A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies (original) (raw)
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European Journal of Biochemistry, 1978
Glycopeptides with complex carbohydrate structure were isolated from delipidated human erythrocyte membranes after digestion with pronase. The poly(glycosyl)peptides isolated (apparent molecular weight 4000--13000) are suggested to contain 20--70 sugar residues in an alkali-stable saccharide chain linked through N-acetylglucosamine to asparagine. The main sugar components are galactose and N-acetylglucosamine, which together account for 80% of total sugars. That the compounds isolated are glycopeptides and not glycolipids is concluded from the following findings: only trace amounts of glucose and fatty acids were present, and no long-chain (sphingosine) bases could be detected; on the other hand, the amounts of mannose and amino acids found are compatible with an N-glycosidic poly(glycosyl)peptide structure. The structure of the poly(glycosyl)peptides was studied using methylation analysis, exoglycosidase treatments, acid hydrolysis of the native as well as the N-deacetylated glycopeptides, and chromium trioxide oxidation. The studies indicate that the poly(glycosyl)peptides contain a repeating-3)galactosyl(beta1-4)N-acetylglucosaminyl(beta1-structure with branch points at the C-6 of the galactose residues. The saccharide chains are terminated in N-acetylglucosaminyl, galactosyl, N-acetylneuraminyl(alpha2-3 and 6)galactosyl and fucosyl(alpha1-2)galactosyl residues, and they also contain blood group A and B determinants.
Carbohydrate antigen profiles of human erythroleukemia cell lines HEL and K562
Blood, 1983
The expression of major carbohydrate antigens carried by polylactosaminyl chains in human erythroleukemia cell lines, K562 and HEL, was investigated by applying monoclonal antibodies recognizing specific carbohydrate determinants. The two cell lines showed common differences in their glycolipid compositions: (1) the presence of significant amounts of ganglio-series glycolipids, which are absent in normal erythrocytes; and (2) a remarkable reduction in the amount of globo-series glycolipids, which are the major glycolipids in normal human erythrocytes. A variety of differences were also detected in the carbohydrate antigens carried by lacto-series glycolipids and glycoproteins having related carbohydrate chains. K562 cells were i+H- X+, with a minor population of I+ cells. HEL cells were I-i+H+X-. The presence of the I+ population in K562 cells is particularly noteworthy, since I-antigen is characteristic of adult mature erythrocytes and is absent in most human leukemic cell lines. S...
European Journal of Biochemistry, 1981
The distribution of blood-group A and B determinants was studied by isolating blood-group ABH-active polyglycosyl peptides from delipidated human blood-group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to BsI lectin. The glycopeptides bound were further fractionated using the blood-group-A-specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood-group-B-specific isolectin from B. simplicifolia (BsIB4) whereas the Vc-unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains. The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N-acetylgalactosamine was found only in the BsI-bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3-O-linked galactose, which was enriched 7.5-fold in the BsI-bound glycopeptides and 3,6-O-linked galactose, which also enriched in the BsI-bound glycopeptides suggesting that these have a more branched structure than the BsI-unbound glycopeptides. Glycopeptides derived from bands 3 and 4.5 were prepared from A1B-blood-group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI-lectin from both samples. 70% of the BsI-bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.
Carbohydrate antigen profiles on human erythroleukemia cell lines HEL and K562
Blood
The expression of major carbohydrate antigens carried by polylactosaminyl chains in human erythroleukemia cell lines, K562 and HEL, was investigated by applying monoclonal antibodies recognizing specific carbohydrate determinants. The two cell lines showed common differences in their glycolipid compositions: (1) the presence of signifucant amounts of ganglio-series glycolipids. which are absent in normal erythrocytes; and (2) a remarkable reduction in the amount of globo-series glycolipids. which are the major glycolipids in normal human erythrocytes. A variety of differences were also detected in the carbohydrate antigens carried by lacto-series glycolipids and glycopro-R EMARKABLE ALTERATION of cell surface carbohydrates associated with malignant transformation has been noted for many years."2 Recently, From Biochemical Oncology.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1985
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 glycusphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gait1-, 4GicNAc~I ~ 3Gait1 ~ 4GIcNAcfll ~ 3Gait1 ~ 4GlcNAc~1-, 3Gait1-> 4GIc-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: Galfll-, 4(Fucal ~ 3)GIcNAc and Fucal ~ 2Galfll-*4(Fucal ~3)GIcNAc, 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-Nacetyilactosamine type that are susceptible to degradation with endo-fl-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.
2002
Glycoproteins are generally composed by a population of closely related glycosylation variants having in common the same polypeptide chain but varying by thei r carbohydrate moiety. This carbohydrate-mediated heterogeneity is often called the microheterogeneity of glycoproteins and the different variants referred to as glycoforms . This heterogeneity arises from the presence of multiple glycannic structures attached a t one glycosylation site . The carbohydrate groups are covalently attached to the polypeptidic backbone through the amide nitrogen of an asparagine residue (N-glycosylation) o r through an 0-linkage with a serine, threonine and sometimes a hydroxyproline residu e (O-glycosylation) . N-linked oligosaccharides may be classified on the basis of th e nature of monosaccharides which constitute the oligosaccharides . High-mannose-type oligosaccharides only consist in mannose and N-acetylglucosamine residues and ar e not charged . Complex-type oligosaccharides may be negatively charged and contai n galactose, fucose and sialic acid residues in addition to the monosaccharides foun d in the high-mannose chains . Hybrid structures are intermediate structures between th e high-mannose and the complex-type oligosaccharides . The three families of oligosaccharides share the same pentasaccharide structure (GlcNac-GlcNac-Man 3 ) by whic h the oligosaccharide is attached to the protein .
Glycobiology, 2011
Glycolipids from the red cells of a rare blood group A subgroup individual, expressing the blood group A 3 phenotype with the classical mixed-field agglutination phenomenon, A 2(539G>A) /O 1 genotype, and an unusual blood group A glycolipid profile, were submitted to a comprehensive biochemical and structural analysis. To determine the nature of blood group A glycolipids in this A 3 phenotype, structural determination was carried out with complementary techniques including proton nuclear magnetic resonance (1D and 2D), mass spectrometry (MS) (nano-electrospray ionization/quadrupole time-of-flight and tandem mass spectrometry) and thin layer chromatography with immunostaining detection. As expected, total blood group A structures were of low abundance, but contrary to expectations extended-A type 2 and A type 3 glycolipids were more dominant than A hexaglycosylceramides based on type 2 chain (A-6-2 glycolipids), which normally is the major A glycolipid. Several para-Forssman (GalNAcĪ²3GbO 4) structures, including extended forms, were identified but surmised not to contribute to the classic mixed-field agglutination of the A 3 phenotype. It is proposed that the low level of A antigen combined with an absence of extended branched glycolipids may be the factor determining the mixed-field agglutination phenomenon in this individual.