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.

Cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-N-acetyllactosamine series.

Roles of cell surface carbohydrates containing the 3-fucosyl-N-acetyllactosamine and poly-N-acetyllactosamine sequences (SSEA-1 and I antigens, respectively) in the compaction of mouse embryos have been investigated using the endo-beta-galactosidase of Bacteroides fragilis to modify the surface of cleavage-stage embryos. Treatment with this enzyme abolished SSEA-1 activity and diminished I antigen activity on the embryonic cell surface. Embryos cultured in the presence of endo-beta-galactosidase from the 2- to 4-cell stage onwards, or treated with the enzyme at the compacting 8-cell stage, continued to compact and proceeded to form blastocysts at the normal rate.

Monoclonal antibody (EGR/G49) reactive with the epidermal growth factor receptor of A431 cells recognizes the blood group ALeb and ALey structures.

The carbohydrate specificity of the monoclonal antibody EGR/G49, raised against the epidermal growth factor (EGF) receptor of A431 cells, has been investigated by assessing its interactions with glycoproteins and erythrocytes derived from individuals of known blood group ABH, Lewis and secretor types, and by inhibition of binding assays using structurally defined oligosaccharides. The results indicate that this antibody reacts with the difucosylated blood group structures ALeb and ALey: (formula; see text) This antibody differs from the previously described anti-EGF receptor antibody. TL5, which is directed at the terminal blood group A trisaccharide structure and reacts poorly with the ALeb/Ley structures.

Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody.

A monoclonal antibody (NIBy 142-36/8) raised against the soluble galactose-binding lectin of bovine heart muscle has been tested by solid-phase vinyl-plate radiobinding and nitrocellulose immunoblotting with homogenates of various bovine tissues, and the muscle tissues of pig, rabbit, chicken and rat. Muscle lectins of chicken, rabbit and rat differed from those of man and pig in their lack of reactivity with the 36/8 antibody. There was a good correlation of haemagglutinating activities and immunoreactivities of the bovine tissue homogenates, suggesting that the soluble galactose-binding protein is a major haemagglutinin in various tissues.

Structural analysis of the O-glycosidically linked core-region oligosaccharides of human meconium glycoproteins which express oncofoetal antigens.

Glycoproteins were extracted from meconium samples of group O neonates of secretor type by pronase digestion followed by precipitation in 67% aqueous ethanol and separated into Ii antigen enriched and depleted fractions by affinity chromatography. The latter fraction strongly expressed the oncofoetal antigens recognised by natural antibodies in mouse sera and the hybridoma antibody FC 10.2, and this activity was enhanced after mild acid hydrolysis to remove sialic acid and fucose residues.

Differences in the fine specificities of monoclonal (Class A) antibodies to human myeloid cells.

Among 13 monoclonal antibodies to human myelomonocytic cells, six could be assigned to a group designated Class A with the following properties: (a) they react almost exclusively with granulocytes among cells of the peripheral blood, (b) they resemble the previously described anti-granulocyte antibodies, VEP8 and VEP9, and the anti-mouse embryo, anti-SSEA-1, in their strong reactions with human meconium glycoproteins and ovarian cyst mucins of non-secretor type and (c) they recognize the carbohydrate antigen 3-fucosyl-N-acetyllactosamine (alpha 1----3fucosylated Type 2 blood group chains). The binding of these anti-myeloid antibodies is more strongly inhibited by lacto-N-fucopentaose III than by the trisaccharide-fucosyl-N-acetyllactosamine, in contrast to anti-SSEA-1 which is more strongly inhibited by the trisaccharide. These observations suggest that the myeloid Class A antibodies recognize additional determinants on the neolacto (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4) backbone of the pentasaccharide which occurs on the glycoproteins and glycolipids of myeloid cells.

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin.

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.

Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens.

The hope that hybridoma antibodies would reveal unique cell surface antigens during embryogenesis, differentiation and oncogenesis has been replaced by the realization that such antigens are mainly carbohydrate structures of glycoproteins and glycolipids occurring in many cell types. These findings either may reflect limitations in the methods of selection of hybridoma antibodies or may point to important roles for the diverse carbohydrate structures as receptors for regulators of cell growth and differentiation.

Blood group antigens A, B, H, Lea, Leb, and I(Ma) in resting and tetragastrin stimulated gastric juice of patients with non-neoplastic diseases of the stomach.

In view of the anomalous expression of blood group and related antigens in the gastric mucosae of patients with malignant and premalignant diseases of the stomach, and the potential clinical value of their measurement, a preliminary study has been performed on the blood group antigens A, B, H, Lea, Leb, and I(Ma) in glycoprotein rich extracts of the resting and tetragastrin stimulated gastric juice of patients without evidence of gastric cancer. The aim has been to assess whether the antigenic profiles known to distinguish the gastric mucosae of secretors from those of non-secretors are reflected in the glycoproteins of gastric juice. Antigenic profiles which distinguish secretors from non-secretors were observed in the stimulated rather than the resting gastric juice as follows: the A, B or H antigens but not I(Ma) were strongly expressed in the glycoproteins of secretors, while I(Ma) was the antigen characteristic of non-secretors.

Evidence for sialylated type 1 blood group chains on human erythrocyte membranes revealed by agglutination of neuraminidase-treated erythrocytes with Waldenström's macroglobulin IgMWOO and hybridoma antibody FC 10.2.

Haemagglutination studies have been performed with untreated and neuraminidase-treated human erythrocytes of the three Lewis antigen types Le(a-b-), Le(a+b-) and Le(a-b+) using two monoclonal antibodies, IgMWOO and FC 10.2, which were previously shown to recognize the type 1 based blood group chains: Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc/GlcNAc (for explanation of abbreviations see table IV legend). Both antibodies behaved as cold agglutinins with neuraminidase-treated but not with untreated erythrocytes of the three Lewis antigen types.

Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of I and i antigen types.

The role of sialoglycolipids (gangliosides) as receptors for the human pathogen Mycoplasma pneumoniae was investigated by using purified gangliosides of known carbohydrate structures as inhibitors of the binding of 51Cr-labeled erythrocytes to sheet cultures of M. pneumoniae. We found that sialoglycolipids with long carbohydrate backbones of the poly-N-acetyllactosamine type were more potent inhibitors of M.

Carbohydrate antigens in human cancer.

Studies with naturally occurring and hybridoma-derived monoclonal antibodies have shown that surface and secreted antigens which distinguish human tumour cells from their normal counterparts are predominantly carbohydrate structures. Many of these belong to a family which includes the major blood-group antigens. In fact, the blood-group genes and the related secretor gene account for the individual and tissue-specific patterns of expression of several carbohydrate antigens as normal or as tumour-associated antigens.

Comparison between murine natural antibodies and natural killer cells: recognition of separate target structures as revealed by differential in vitro expression and dependence on glycosylation.

The specificity of complement-fixing, cytotoxic antibodies against the YAC lymphoma in sera of normal young adult (A X C57BL)F1 mice was studied. In vivo-maintained, immunoselected sublines of the YAC lymphoma expressed low amounts of the natural antibody (NAb) target structure. These cell lines were also resistant to natural killer (NK) cell-mediated lysis.

The carbohydrate specificities of the monoclonal antibodies 29.1, 455 and 3C1B12 to the epidermal growth factor receptor of A431 cells.

Sixteen hybridoma-derived antibodies to the epidermal growth factor receptor of A431 cells were studied with respect to their reactions with blood group-related carbohydrate structures. Twelve of these were assessed as recognizing carbohydrate determinants on the basis of their immunostaining of reference blood group substances on nitrocellulose paper. Three of these antibodies were further investigated by inhibition of binding assays with glycoproteins and structurally defined oligosaccharides or by haemagglutination of erythrocytes before and after treatment with endo-beta-galactosidase.

Further studies of the specificities of monoclonal anti-i and anti-I antibodies using chemically synthesized, linear oligosaccharides of the poly-N-acetyllactosamine series.

The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells.

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type.

Cryptic I antigen activity and Mycoplasma pneumoniae-receptor activity associated with sialoglycoprotein GP-2 of bovine erythrocyte membranes.

The 250-kDa sialoglycoprotein of bovine erythrocyte membranes, GP-2, has been found to be an exceptionally rich source of branched sialo-oligosaccharides of poly-N-acetyllactosamine (I antigen) type with receptor activity for the human pathogen Mycoplasma pneumoniae. Desialylated GP-2 is the most potent I-active substance thus far tested. Since this glycoprotein is hydrophobic and can be readily re-incorporated into cell membranes, it should be useful in future studies of the mechanism of production of autoantibodies to the I antigen which commonly arise following human infection with M.

Species differences in the expression of carbohydrate differentiation antigens on mammalian blood cells revealed by immunofluorescence with monoclonal antibodies.

Following recent observations using monoclonal antibodies that carbohydrate structures behave as differentiation antigens of man and mouse, we have made a preliminary survey of the expression of 8 monoclonal antibody-defined carbohydrate antigens on blood cell smears of man, baboon, mouse, rat, rabbit, pig, and dog. There are considerable species differences in the patterns of antigen expression. However, certain generalizations can be made as follows: the i and I antigens, associated with linear and branched carbohydrate chains consisting of repeating N-acetyl-lactosamine sequences (Gal beta 1-4GlcNAc, termed Type-2 backbone sequences) are widely distributed among granulocytes and lymphocytes of all the species studied, and on erythrocytes, monocytes, and platelets of some of them.

The effect of mild alkali and alkaline borohydride on the carbohydrate and peptide moieties of fetuin.

In the light of recent reports, based on radioactive labelling studies, that substantial amounts of N-linked oligosaccharides are released from protein under the mild-alkaline borohydride degradation conditions that are usually used to release O-linked oligosaccharides, we have investigated by chemical methods the effects of alkali alone and alkaline borohydride on the carbohydrate and peptide moieties of fetuin. The chromatographic profiles on Sephadex G50 columns, of the hexose- and ninhydrin-positive components of the native and Pronase-treated glycoprotein have been compared with those obtained after treatment with mild alkali alone (0.05 M-NaOH, 50 degrees C, 16 h) or mild-alkaline borohydride (0.

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins.

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs.

Endo-beta-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series.

The specificities of the endo-beta-galactosidases of Bacteroides fragilis and Escherichia freundii towards linear and branched oligosaccharides of the lacto-N-glycosyl series were investigated using as substrates glycolipids containing (a) linear neolactotetra - or hexaosyl sequences, (b) branched biantennary neolactooctaosyl sequences, and (c) triantennary neolactononaor dodecaglycosyl sequences. Glycolipid and oligosaccharide hydrolysis products were identified by tlc and/or paper chromatography. The rate of hydrolysis was assessed in time course experiments in which the oligosaccharides released were quantified as 3H-labeled alditols.

Production and characterization of monoclonal antibodies to beta-galactoside-binding lectin of bovine heart muscle. Direct evidence that haemagglutinating activity is associated with a 13kDa protein.

With the aim of obtaining monospecific antibodies against the beta-galactoside-binding lectin of bovine heart muscle, spleen cells from Lou rats immunized with lectin were fused with the rat myeloma line Y3.Ag1.2.

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue.

Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type.

Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide.