No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Permanent mixed-field polyagglutinable erythrocytes lack galactosyltransferase
... Tn syndrome or permanent mixed-field polyagglutinability (PMFP)' is a human disorder characterized by the exposure of truncated 0-glycans, in particular the Tn antigen (GalNAca 1-O-Ser/Thr) (1) due to a selective deficiency of UDP-Gal:Gal-NAca l-O-Ser/Thr #1-3 galactosyltransferase (f3Gal-T) activ-ity (2,3) in a variable proportion of hematopoietic cells (4). These patients, who are also said to have blood group Tn (5), occasionally develop hemolytic anemia, thrombopenia, and leukopenia most likely mediated by anti-Tn natural antibodies present in normal serum (6). ...
... gens detected on the cell surface to the induction of f33Gal-T activity, cellular enzyme activity was determined. Detergent cell lysates from 5-azaC-treated PMFP T cells and untreated control cells were assayed using asialo-OSM as acceptor substrate (2,4,27). As depicted in Fig. 6, no activity could be detected in untreated PMFP T cells which is in accordance with the lack ofdetectable TF epitopes (Fig. 3 J). ...
... More than a decade ago, deficiency of 33Gal-T activity was shown to be the underlying enzymatic defect causing PMFP (2,3). A somatic mutation occurring at the stem cell level was suggested to be responsible for the loss of gene activity ( 1,9) but thus far, the molecular nature of,3Gal-T deficiency has not been analyzed due to a major experimental limitation: the enzyme deficiency was first reported to occur in erythrocytes (2) and thrombocytes (3). ...
A human hematopoietic disorder designated as Tn syndrome or permanent mixed-field polyagglutinability has been ascribed to a stem cell mutation leading to a specific deficiency of UDP-Gal:GalNAc alpha 1-O-Ser/Thr beta 1-3 galactosyltransferase (beta 3 Gal-T) activity in affected cells. To test for the possibility that an allele of the beta 3Gal-T gene might be repressed instead of mutated, we have investigated whether 5-azacytidine or sodium n-butyrate, both inducers of gene expression, would reactivate expression of beta 3Gal-T in cloned enzyme-deficient T cells derived from a patient affected by the Tn syndrome. Flow cytometry revealed that a single treatment induced de novo expression of the Thomsen-Friedenreich antigen (Gal beta 1-3GalNAc-R), the product of beta 3Gal-T activity. In addition, a sialylated epitope on CD43 (leukosialin), which is present on normal but not on beta 3Gal-T-deficient T cells, was also reexpressed. Although no beta 3Gal-T activity was detectable in untreated Tn syndrome T cells, after exposure to 5-azaC,beta 3Gal-T activity reached nearly normal values. Both agents failed to reactivate beta 3Gal-T in Jurkat T leukemic cells, which also lack beta 3Gal-T activity. These data demonstrate that Tn syndrome T cells contain an intact beta 3Gal-T gene copy and that the enzyme deficiency in this patient is due to a persistent and complete but reversible repression of a functional allele. In contrast, the cause of beta 3Gal-T deficiency appears to be different in Jurkat T cells.
... Together with serological data, Dahr concluded that the Tnantigen consists of terminal K-GalNAc. Subsequent work by Hesford et al. [27] con¢rmed the presence of terminal KGalNAc residues on glycoproteins derived from Tn(+) erythrocytes of the case RR: Tn(+) erythrocyte membranes could be used as acceptor Table 2 Reagents used to detect the Tn-antigen : monoclonal antibodies strates for the newly identi¢ed erythrocyte membrane 1,3GT [28]: Normal donor erythrocyte membranes solubilized in Triton X-100 did not incorporate labeled galactose from [ 14 C]Gal, neither did Tn(+) erythrocyte membranes incorporate label. However, mixing normal with Tn(+) membrane lysates led to signi¢cant incorporation of label into glycophorin as shown by autoradiography of electrophoretically separated erythrocyte membranes. ...
... 7% of all red blood cells exhibit the TF-antigen , thus appear normal. 93% have been shown to be Tn(+), thus not reactive with the mAb [16,28]. BBADIS 61874 13-9-99 found for erythrocytes (seeFig. ...
... This Fig. 5. L1,3 Galactosyltransferase activity in 6 di¡erent Tn(+) and 3 TF(+) T cell clones. 1,3GT activity is given as nmol/min per mg of protein and was measured using asialo-OSM as sub- strate [28]. Reproduced from Thurnher et al. [43] by copyright permission from Eur. ...
The idiopathic Tn-syndrome, formerly called 'permanent mixed-field polyagglutinability', is a rare hematological disorder characterized by the expression of the Tn-antigen on all blood cell lineages. The immunodominant epitope of the Tn-antigen is terminal alpha-N-acetylgalactosamine, O-glycosidically linked to protein. Normally this residue is 3'-substituted by 5-galactose thereby forming the core 1 structure known as the Thomsen-Friedenreich (TF) antigen (Galbeta1 ==> 3GalNAcalpha1 ==> Thr/Ser). The cause of the exposure of the Tn-antigen appears to be due to the silencing of the gene expression of beta1,3galactosyltransferase, since treatment of deficient Tn(+) lymphocyte T clones with 5'azacytidine or Na butyrate leads to reexpression of enzyme activity and the sialylated TF-antigen. The Tn-syndrome is acquired and permanent and affects both sexes at any age. Its origin is unknown. Pluripotent stem cells are affected since all lineages are involved but each one to a variable extent. Therefore, normal cells co-exist with Tn-transformed cells. Clinically, patients suffering from the Tn-syndrome appear healthy. Laboratory findings usually reveal moderate thrombocyto- and leukopenia and some signs of hemolytic anemia not warranting any treatment.
