Oncofetal expression of Lex carbohydrate antigens in human colonic adenocarcinomas. Regulation through type 2 core chain synthesis rather than fucosylation

The mechanism of expression of a series of glycolipid antigens carrying the Lex determinant structure, Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----, and characterized by oncofetal expression in fetal colon and colonic adenocarcinomas has been studied in human fetal and adult proximal colon tissue. Results presented from TLC immunostain analysis of neutral glycolipids isolated from normal adult colonic mucosa have indicated the presence of only barely detectable quantities of both an Lex-active glycolipid that co-migrated with III3V3Fuc2nLc6 and its precursor nLc6. These structures were found in large quantities in glycolipid fractions from human adenocarcinoma tumors and human small cell lung carcinoma NCI-H69 cells. In contrast, type 1 chain-based Lea antigen structures were found in both normal mucosa and adenocarcinomas. Analysis of gangliosides of normal colonic mucosa by TLC immunostain indicated the presence of a series of type 2 chain-based gangliosides; however, sialyl-Lex was not detected. The ability of normal colonic mucosa to synthesize type 2 chain core structures was demonstrated by the presence of a beta 1----4 galactosyltransferase activity with Lc3 as an acceptor in an amount equivalent to 60-65% of the total galactosyltransferase activity. An alpha 1----3 fucosyltransferase was also found to be expressed in significant quantity in adult colonic mucosa. Kinetic studies indicated that this is most probably the alpha 1----3/4 fucosyltransferase suggested to be a product of the Lewis gene (Le). Thus, although normal adult colonic mucosa contained the enzymes to synthesize Lex and sialyl-Lex structures, these antigens were not found. Tissue immunofluorescence studies indicated that type 2 chain precursors and the alpha 1----3/4 fucosyltransferase were found in different cell populations in adult proximal colonic mucosa. However, both type 2 chain core structures and their fucosylated derivatives were found to be associated with epithelial cells of fetal colon. These results indicate that oncofetal expression of Lex antigens in fetal colonic epithelium and in adenocarcinomas but not in normal adult mucosa is due to the retrogenetic expression of type 2 chain precursors which are not found in normal adult colonic epithelial cells.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

The LEC11 Chinese hamster ovary mutant synthesizes N-linked carbohydrates containing sialylated, fucosylated lactosamine units. Analysis by one- and two-dimensional 1H NMR spectroscopy

Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Lex determinant (Gal beta 1,4(Fuc alpha 1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Lex determinant (NeuAc alpha 2,3Gal beta 1,4(Fuc alpha 1,3)GlcNAc), and both mutants have been shown to possess a distinct alpha(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Lex to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Lex determinant and that G from LEC11/VSV also possesses sialylated Lex. Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of alpha(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking alpha(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

Biosynthesis of the sialyl-Lex determinant carried by type 2 chain glycosphingolipids (IV3NeuAcIII3FucnLc4, VI3NeuAcV3FucnLc6, and VI3NeuAcIII3V3Fuc2nLc6) in human lung carcinoma PC9 cells

The pathway for synthesis of three glycosphingolipids bearing a common sialyl-Lex determinant (NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNac beta 1----R) from their type 2 lactoseries precursors has been studied using the 0.2% Triton X-100-soluble fraction from human lung carcinoma PC9 cells. Two enzymes were found to be required for their synthesis: (i) an alpha 1----3 fucosyltransferase, the properties of which have been characterized as being similar to the enzyme from human small cell lung carcinoma NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627); and (ii) an alpha 2----3 sialyltransferase that was efficiently solubilized by 0.2% Triton X-100 and required divalent metal ions and 0.3% Triton CF-54 for optimal activity at pH 5.9 in cacodylate buffer. Biosynthesis of the sialyl-Lex determinant was shown to proceed via sialylation of nLc6 and nLc4, followed by alpha 1----3 fucosylation at the penultimate GlcNAc residues, based on the following: (i) transfer of NeuAc by PC9 cell sialyltransferase was found only when the nonfucosylated acceptors nLc4 and nLc6 were added, and none of the glycolipids with Lex structure (III3FucnLc4; V3FucnLc6; III3V3Fuc2nLc6) were sialylated; and (ii) the PC9 cell fucosyltransferase was active with both neutral and ganglioside neolacto (type 2 chain) acceptors. Transfer of fucose to VI3NeuAcnLc6 yielded mono- and difucosyl derivatives, whereas only a monofucosyl derivative was obtained when VI6NeuAcnLc6 was the acceptor. This is most probably due to different conformations at the terminus of the two acceptor gangliosides. The fucosyltransferase was incapable of transferring fucose to sialyl 2----3 lactotetraosylceramide (IV3NeuAcLc4).     

Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay

The study of the expression of Gal alpha 1----3Gal beta 1----4GlcNAc residues on mammalian glycoconjugates is of particular interest since as many as 1% of circulating IgG antibodies in man (the natural anti-Gal antibody) interact specifically with this carbohydrate residue. In recent studies, we have found that Gal alpha 1----3Gal beta 1----4GlcNAc residues are abundant on red cells and nucleated cells of nonprimate mammals, prosimians, and New World monkeys, but their expression is diminished in Old World monkeys, apes, and humans. In the present work, we have analyzed the expression of these residues on secreted mammalian glycoproteins. For this purpose, we have developed a radioimmunoassay (RIA) which enables the quantification of Gal alpha 1----3Gal beta 1----4GlcNAc residues on the secreted glycoproteins. Purified biotinylated anti-Gal was used as the antibody in the RIA, and bovine thyroglobulin enriched for Gal alpha 1----3Gal beta 1----4GlcNAc residues served as a solid-phase antigen. In this study, it is reported for the first time that the evolutionary pattern of Gal alpha 1----3Gal beta 1----4GlcNAc residue distribution in in vivo secreted glycoproteins is similar to that observed in membranes of cell lines and of red cells. Thyroglobulin, fibrinogen, or IgG molecules from nonprimate mammals and from New World monkeys express varying amounts of Gal alpha 1----3Gal beta 1----4GlcNAc residues ranging between 0.01 and 11 residues per molecule, whereas no such residues are present on any of these glycoproteins of human or Old World monkey origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of type 1 and 2 lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed beta 1----3N-acetylglucosaminyltransferase

Human colonic adenocarcinoma tissue and derived cell lines have been characterized by an abundance of different type 1 and 2 lacto series glycolipid antigens which are either low or not found in normal colonic mucosa. The enzymatic basis for the expression of contrasting glycolipid compositions between adenocarcinomas and normal colonic mucosa, as well as between derived cell lines, has been studied. The following results were of particular interest. (i) Abundant activities of beta 1----4galactosyltransferase associated with synthesis of both lactosylceramide and lactoneotetraosylceramide, beta 1----3galactosyltransferase for synthesis of lactotetraosylceramide, and an alpha 1----3/4fucosyltransferase responsible for synthesis of Lex and Lea antigens were found in normal colonic mucosa or in a normal mucosal epithelial cell line HCMC, or in both. Variable levels of these activities were found in adenocarcinoma tissues and in various established adenocarcinoma cell lines. In striking contrast, significant activity of a beta 1----3N-acetylglucosaminyltransferase responsible for synthesis of lactotriaosylceramide (Lc3) was found in various cases of colonic adenocarcinoma and cell lines, but was undetectable in normal colonic epithelial cells. (ii) In situ transfer of galactose to Lc3 was performed on histologic sections by preincubation of the tissue with acceptor glycolipid followed by incubation with UDP-galactose. The biosynthesized glycolipid was revealed by indirect immunofluorescence with the monoclonal antibody 1B2 which defines lactoneotetraosylceramide antigen. In these studies, histologic sections prepared from frozen normal proximal colon tissue were shown to lack native type 2 chain structures. However, transfer of galactose from UDP-galactose could be demonstrated in the epithelial cells of normal proximal colon after incorporation of Lc3 into the membranes, indicating the ability of normal colonic epithelial cells to synthesize type 2 chain core structures if the precursor Lc3 is available. In contrast, adenocarcinoma tissues showed significant native immunofluorescence with the antibody. These data suggest that an accumulation of both type 1 and 2 chain lacto series glycolipids with alpha 1----3- or alpha 1----4fucosyl substitution in human adenocarcinoma is due to enhanced beta 1----3N-acetylglucosaminyltransferase rather than enhancement of other enzymes. This enzyme may play a key role in regulating the level of various types of lacto series tumor-associated antigens with the lacto type 1 or 2 chain.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Assignment of the gene for human arylsulfatase B, ARSB, to chromosome region 5p11----5qter

The structural gene coding for human arylsulfatase B, ARSB, is assigned to 5p11----5qter by analysis of somatic cell hybrids isolated from two separate fusions of human fibroblasts carrying a translocation involving chromosome 5 with the Chinese hamster cell line a3.     

Purification and properties of N-acetylglucosaminide alpha 1----3-fucosyltransferase from embryonal carcinoma cells

A membrane-bound alpha-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on GDP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65 000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to alpha-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of beta-galactoside alpha 1----2-fucosyltransferase and N-acetylglucosaminide alpha 1----4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above.     

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.     

The use of cell surface antigens to characterize and select for fragments of human chromosomes retained by interspecies hybrids

We have used a mouse cell transformant generated by human chromosome-mediated gene transfer (CMGT) to explore the use of cell surface antigens in the identification of fragments of human chromosomes retained by somatic cell hybrids. The transformed line, 21-30b, contained an intact rear-ranged human chromosome, and could be shown by isozyme analysis to contain genetic material from chromosomes 9 and X. By using the transformant as an immunogen in mice, it was also possible to produce antiserum to human-specific surface antigens. Using genetically characterized human X rodent hybrid lines, the genes controlling expression of these antigens could be localized to 11per----11p13, segregating concordantly with surface antigen S3. These conclusions were possible despite the fact that the presence of chromosome 11 in the transformant was not detectable by the presence of chromosome specific isozyme LDH-A or surface antigens W6/34 and 4F2. Finally, the fluorescence-activated cell sorter (FACS) was used to fractionate the transformant cells into antigen positive and negative subpopulations. This resulted in the isolation and characterization of four additional chromosome rearrangements involving interspecies chromosome translocations. This work demonstrates the value of chromosome-specific surface antigens and the FACS in the evaluation of human chromosome fragments retained by interspecies hybrids.     

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

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

Novel fucolipids of human adenocarcinoma: characterization of the major Ley antigen of human adenocarcinoma as trifucosylnonaosyl Ley glycolipid (III3FucV3FucVI2FucnLc6)

A series of glycolipid antigens with Ley determinant (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc) defined by monoclonal antibody AH6 (Abe, K., McKibbin, J. M., and Hakomori, S. (1983). J. Biol. Chem. 258, 11793-11797) have been detected in human colonic carcinoma cases. Three Ley-active components have been identified as follows. The simplest compound was characterized as Ley hexaosylceramide (lactodifucohexaosylceramide, III3FucIV2FucnLc4), which was previously isolated and was found as the major component in six out of eight cases of colonic adenocarcinoma but as only a very minor component in two cases. The second component was a very minor component in all eight cases, and its structure was identified by 1H NMR spectroscopy as an extended Ley (lactodifucooctaosylceramide, V3FucVI2FucnLc6; Structure 1 below). The third, major component, common in all eight cases, has been identified as trifucosyl Ley (lactotrifucononaosylceramide, III3FucV3FucVI2FucnLc6, Structure 2 below) based on 1H NMR spectroscopy, methylation analysis, and direct-probe electron-impact mass spectrometry. (formula; see text)