erythrocyte skeletal proteins are known to play an important role in determining membrane deformability. In order to see whether transmembrane proteins also influence deformability and, if so, whether this influence is mediated by an interaction with the membrane skeleton, we examined the effect on deformability of ligands specific for transmembrane proteins. We found membrane deformability markedly reduced in erythrocytes that were pretreated with glycophorin A-specific ligands. In contrast, ligands specific for band 3 and A and B blood group antigens had no effect. The increase in membrane rigidity appeared to depend upon a transmembrane event and not upon a rigidity-inducing lattice on the outside surface of the cell in that a monovalent Fab of antiglycophorin IgG caused decreased deformability. We therefore looked for a ligand-induced association of glycophorin and the skeletal proteins and found, in Triton X-100-insoluble residues, a partitioning of glycophorin with the skeletal proteins only after preincubation with a ligand specific for glycophorin. We then studied cells and resealed membranes with skeletal protein abnormalities. In spectrin-deficient and protein 4.1-deficient erythrocytes and in 2,3-diphosphoglycerate-treated resealed membranes, the antiglycophorin IgG was only one-third as effective in decreasing deformability as it was in normal cells. Thus, normal skeletal proteins appear to be essential for liganded glycophorin to affect membrane deformability maximally. Taken together, these observations indicate that there is a ligand-induced interaction between glycophorin A and skeletal proteins and that this interaction can directly influence membrane deformability.
Antigen-Antibody Complex↗
Excessive binding of the natural anti-alpha-galactosyl IgG to sickle red cells: enhancement of red cell destruction by a physiological process.
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Anemia, Sickle Cell↗
Fluorescent cytoplasm and Heinz bodies of hemoglobin Köln erythrocytes: evidence for intracellular heme catabolism.
Hb Köln, one of the common mutant hemoglobins responsible for unstable hemoglobin disease, was found to be degraded to a fluorescent yellow pigment (FYP) in circulating erythrocytes. FYP is responsible for a strong green fluorescence observed in the cytoplasm and is particularly abundant in the Heinz bodies of Köln RBC. Front face fluorometry and fluorescence microscopy showed that Heinz bodies emit 10% to 20% of the fluorescence of RBCs. Hb-free FYP was obtained by means of a cellulose column separation of the cytoplasm or from a precipitate formed during the incubation of Köln RBC cytoplasm at 50 degrees C. The absorption and emission spectra of FYP are consistent with those of dipyrroles.
Adult↗
Erythrocyte deformability changes in autoimmune hemolytic anemia during development of NZB mice and their (NZB/NZW)F1 hybrid.
NZB and B/W hybrid mice develop compensated hemolytic anemia during the first year of their life. By the age of 3-5 months, their erythrocytes show evidence of spherocytosis, increased osmotic fragility and decreased whole cell deformability, as measured by ektacytometry, a laser diffraction technique. The presence of spherocytes with decreased surface area/volume ratio was confirmed by scanning electron microscopy and osmotic gradient ektacytometry. Whereas these abnormalities persisted and worsened in the NZB mice with further growth, they gradually improved and reverted to normal by the age of 12 months in B/W mice. This spontaneous improvement seems to be due to the accumulation of red cell membrane lipids reflecting the lipemia of immune complex nephritis in B/W mice. The implications of these findings in the modulation of autoimmune hemolytic anemia are discussed.
Age Factors↗