... The apparent underlying defect of 0-glycosylation in the T cell leukemia cell line Jurkat is a greatly decreased level of UDP-Gal:GalNAc /31+3galactosyltransferase activity (Table III). This defect is similar to the altered 0-glycan biosynthesis described for the Tn syndrome (Berger and Kozdrowski, 1978;Cartron et al., 1978). The exposed GalNAc residues in Jurkat cells are not acceptors for the ot2+6 sialyltransferase since no significant amount of NeuNAcaB+ 6GalNAc was detected, although an enzymatic activity could be measured using asialo-BSM as acceptor substrate. ...
Glycoproteins from the human T leukemia cells Jurkat were found to bind to the GalNAc alpha 1----Ser/Thr-specific lectin from Salvia sclarea seeds. The analysis of the O-linked saccharides of immunopurified leukosialin, the major [3H]glucosamine-labeled glycoprotein in Jurkat cell lysate, revealed the presence of mainly GalNAc alpha 1----Ser/Thr with only minor amounts (approximately 17%) of more complex O-glycans. A comparison between Jurkat and K562 cell glycosyltransferase involved in the biosynthesis of O-linked carbohydrates showed that a markedly lower activity of UDP-Gal:GalNAc alpha 1----Ser/Thr beta 1----3galactosyltransferase is apparently responsible for the presence of truncated O-glycans in the Jurkat cell line. The O-glycosylation defect makes Jurkat cells an ideal model to study the initiation of O-linked saccharides. Pulse-chase experiments with [35S] methionine showed that the addition of GalNAc to leukosialin is responsible for the decreased mobility of the mature glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, no biosynthetic intermediates between the O-glycan-free precursor and the fully O-glycosylated form could be detected either with an anti-leukosialin antiserum or with the GalNAc-specific lectin. Lowering the chase temperature to 15 degrees C completely inhibited the transfer of GalNAc to the peptide core indicating that O-glycan initiation takes place in the first Golgi elements and not in transitional vesicles between endoplasmic reticulum and Golgi. In addition, treatment of the cells with monensin did not inhibit GalNAc transfer to leukosialin apoprotein. These results indicate that the initiation of O-glycosylation in Jurkat cells starts in the cis-Golgi stacks.
... Indeed, the testing of galactosyltransferase activity in the erythrocyte membrane of Tnsyndrome patients confirmed this hypothesis. With this experiment, the first disease of glycosylation was established [9]. ...
... Therefore, usually only a part of erythrocytes (descendants of the mutated cells) of a given individual shows the Tn characteristics. Tn erythrocytes (found to be different from TF by means of lectins) contain glycophorins with defective ,Qglycosidic chains, lacking sialic acid and galactose residues (Dahr ~t a1., 1975b) This defect is caused by the lack of galactosyltransferase involved in biosynthesis of these chains (Berger & Kozdrowski, 1978) The glycophorin variant with more complex oligosaccharide chains is present in erythrocytes carrying a rare antigen Cad. Cad is inherited as an autosomal dominant character and was first recognized by an unexpected strong reactivity of blood group 0 or B erythrocytes with (vaith & Uhlenbruck, 1978;Dahr et al., 1975b;Blanchard et al., 1983;Adamany ~t al., lL983 (Adamany ~ al., 1983). ...
The name of glycophorin was given by Marchesi et
al. (1972) to the major sialoglycophorin of human erythrocyte membranes, known earlier as the glycoprotein carrying blood group M and N determinants and receptors for agglutinins of influenza viruses (Baranowski et
al., 1959; Romanowska 1959; Klenk & Uhlenbruck, 1960; Kathan et
al., 1961; Springer et
al., 1966). Fractionation of the erythrocyte membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualization of sialoglycoproteins with periodic acid-Schiff (PAS) reagent give a complex pattern of bands which may differ in details, depending on electro- phoretic conditions. It has been now accepted that there are at least four distinct sialoglycoproteins in human erythrocyte membranes. Furthmayr et
al. (1975) designated three of them as glycophorin A, B and C, in order of their decreasing amount in the membrane. Anstee et
al. (1979) denoted them glycoprotein α, β, γ and δ, in order of their decreasing molecular weight. Dahr et al. (1978c) used other designations. The more numerous bands seen in SDS-PAGE correspond to monomers of the sialoglycoproteins and to homo- and heterodimers (and higher oligomers) formed by the most abundant glycophorins A and B (Fig. 1).
The phenomenon of polyagglutination is characterised by the agglutinability of erythrocytes and some other blood cells by almost all sera from normal human adults, independent of standard blood groups [15,63]. This unusual reactivity is due to the recognition of secondarily altered erythrocyte membrane antigens by agglutinins normally present in human sera. The receptors are essential constituents of oligosaccharide chains found in membrane glycoconjugates. They are normally hidden on human erythrocytes but become exposed in polyagglutinable cells. The following types of polyagglutination will be discussed in this chapter(1): forms of acquired polyagglutination (T, Tk, Tx, and Th) which develop as a result of bacterial infections; the transformation is usually transient and disappears after recovery of the patient. In these cases the antigens are uncovered by various enzymes released in the blood stream by viruses or microorganisms. Because some bacteria strains produce several enzymes, the simultaneous appearance of two or three of these characteristics may be observedTn, another form of polyagglutination which is caused by somatic mutations in the haematopoietic stem cells; these mutations involve genes responsible for the biosynthesis of glycoconjugates. The resulting disorder in glycosylation of the membrane constituents leads to a changed antigen pattern of the erythrocyte membranethe characters VA and NOR, which have not yet been classified. forms of acquired polyagglutination (T, Tk, Tx, and Th) which develop as a result of bacterial infections; the transformation is usually transient and disappears after recovery of the patient. In these cases the antigens are uncovered by various enzymes released in the blood stream by viruses or microorganisms. Because some bacteria strains produce several enzymes, the simultaneous appearance of two or three of these characteristics may be observed Tn, another form of polyagglutination which is caused by somatic mutations in the haematopoietic stem cells; these mutations involve genes responsible for the biosynthesis of glycoconjugates. The resulting disorder in glycosylation of the membrane constituents leads to a changed antigen pattern of the erythrocyte membrane the characters VA and NOR, which have not yet been classified.
