Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked alpha1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer. Treatment of NOR2 with alpha-galactosidase gave a glycolipid migrating between NOR1 and NOR2, which did not react with either GSL-IB4 or anti-NOR antibodies but did react with GalNAc-specific soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.     

Characterization of porcine kidney neutral glycosphingolipids: identification of a carbohydrate antigen recognized by human natural antibodies

We have investigated the glycosphingolipids of pig kidney with a special interest in identifying compounds which may be involved in the rejection of tissue in xenotransplantation. Nine neutral glycosphingolipids have been characterized in porcine kidney and structurally characterized by a combination of techniques including¹H-NMR, permethylation analysis and thin-layer chromatography (TLC) immunostaining with carbohydrate sequence-specific monoclonal antibodies. The major components are members of the globo family and represent the human p^k and P antigens. Three other compounds were found to contain a neolacto core structure; the major neolacto compound carries a nonreducing terminal epitope (Gal1-3Gal1-4GlcNAc) recognized by the naturally-occurring human antibody, anti-Gal, and a second neolacto compound carries the blood group A trisaccharide (GalNAc1-3(Fuc1-2)Gal). These results are discussed with respect to tissue transplantation.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

Nuclear ribonucleoproteins recognized by human antinuclear antibodies in retrovirus-infected cells

Antibodies present in sera of patients with auto immune diseases (systemic Lupus erythematosus, mixed connective tissue disease) were used to react with nuclear ribonucleoproteins (HnRNPs) from normal cells and cells infected with retroviruses. Only antibodies directed against Sm and RNP antigens precipitated particles with definite spectra of small nuclear RNAs (SnRNA) and proteins. No difference could be found between infected and uninfected cells, suggesting that virus replication is dependent on normal cellular fonctions.     

Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega

A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and alpha-hydroxy fatty acids. Normal C16:0, C18:0, and 2-hydroxy C18:0 are the predominant fatty acids.     

Characterization of membrane antigens on human cytomegalovirus-infected fibroblasts recognized by human antibodies.

The antigens on the surface of human cytomegalovirus (HCMV)-infected fibroblasts which are recognized by human HCMV antibody-positive sera were characterized. Three HCMV-induced polypeptides, with apparent molecular masses of 53 to 63, 94, and 94 to 120 kilodaltons, were precipitated from 125I-surface-labeled cell extracts with different sera obtained from healthy individuals. Renal transplant recipients who were suffering from active HCMV infections recognized the same set of antigens. By the use of monoclonal antibodies, these antigens were identified as polypeptides belonging to the gcI and gcIII families of HCMV glycoproteins.     

Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.     

Definition of the immunogenic forms of modified human LDL recognized by human autoantibodies and by rabbit hyperimmune antibodies

Humans and laboratory animals recognize human modified LDL as immunogenic. Immune complexes (ICs) isolated from human sera contain malondialdehyde-modified LDL (MDA-LDL) and N (epsilon)(carboxymethyl)lysine-modified LDL (CML-LDL) as well as antibodies reacting with MDA-LDL, copper-oxidized LDL (OxLDL), CML-LDL, and advanced glycosylation end product (AGE)-modified LDL. OxLDL and AGE-LDL antibodies isolated from human sera recognize the same LDL modifications and do not react with modified non-LDL proteins. Rabbit antibodies have different reactivity patterns: MDA-LDL antibodies react strongly with MDA-LDL and MDA-BSA but weakly with OxLDL; OxLDL antibodies react strongly with OxLDL and weakly with MDA-LDL; CML-LDL antibodies react with CML-LDL > CML-BSA > AGE-LDL > OxLDL; AGE-LDL antibodies react strongly with AGE-LDL, react weakly with OxLDL, and do not react with CML-LDL. Thus, human and rabbit antibodies seem to recognize different epitopes. Capture assays carried out with all rabbit antibodies showed binding of apolipoprotein B-rich lipoproteins isolated from ICs, suggesting that laboratory-generated epitopes are expressed by in vivo-modified LDL, although they are not necessarily recognized by the human immune system. Thus, the definition of immunogenic forms of modified LDL eliciting human autoimmune responses requires the isolation and characterization of autoantibodies and modified LDL from human samples, whereas rabbit antibodies can be used to detect in vivo-modified human LDL.     

Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies

Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.     

Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes.

We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from Colo 320 cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins.     

Rodent islet cell antigens recognized by antibodies in sera from diabetic patients

Rat islets, rat insulinoma cells and islets from three different mouse strains were labelled with 35S-cysteine and/or 35S-methionine. Detergent lysates of the cells were subjected to immunoprecipitation with sera from 5 newly diagnosed diabetic children and 5 control sera. The immunoprecipitates were analysed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography. One of the sera immunoprecipitated a protein of Mr 64K from lysates of rat islets, rat insulinoma cells, A. TH and NMRI but not CBA/H mouse islets. This protein was not consistently immunoprecipitated by the other diabetic sera, however, it was never found with control sera nor was it detected in rodent lymphocytes. Some proteins of lower molecular weight (59K, 57K, 40K, 29K) were specifically immunoprecipitated by one or more diabetic sera from some of the rodent islet cell preparations. It is concluded that rodent islet cells contain a protein of Mr 64K which may be antigenically related to a 64K protein previously detected in immunoprecipitates of human islet cells with the same diabetic sera. The variable results with rat and mouse islet cell material suggest that the level of cross-reactivity is low. Further studies are needed to clarify whether the lower molecular components detected in some immunoprecipitates represent other antigenic determinants or degradation products of the 64K protein.     

Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.     

Characterization of a human granulocyte differentiation antigen (CDw15) commonly recognized by monoclonal antibodies

Many different anti-human granulocyte monoclonal antibodies recognize the same carbohydrate antigen which contains the trisaccharide 3-fucosyl-N-acetyllactosamine. The antigen is expressed mainly on two cell surface glycoproteins of molecular weights around 105 K and 160 K which are apparently not members of the LFA-1 family of proteins. Although specific for granulocytes in blood, the antigen is expressed on a wide range of non-haemopoietic cell types.     

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals

Incubation of human erythrocytes with either uranyl ions (UO₂²⁺) or rare earth metals (La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ and Yb³⁺) at 37°C for 30–45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca²⁺, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca²⁺- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35–45%) the fusion induced by UO₂²⁺ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.