The phenomenon of polyagglutination is characterised by the agglutinability of erythrocytes and some other blood cells by almost all sera from normal human adults, independent of standard blood groups [9,62]. This unusual reactivity is due to the fact that antibodies normally present in human sera bind to secondarily altered erythrocyte membrane antigens.
Since cellular glycosylation appears to be a function of growth and differentiation, it is not surprising that in disease there are profound alterations of carbohydrate structures and the enzymes involved in their synthesis (Table 4). These complex alterations are difficult to classify, but based on many studies in this field we begin to understand emerging patterns.
Glycoproteins are a diverse group of biopolymers containing one or more carbohydrate chains linked covalently to a polypeptide backbone. The carbohydrate chains are classified according to the linkage between sugar and amino acid. This chapter will deal with the biosynthesis of mammalian and avian protein-bound oligosaccharides linked to polypeptide via asparagine-N-acetyl-D-glucosamine (Asn-GlcNAc, N-glycosidic) and serine(threonine)-N-acetyl-D-galactosamine [Ser(Thr)-GalNAc, O-glycosidic] linkages. Several recent reviews have considered various aspects of this topic (Schachter, 1978; Schachter and Roseman, 1980; Schachter and Williams, 1982; Beyer et al., 1981).
The Lutheran system consists of 20 antigens, including four pairs of antithetical antigens representing SNPs in the Lutheran gene, BCAM. The Lutheran glycoproteins (CD239) are a pair of isoforms that belong to the immunoglobulin superfamily. Their function is unknown, but they bind the extracellular matrix glycoprotein laminin. Red cells of the dominantly inherited In(Lu) and X-linked XS2 phenotypes have very low levels of Lutheran antigens and some other blood group antigens. Both result from mutations in erythroid transcription factor genes: In(Lu) from heterozygosity for inactivating mutations in KLF1, the gene for EKLF; XS2 from hemizygosity for a mutation in GATA1.
This chapter discusses the biosynthesis of Oligosaccharides (O-glycans) of the N-acetylgalactosamine- α-Ser/Thr linkage type. The synthesis of the frame-work of oligosaccharide chains regulates the expression of functional terminal carbohydrate structures. Thus, a control on the early steps of the biosynthetic pathways proves to have a great impact on the structures, properties, and biological functions of O-glycans. O-Glycosylation commonly changes during development, differentiation, growth, and in disease. In addition, various tissues and species express characteristic O-glycans associated with various biological functions. Genetic defects in the biosynthetic pathways of O-glycans are rare; possibly because the development of a multicellular organism is dependent on interactions with cellular carbohydrate.The sugars commonly found in O-glycans are GalNAc, Gal, GlcNAc, sialic acid, and fucose. Gal, GlcNAc and GalNAc may be sulfated and contribute with sialic acid to the acidity of O-glycans. O-glycans are found on many mammalian and non-mammalian soluble and membrane-bound glycoproteins, and on proteoglycans.
Polyagglutinable erythrocytes expressing the rare human blood group phenotype Tn were tested for expression of both Tn (GalNAcα1→O-Ser/Thr) and sialosyl-Tn (NeuAcα2→6GalNAcα1→O-Ser/Thr) antigens by agglutination with a panel of well-characterized monoclonal antibodies specifically directed to Tn and sialosyl-Tn antigens, respectively. Tn erythrocytes from 4 patients were strongly agglutinated by both sialosyl-Tn and Tn-specific monoclonal antibodies, indicating coexpression of sialosyl-Tn and Tn antigens on the cell membrane surface of Tn polyagglutinable erythrocytes. Human polyclonal anti-Tn antisera were found to express weak and variable anti-sialosyl-Tn antibodies in conjunction with the anti-Tn antibodies. The Tn phenotype thus involves not only the classically known Tn antigens but also sialosyl-Tn antigens. However, the human anti-Tn antibodies may be directed mainly toward the Tn antigen (GalNAcα1→O-Ser/Thr).
The human hematopoietic disorder named Tn syndrome has been ascribed to an acquired stem cell mutation resulting in loss of β-1,3-galactosyltransferase activity in affected Tn+ cells of the hematopoietic lineages. Recently, we could demonstrate that this deficiency is due to a repression of a functional allele of the β-1,3-Gal-T gene since treatment of Tn+ T-lymphocytes from a patient (R.R.) afflicted with the Tn-syndrome with 5-azacytidine or Na n-butyrate resulted in re-expression of the Thomsen–Friedenreich (TF) antigen, the product of β-1,3-Gal-T activity [M. Thurnher, S. Rusconi, E.G. Berger, Persistent repression of functional allele can be responsible for galactosyltransferase deficiency in Tn syndrome, J. Clin. Invest. 91 (1993) 2103–2110]. To reduce these observations to a common pathogenetic mechanism responsible for the Tn-syndrome, more Tn patients need to be investigated. Here, we describe similar Tn+ T-lymphocytes cultured ex vivo from patient M.Z. whose Tn+ syndrome was newly recognized. Tn+ and TF+ T-lymphocyte cultures were characterized by flow cytometry and measurement of β-1,3-Gal-T and shown to be deficient in Tn+ cells. Furthermore, Tn+ cells were treated with 5-azacytidine and Na n-butyrate as described before. Reoccurrence of β-1,3-Gal-T activity dependent epitopes on the cell surface of Tn+ cells was shown by flow cytometry. These support the notion of β-1,3-Gal-T gene repression as a common pathogenetic mechanism underlying the Tn-syndrome.
Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is . Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.
Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.
To evaluate whether exposure of Tn determinants at the surface of human erythrocytes, platelets, and granulocytes could arise from a somatic mutation in a hemopoietic stem cell, burst-forming unit erythroid (BFU-E) colonies, colony-forming unit granulocyte-macrophage (CFU-GM), and colony-forming unit-eosinophil (CFU-Eo) were grown from a blood group O patient with a typical Tn syndrome displaying two distinct populations (Tn+ and Tn-) of platelets, granulocytes, and erythrocytes. A large number of colonies was observed. Individual colonies were studied with a fluorescent conjugate of Helix pomatia agglutinin (HPA). A sizeable fraction of each of the erythroid and granulocytic colonies appeared to consist exclusively of either HPA-positive or HPA-negative cells, thereby demonstrating the clonal origin of those exhibiting the Tn marker. Similar results were obtained from a second patient. These findings establish that the HPA labeling of Tn cells is an accurate marker permitting assessment of the clonality of the human megakaryocyte (MK) colony assay. For the study of MK cultures a double-staining procedure using the HPA lectin and a monoclonal antiplatelet antibody (J-15) was applied in situ to identify all MK constituting a colony. Our results, obtained in studies of 133 MK colonies, provide definitive evidence that the human MK colony assay is clonal because all MK colonies were exclusively composed of Tn+ and Tn- MK. Furthermore, the distribution of MK within a single colony was shown to be seminormal with a mean at 6 MK, isolated MK typically being absent in culture.
To delineate the extent of O-galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen-Friedenreich (TF) antigen (Gal beta 1----3GalNAc alpha 1-O-Ser/Thr) and its precursor the Tn antigen (GalNAc alpha 1-O-Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed-field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn+ lymphocytes mainly belonged to the CD3+ subset, but were also CD19+ or CD16+. Both Tn+ and TF+ T cell clones from patient R. R. were established and shown to belong to the CD4+ or CD8+ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn+ clones were deficient in UDP-Gal: GalNAc alpha 1-O-Ser/Thr beta 1----3 galactosyltransferase (beta 3Gal-T) activity; by contrast this activity was present in all lysates from TF-expressing clones. UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (GalNAc-T) and UDP-Gal: GlcNAc-R beta 1----4 galactosyl-transferase (beta 4Gal-T) exhibited similar activities in both Tn+ and TF+ T cell clones. As a consequence of defective O-galactosylation in Tn+ T cells, cell surface sialic acid of Tn+ clones was reduced by greater than 50% when compared to TF+ clones as demonstrated by sialic acid-specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O-galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn+ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O-linked carbohydrates for proper T cell function.
Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this 'artificial' Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc alpha- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.
To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.
Many studies have suggested that malignant transformation is associated with fundamental changes in the cell surface; similar changes have been described for normal stem cells and cells of embryonic or fetal origin. There is now evidence that the tumor cell secretes or sheds glycoproteins and glycosyltransferases into the surrounding medium and into serum. There are claims that some of these serum glycoproteins and glycosyltransferases are associated with, or specifically related to, the extent of tumor growth and may serve as a cancer marker. A cancer-associated galactosyltransferase isoenzyme (GT-II) has been described and purified. Different isoelectric forms of fucosyltransferase have also been described as indicative of malignancy. The articles to be published in CRC Critical Reviews in Clinical Laboratory Sciences will analyze the evidence for the association of these membrane factors with tumor growth. In order to better understand the possible significance of altered glycoproteins and of increased or different forms of glycosyltransferases during tumor growth, recent data on glycoprotein synthesis will be discussed including the new concepts on the control of glycoprotein synthesis through lipid intermediates. The possible mechanisms whereby malignant transformation could alter glycoprotein synthesis will be discussed with particular emphasis on the significance of these alterations to the biology of the malignant cell. Changes in surface membrane glycoproteins have long been implicated in the ability of a cell to metastasize. Secretion and/or shedding of the cell surface may also be important in the process of metastasis and in altering the host immune response. Detection and the study of these "shed" materials in patients appear to be indicating a new approach to cancer biology detection and therapy.
125I-Helix pomatia and 125I-wheat germ lectins were used as probes to visualise membrane glycoproteins of circulating blood cells from individuals with Tn syndrome. Cells were solubilized in Triton X-100 and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis prior to incubation with iodinated lectins. Normal platelets, granulocytes and T lymphocytes have a single major sialoglycoprotein of mol. wt. 143,000, 115,000 and 115,000 respectively. In Tn platelets, granulocytes and T lymphocytes there was a marked reduction in lectin binding in the region of the sialoglycoproteins but new lectin binding components of lower mol. wt. (125,000, 98,000 and 98,000 respectively) were apparent. It is suggested that these new lectin binding components are the sialoglycoproteins with deficient glycosylation resulting from the known deficiency of beta-3-galactosyltransferase in Tn syndrome.
Anti-human galactosyltransferase (E.C. 2.4.1.22) antibodies were elicited in rabbits and purified on a galactosyltransferase-agarose column. Purified antibodies were used to localize galactosyltransferase in acetone-fixed HeLa cells and human lung fibroblasts. Both protein A-peroxidase developed with 3-amino 9-ethylcarbazole and swine anti-rabbit IgG-fluorescein isothiocyanate served to detect binding of anti-galactosyltransferase antibodies. In cells of confluent cultures, anti-galactosyltransferase staining appeared as a concise triangular structure in the juxtanuclear region with one angle oriented toward the bulk of the cytoplasm. The stained structure appeared as a dense cap on the nucleus in HeLa cells and as a more extended granular structure in fibroblasts. In cells of sparse cultures, specific anti-galactosyltransferase staining appeared in both HeLa cells and fibroblasts as a granular, extended structure, which was occasionally perinuclear. There was no evidence of cell surface localization of galactosyltransferase by light microscopy. The positively stained structures are interpreted to be part of the Golgi complex.
Sepharose 4B-immobilized desialylated ovine submaxillary mucin was used as an acceptor for galactose transfer from UDP-galactose, catalyzed by a Triton X-100-solubilized galactosyltransferase from human erythrocyte ghosts. The product could be cleaved from the insoluble acceptor substrate by alkaline borohydride treatment and identified on Bio-Gel P-2 as a disaccharide. The nature of the glycosidic bond of the isolated material was elucidated by periodate oxidation/NaB[3H]4 reduction/acid hydrolysis and subsequent identification of the aminopolyol formed as L-threosaminitol. Specific cleavage of the enzymatic product by beta-galactosidase indicated a beta-configuration for incorporated galactose. These data permit classification of the enzyme as UDP-galactose: alpha-D-N-acetylgalactosaminyl-protein beta (1 leads to 3) transferase. Furthermore, in the presence of Triton X-100, the enzyme from normal erythrocytes catalyzed transfer of galactose to the glycan moieties of asialo-agalacto-glycophorin in Tn-erythrocytes from a patient with permanent mixed-field polyagglutinability.
Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.
Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from 125I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 107 and 6.61 × 107m−1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 107 and 1.17 × 107m−1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 107m−1). The number of Vg binding sites increases from 0.085 × 105 to 0.8 × 105 (high affinity) and from 2.10 × 105 to 6.25 × 105 (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 105 to 0.51 × 105 (high affinity) and 0.13 × 105 (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 105 Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.
The 2 types of erythrocytes from a person with persistent mixed-field polyagglutinability (Tn abnormality) were separated from each other by preparative cell electrophoresis. Surface labelling using the galactose oxidase/NaB3H4 technique followed by polyacrylamide gel electrophoresis showed a strong labelling in the glycophorin A region of Tn positive erythrocytes indicating exposed galactosyl N-acetyl/galactosaminyl residues. Tn positive cell membranes were labelled by the galactose oxidase/NaB3H4 technique and solubilized in non-ionic detergent. After chromatography on Helix pomatia lectin-linked Sepharose, glycophorin A was immunoprecipitated from the sugar eluate using specific antiserum. Glycophorin A from Tn negative cells and normal red blood cells did not bind to Helix pomatia lectin but to Lens culinaris lectin-Sepharose. Glycophorin A and band 3 were isolated by preparative gel electrophoresis from normal cells and the two red cell populations of the Tn individual. Pronase treatment of labelled glycophorin A followed by gel filtration revealed a more efficient proteolysis in molecules isolated from Tn positive cells. Mild alkaline treatment of galactose oxidase/NaB3H4 or periodate/NaB3H4 labelled glycophorin A liberated 3 different oligosaccharides from Tn positive cells. No significant difference was found between the oligosaccharides of band 3 protein from normal and Tn positive cells and the amounts of glycophorin A were identical in both cell types when determined by radioimmunoassay.
La polyagglutinabilité acquise de type Tn s'accompagne de la présence à la surface des érythrocytes d'un récepteur spécifiquement reconnu par la lectine de Salvia sclarea.
Sur le plan hématologique, la transformation Tn est souvent associée à une leucopénie et une thrombopénie, suggérant l'existence du récepteur spécifique à la surface des leucocytes et des plaquettes sanguines.
Par la méthode d'électrophorèse en gel de polyacrylamide, nous avons montré que les érythrocytes et les plaquettes Tn solubilisées dans le SDS présentent effectivement des anomalies structurales de leurs glycoprotéines (GP) de surface, glycophorine et GP Ib respectivement, se traduisant par un effondrement de la coloration à l'acide périodique-Schiff (PAS). Cependant, les incorporations normales d'iode 125I (catalysée par la lactopéroxydase) et de tritium 3H (traitement à la galactose oxydase suivi d'une réduction au borohydrure de sodium tritié) sur les protéines et les glucides de la surface cellulaire respectivement, démontrent que l'altération du profil PAS des GP de membranes Tn ne traduit qu'une modification de la glycosylation de ces macromolécules.
D'ailleurs, nous avons mis en évidence dans ces cellules un déficit sélectif d'un même enzyme, l'UDPGal : Gal-NAc-β-3-D-galactosyltransférase ou enzyme T, qui intervient dans la biosynthèse des chaînes glucidiques alcali-labiles des GP.
Un tel bloc métabolique empêche la galactosylation et la sialylation des résidus N-acétylgalactosamine fixés par une liaison α-O-glycosidique sur les GP (déterminants Tn : GalNAc-α-O-Ser (Thr)) et laisse les déterminants accessibles aux réactifs anti-Tn et à la lectine de Salvia sclarea en particulier. Il en résulte aussi une diminution du contenu en acide sialique et en galactose bien démontrée au niveau des érythrocytes et très vraisemblable au niveau des plaquettes Tn.
Ces résultats suggèrent une communauté structurale entre la partie glucidique de la glycophorine érythrocytaire et la GP Ib des plaquettes. Contrairement à ce qui est observé dans le syndrome de Bernard-Soulier, l'altération de la GP Ib des plaquettes Tn n'entraîne pas de troubles de l'hémostase, puisque toutes les fonctions plaquettaires testées ont été trouvées normales, en particulier l'aggrégabilité à la ristocétine. Les résultats obtenus démontrent clairement que l'activation Tn résulte d'une modification survenue au niveau des cellules souches pluripotentes de la moelle osseuse (les granulocytes sont également affectés) à la suite d'une mutation somatique ou peut être de l'incorporation d'une particule virale dans le génome.
Il est enfin évident que seules quelques cellules souches sont modifiées, puisque le sang circulant contient un mélange séparable de cellules (hématies et plaquettes) dont certaines possèdent le récepteur Tn et d'autres ne le possèdent pas.
The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown. Mesangial IgA is of the IgA1 subclass, and since no consistent antigenic target for the IgA1 has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or IgA2. Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgA1, and the O-linked sugars of IgA1 and C1 inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.
We report our discovery that many glycoproteins synthesized by Chinese hamster ovary (CHO) cells contain fucose in O-glycosidic linkage to polypeptide. To enrich for the possible presence of O-linked fucose, we studied the lectin-resistant mutant of CHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferase I and are therefore unable to synthesize complex-type N-linked oligosaccharides. Lec1 cells were metabolically radiolabelled with [6-3H]fucose and total glycoproteins were isolated. Glycopeptides were prepared by proteolysis and fractionated by chromatography on a column of concanavalin A (Con A)-Sepharose. The sets of fractionated glycopeptides were treated with mild base/borohydride to effect the beta-elimination reaction and release potential O-linked fucosyl residues. The beta-elimination produced [3H]fucitol quantitatively from [3H]fucose-labelled glycopeptides not bound by Con A-Sepharose, whereas none was generated by treatment of glycopeptides bound by the lectin. The total [3H]fucose-labelled glycoproteins from Lec1 cells were separated by SDS-PAGE and detected by fluorography. Treatment of selected bands of detectable glycoproteins with mild base/borohydride quantitatively generated [3H]fucitol. Pretreatment of the glycoproteins with N-glycanase prior to the SDS-PAGE method of analysis caused an enrichment in the percentage of radioactivity recovered as [3H]fucitol. Trypsin treatment of [3H]fucose-labelled intact CHO cells released glycopeptides that contained O-linked fucose, indicating that it is present in surface glycoproteins. These findings demonstrate that many glycoproteins from CHO cells contain O-linked fucosyl residues and raise new questions about its biosynthesis and possible function.
Glycoproteins are widely distributed among species in soluble and membrane-bound forms, associated with many different functions. The heterogenous sugar moieties of glycoproteins are assembled in the endoplasmic reticulum and in the Golgi and are implicated in many roles that require further elucidation. Glycoprotein-bound oligosaccharides show significant changes in their structures and relative occurrences during growth, development, and differentiation. Diverse alterations of these carbohydrate chains occur in diseases such as cancer, metastasis, leukemia, inflammatory, and other diseases. Structural alterations may correlate with activities of glycosyltransferases that assemble glycans, but often the biochemical origin of these changes remains unclear. This suggests a multitude of biosynthetic control mechanisms that are functional in vivo but have not yet been unraveled by in vitro studies. The multitude of carbohydrate alterations observed in disease states may not be the primary cause but may reflect the growth and biochemical activity of the affected cell. However, knowledge of the control mechanisms in the biosynthesis of glycoprotein glycans may be helpful in understanding, diagnosing, and treating disease.
Previously, β1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol
22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAcα1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Acα2,6GalNAcα1-O-R) or TF (Galβ1-3GalNAcα1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.
Glycoproteins are proteins that carry N- and O-glycosidically-linked carbohydrate chains of complex structures and functions. N-glycan chains are assembled in the endoplasmic reticulum and the Golgi by a controlled sequence of glycosyltransferase and glycosidase processing reactions involving dolichol intermediates. The assembly of O-glycans occurs in the Golgi and does not involve dolichol. For most reactions, families of glycosyltransferases exist; the expression of the individual enzymes within a family is often subject to complex regulation. The biosynthesis of N- and O-glycan is controlled at the level of gene expression, mRNA, enzyme protein activity and localization, and through substrate and cofactor concentrations at the site of synthesis. This complex regulation results in many hundreds of structures, the range of which varies in different species, cell types, tissue types, states of development and differentiation. In diseased cells, the relative proportions of these structures are often characteristically different from normal, and may be useful for the assessment of the stage of the disease and for diagnosis. Knowledge of disease-specific glycoprotein structures and their functions may be used therapeutically, in immunotherapy, in blocking cell adhesion or interfering with other binding or biological processes. Recently, some of the mechanisms underlying glycoprotein alterations in disease have been elucidated. This opens the possibility of an active interference in the disease process. The functions of glycans in diseased cells will become more clear with the tools of molecular biology and transgenic animal models.
Galactose is transferred via several linkages to acceptor structures by galactosyltransferase enzymes. In prokaryotes, galactose is mainly found on lipopolysaccharides and capsular polysaccharides. In eukaryotes, galactosyltransferases, which are localized in the Golgi apparatus, are involved in the formation of several classes of glycoconjugates and in lactose biosynthesis. Although they sometimes catalyze identical reactions, prokaryotic and eukaryotic galactosyltransferases share only little structural similarities. In mammals, 19 distinct galactosyltransferase enzymes have been characterized to date. These enzymes catalyze the transfer of galactose via beta1-4, beta1-3, alpha1-3 and alpha1-4 linkages. The present review focuses on the description of these mammalian galactosyltransferases.
A sialyltransferase that catalyzes the synthesis of mucin from cytidine 5′-monophospho-N-acetylneuraminic acid and sialidase-treated sheep submaxillary mucin has been isolated from sheep submaxillary glands. The partially purified transferase incorporated approximately 70% of the sialic acid cleaved from the mucin by sialidase. Kinetic studies showed that the approximate Km values were 1.9 mm for the sialidase-treated mucin (calculated in terms of the concentration of N-acetylgalactosamine acceptor sites), and 0.57 mm for the CMP-N-acetylneuraminic acid. No metal ion requirement could be demonstrated for the reaction. The purified enzyme could utilize CMP-N-glycolylneuraminic acid in place of CMP-N-acetylneuraminic acid.
Active acceptors included polymers containing terminal N-acetylgalactosamine residues such as sialidase-treated mucins from the submaxillary glands of sheep, pig, and cow, as well as fetuin, a glycopeptide from milk, and erythrocyte hemagglutination inhibitor. Crude extracts contained an endogenous acceptor for sialic acid, which was separated from the sialyltransferase and partially characterized as an “incomplete” mucin. A tissue survey showed the presence of the sialyltransferase in extracts of submaxillary glands of sheep, pig, and cow, and each of the extracts was active with the mucins obtained from any of the species. The requirements for acceptor activity are discussed.
The ovine submaxillary mucin synthesized by the purified enzyme was purified and characterized, and periodate oxidation studies showed that the sialic acid was linked to the N-acetylgalactosamine residue at C-6.
Two immunochemically distinct mucins have been isolated from pig submaxillary glands. The glands were combined according to the ability of aqueous extracts of these glands to inhibit hemagglutination of human type A erythrocytes. Mucin isolated from the glands containing blood group A activity is designated A⁺ pig submaxillary mucin (A⁺-PSM), while mucin isolated from the remaining glands is designated A⁻ pig submaxillary mucin (A⁻-PSM). The carbohydrate composition of both mucins is similar and comprises N-acetylgalactosamine, fucose, galactose, and N-glycolylneuraminic acid.
Treatment of these mucins with alkaline borohydride resulted in the release of a series of reduced oligosaccharides and the monosaccharide, 2-acetamido-2-deoxy-d-galactitol (N-acetylgalactosaminitol). Conditions are reported which give more than 90% cleavage of the sugar residues from the protein chain. The most complex oligosaccharide (designated oligosaccharide I) was a pentasaccharide, 2-acetamido-2-deoxy-α-d-galactopyranosyl (1 → 3)-[α-l-fucopyranosyl-(1 → 2)]-β-d-galactopyranosyl-(1 → 3)-[N-glycolylneuraminyl-(2 → 6)]-2-acetamido-2-deoxy-d-galactitol. In addition, the following oligosaccharides were isolated (structures are given as related to oligosaccharide I): oligosaccharide II, I minus N-acetylgalactosamine; oligosaccharide III, a disaccharide N-glycolylneuraminyl → N-acetylgalactosaminitol; oligosaccharide IV, I minus N-glycolylneuraminic acid; oligosaccharide V, II minus N-glycolylneuraminic acid. N-Acetylgalactosaminitol (Fraction VI) was the only detectable monosaccharide. Rabbit antiserum to human type A erythrocyte stroma precipitated A⁺-PSM, but not A⁻-PSM. Oligosaccharides I and IV, found only in A⁺-PSM, are potent inhibitors of the anti A-A⁺-PSM precipitation, but oligosaccharides II, III, and V and a monosaccharide (Fraction VI) are completely inactive.
Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition.
Human serum and hemoglobin-free erythrocyte membranes were found to contain a galactosyltransferase which catalyzes the transfer of galactose from UDP-galactose to specific large and small molecular weight acceptors. The requirements for enzyme activity were found to be similar for the enzymes from both sources. However, the membrane-bound enzyme depended on a detergent for maximal activity. Mn(++) was an absolute requirement for transfer and uridine nucleoside phosphates were inhibitors. The most effective acceptor for galactose was a glycoprotein containing N-acetylglucosamine residues in the terminal position of its oligosaccharide side chains, N-acetylglucosamine was also an acceptor. While the presence of alpha-lactalbumin in the incubation medium resulted in a significant decrease in the transfer of galactose to N-acetylglucosamine, glucose, which was not an acceptor for galactose in the absence of alpha-lactalbumin, became an excellent acceptor. The serum enzyme catalyzed the transfer of 54 nmoles of galactose per milliliter of serum per hour and its apparent K(m) for UDP-galactose was 7.5 x 10(-6)M. The membrane enzyme had a similar apparent K(m). Using a quantitative assay system the enzyme was found to be present in all individuals studied, regardless of their blood type, secretor status, or sex.
The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and found to have a pronounced influence upon hemoglobin binding in the ghosts. Buffer concentrations between 10 and 20 ideal milliosmolar (imOsm), at pH values 5.8 – 8.0, resulted in maximum hemoglobin removal from ghosts. The pH optimum for hemoglobin binding to ghosts was between 5.8 and 5.9 in a 20 imOsm buffer. The influence of these variables suggest an electrophysical interaction of hemoglobin with membrane constituents.This study provides a basis for comparison of existing methods for ghost preparation, as well as a means for prediction of the conditions required for preparation of ghosts containing any desired amount of hemoglobin. Conditions were found that allowed the preparation of hemoglobin-free ghosts by single-stage hemolysis and washing. Hemoglobin-free ghosts were prepared in 20 imOsm phosphate buffer at pH 7.4. Essentially all the lipid was recovered in the ghosts, but non-hemoglobin nitrogen-containing substances were lost.The pyridine hemochromogen method for hemoglobin determination was adapted for the measurement of very small quantities of hemoglobin through use of the Soret band (418 mμ) for absorbancy measurements.
A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.
The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.
1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high‐ and low‐molecular‐weight acceptors. Transfer to N ‐acetylglucosamine was measured after the separation of the reaction product N ‐acetyllactosamine from all other radioactive molecules (including galactose) on an ion‐exchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.
2. Both enzymic activities were enriched 60–80‐fold (compared with the post‐nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 95%), presumably because both compete for the same enzyme.
3. The transferase activities were enhanced by the nonionic detergent Triton X‐100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the w/v concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation: Half‐maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50% of the fully activated enzyme could still be sedimented (1 h, 100000 × g ).
4. Galactose transfer to the high‐molecular‐weight acceptor required a higher Triton/phospholipid ratio for half‐maximal activation than did the transfer to the monosaccharide N ‐acetylglucosamine (1 mg/mg compared with 0.5 mg/mg). The degree of activation maximally achieved was much higher with the protein acceptor (400%) than with the sugar (150%). Because both activities are probably due to the same enzyme, it is suggested that these differences in activation reflect properties of the membrane rather than the enzyme, e.g . the presence of a tight diffusion barrier for ovomucoid and the breakdown of this barrier by the detergent.
The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group. This residue covers in the MM antigen the beta-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal alpha-linked N-acetylneuraminic acid residue, is responsible for N specificity. M specificity was readily converted into N specificity by mild acid treatment. N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto. The NN antigen was inactivated by beta-D-galactosidase as well as by removal of N-acetylneuraminic acid. Some of the reactivities of the NN antigen, lost upon beta-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal. The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen-Friedenreich T antigen, the specificity of which is determined by beta-D-galactopyranosyl groups. Beta-D-Galactosidase treatment transformed the T antigen into one possessing Tnactivity. The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only.
The nature of the receptor sites for several agglutinins is characterized by hemagglutination inhibition assays. The inhibitory activity of human erythrocytes glycoproteins, from which sialic acid, sialic acid and galactose or alkali-labile oligosaccharides have been removed, is compared to the inhibitory effect of compounds with known structure. It is shown that the lectin from Arachis hypogea and anti-T bind to alkali-labile galactosyl-residues. Agglutinins from Bauhinia purpurea and variegata (non- or N-specific), Maclura aurantiaca, Iberis amara, sempervirens, umbellata hybrida and umbellata nana (M- or nonspecific), Moluccella laevis (A- plus N-specific), Helix pomatia, Helix aspersa, Helix lucorum and Caucasotachea atrolabiata interact with alkali-labile N-acetylgalactosamine. The results obtained with the anti-A agglutinins from various snails suggest that human erythrocyte glycoproteins contain, besides the alkali-labile tetrasaccharide, a peptide-linked sialyl-N-acetyl-galactosaminyl-residue. The investigations do not allow a precise definition of the receptor sites for the lectins having M- or N-specificity.
Spectrophotometric and gas-liquid chromatographic analyses on the carbohydrate moiety of tryptic erythrocyte glycopeptides from persons with Tn-syndrome reveal a selective lowering of the galactose and sialic acid content, the degree being dependent on the percentage of polyagglutinable cells. Alkaline borohydride specifically releases N-acetylgalactosaminitol, and the amount is correlated to the percentage of pathological acetylgalactosaminitol, and the amount is correlated to the percentage of pathological erythrocytes. It is concluded that the alkali-labile carbohydrate chains of Tn-polyagglutinable red cells solely consist of N-acetylgalactosamine linked to serine or threonine. Experiments with heterophile agglutinins whose specificity is known are in line with the above-mentioned results. As judged from SDS-polyacrylamide gel electrophoresis the three major membrane glycoproteins are affected to a different extent by the defect.
With the use of endless belt fluid electrophoresis, these studies confirm previous reports of two cell populations in PMFP having different mobilities. Our method shows two clearly separated bands, while with the microelectrophoresis method a distinctly bimodal distribution is reported. The abnormal (A) cells show a 33- to 42-percent reduction in mobility and a 47- to 48-percent reduction in sialic acid as compared with standard cells. The serologically normal (N) cell population, on mixture with standard cells and subjected to electrophoresis, exhibits no increase in streak width indicative of increased electrophoretic heterogeneity. This is consistent with the findings in the preceding report of no serological abnormalities in the (N) cells. Results of hematological studies, red cell isozyme determinations and assays of glycolytic enzymes activity were not significantly different between the two cell populations.
These observations lead us to suggest that the basic abnormality in PMFP (A) cells (Tn transformation) represents a point mutation involving a single genetic step in erythrocyte membrane glycoprotein biosynthesis. A block in the transfer of terminal D-galactose to N-acetylgalactosamine in a substantial proportion of (A) cell heterosaccharides is postulated to be common to all of the serological and biophysical aberrations. This would be consistent with the observed reduction of T, NVG, NHmn (also MHmn), electrostatic charge, sialic acid, and increased ‘saline agglutinability’ in incomplete antisera. The defect in galactose linkage could also leave exposed underlying N-acetylgalactosamines with the consequent manifestation of AHmn, ADB, AHP, NBV and Tn.
A sialyltransferase that catalyzes the synthesis of mucin from cytidine 5' monophospho N acetylneuraminic acid and sialidase treated sheep submaxillary mucin was isolated from sheep submaxillary glands. The partially purified transferase incorporated approximately 70% of the sialic acid cleaved from the mucin by sialidase. Kinetic studies showed that the approximate K(m) values were 1.9 mM for the sialidase treated mucin (calculated in terms of the concentration of N acetylgalactosamine acceptor sites), and 0.57 mM for the CMP N acetylneuraminic acid. No metal ion requirement could be demonstrated for the reaction. The purified enzyme could utilize CMP N glycolylneuraminic acid in place of CMP N acetylneuraminic acid. Active acceptors included polymers containing terminal N acetylgalactosamine residues such as sialidase treated mucins from the submaxillary glands of sheep, pig, and cow, as well as fetuin, a glycopeptide from milk, and erythrocyte hemagglutination inhibitor. Crude extracts contained an endogenous acceptor for sialic acid, which was separated from the sialyltransferase and partially characterized as an 'incomplete' mucin. A tissue survey showed the presence of the sialyltransferase in extracts of submaxillary glands of sheep, pig, and cow, and each of the extracts was active with the mucins obtained from any of the species. The requirements for acceptor activity are discussed. The ovine submaxillary mucin synthesized by the purified enzyme was purified and characterized, and periodate oxidation studies showed that the sialic acid was linked to the N acetylgalactosamine residue at C 6.
- D M Carlson
Carlson, D. M. (1968) J. Biol. Chem. 243,616-626.
- R Bretz
- W Stsiubli
Bretz, R. and Stsiubli, W. (1977) Eur. J. Biochem. 77,
181-192.
- Y S Kim
- Perdomo
- J S Whitehead
Kim, Y. S., Perdomo, 3. and Whitehead, J. S. (1972) 3.
Clm. Invest. 51,2024-2032.
- W Dahr
- G Uhlenbruck
- H Knott
Dahr, W., Uhlenbruck, G. and Knott, H. (1975)
J. Immuno8enet. 2,87-100.
- G L Ellman
- D K Courtney
- V Andres
- R M Featherstone
Ellman, G. L., Courtney, D. K., Andres, V. and
Featherstone, R. M. (1961) Biochem. Pharmacol. 7,
88-95.
- T W Keenan
- D J Morrk
Keenan, T. W. and Morrk, D. J. (1975) FEBS Lett.
55,8-13.
- E G Berger
- I Kozdrowski
- M M Weiser
- D H Van Den Eijnden
- W E C M Schiphorst
Berger, E. G., Kozdrowski, I., Weiser, M. M., Van den
Eijnden, D. H. and Schiphorst, W. E. C. M. (1978) 4th
Int. Symp. Glycoconjugates (Jeanloz, R. W, and
Gregory, J. D. eds) Academic Press, New York, in press.
Dodge, J. T., Mitchell, C. and Hanahan, D. J. (1963)
Arch. Biochem. Biophys. 100,119-130.