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alpha-2,8-Sialyltransferase has been purified from human neuroblastoma CHP-134 cells greater than 2900-fold. The key step in the purification was a substrate affinity column utilizing immobilized colominic acid. Several kinetic parameters of the enzyme were defined. Fetuin but not asialofetuin served as substrate. The product of the enzyme reaction was characterized as containing sialyl residues in alpha-2,8-linkage with the use of recombinant sialidases. It is suggested that the purified enzyme is an initiating enzyme for the biosynthesis of polysialic acid since these cells also have the activity of poly alpha-2,8-sialyltransferase and contain polysialic acid. This alpha-2,8-sialyltransferase may be a new member of a family of alpha-2,8-sialyltransferases recently described, since it differs in substrate specificity reported for the cloned and expressed enzymes.

A glycoprotein, M(r) 200,000, which has the biological activity of the neurotoxin-responsive Na+ channel, was isolated from a clonal line of mouse neuroblastoma cells, N-18. The glycoprotein was purified to homogeneity in 18% yield by methods used to purify glycoproteins, which included metabolic labeling of the cells with L-[3H]fucose and binding of the radioactive glycoproteins to WGA- and lentil-Sepharose, and DEAE-cellulose. The glycoprotein has biological activity of neurotoxin-responsive ion flux when reconstituted into artificial phospholipid vesicles. This activity was shown to depend on the presence of sialic acid since treatment of the purified, reconstituted glycoprotein with Vibrio cholerae neuraminidase abolished the response to neurotoxins of 86Rb flux. The [3H]fucose-containing glycopeptides derived by Pronase digestion of the glycoprotein were characterized by affinity to immobilized lectins and contained di-, tri-, and tetra-antennary oligosaccharides in a ratio of 2:4:3. Most of the glycopeptides were sialylated as shown by binding characteristics to immobilized serotonin-Sepharose with and without neuraminidase. The structure of the diantennary oligosaccharides was elucidated by 500-MHz 1H NMR spectroscopy. The Con A-bound fraction contains alpha-NeuNAc-(2-->6)-bound group on the GlcNAc5' antenna and an alpha-NeuNAc-(2-->3)-bound groups on the GlcNAc5 antenna. An alpha-L-fucosyl group is (1-->6)-bound to the Asn core GlcNAc1 residue.

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.

Fucosyl residues linked alpha 1 leads to 3 or 4 to N-acetylglucosamine were found in large amounts on glycopeptides from the membranes of human tumor cells of neurectodermal origin but not on membrane glycopeptides from human fibroblasts. The fucosyl residues were detected by release of radioactive fucose from the glycopeptides with an almond alpha-L-fucosidase specific for fucosyl alpha 1 leads to 3(4)-N-acetylglucosamine. In other studies, the linkage was shown to be alpha 1 leads to 3 by nuclear magnetic resonance analysis (U. V. Santer, M. C. Glick, H. van Halbeek, and J. F. G. Vliegenthart. Carbohydr. Res., 118: in press, 1983). Glycopeptides containing these fucosyl residues from four human neuroblastoma cell lines were defined by binding to immobilized lectins. In addition, the glycopeptides from one human neuroblastoma cell line, CHP-134, were further characterized by enzyme degradation and columns calibrated for size and charge. The antennary position of fucosyl alpha 1 leads to 3-N-acetylglucosamine on the glycopeptides was demonstrated by the use of exoglycosidases and endoglycosidase D, since complete degradation to yield fucosyl-N-acetylglucosaminylasparagine was obtained only after treatment with almond alpha-L-fucosidase prior to the sequential degradation. Fucosyl alpha 1 leads to 3-N-acetylglucosamine was present on most size and charge classes of membrane glycopeptides and therefore was not limited to a few glycoproteins. Since the almond alpha-L-fucosidase cleaves fucosyl residues from glycoproteins, the physiological effects of the increased specific fucosylation on human tumors of neurectodermal origin can be examined.

Molecular characterization of two human neuroblastoma cell lines has revealed that both contain multiple homogeneously staining regions (HSRs), each representing a chromosome site of N-myc amplification. The newly established cell line CHP-382/JK had two cytogenetically distinct populations with several identical chromosomal abnormalities, indicating a common progenitor cell. Each population had one HSR, one on chromosome 5 at q31-34 and the other on chromosome 2 at q31-32. Chromosomal in situ hybridization with the N-myc probe pNb-1 demonstrated that both HSRs contained amplified copies of N-myc. Southern blot analysis confirmed amplification of N-myc sequences in genomic DNA of CHP-382/JK. Chromosomal features of CHP-382/JK shared with other neuroblastoma cell lines were the deletion of 1p and the presence of extra 17q material. In addition, the cells were highly reactive to monoclonal antibody PI 153/3 used to identify human neuroblastoma. CHP-382/JK cells were further characterized as neuronal cells by the expression of neurotoxin-responsive Na+ channels. Another neuroblastoma cell line, CHP-134, contained a single cell population with three HSRs, one in the short arm of each chromosome 7 and one in the long arm of a chromosome 6. All three HSRs contained amplified copies of N-myc as shown by in situ hybridization with the N-myc probe pNb-1. One of the 7p HSRs was acquired during culture of CHP-134 cells, whereas the 2q HSR of CHP-382/JK was lost. Such findings highlight the continued process of N-myc amplification and transposition in vitro. To our knowledge, amplification of N-myc in multiple HSRs has not been documented previously in neuroblastoma cell lines.

Neurites were prepared by a novel method from differentiating mouse neuroblastoma cells. When electrically differentiated cells were labeled metabolically with L-[3H]fucose or D-[3H]glucosamine, both the neurites and the surface membranes showed the presence of a glycoprotein of apparent Mr = 200,000. In contrast, the level of this glycoprotein was reduced in the surface membranes from nondifferentiated cells and a radioactive glycoprotein of similar molecular weight was found in the growth medium. The method for the isolation of neurites is of potential usage in distinguishing specific proteins associated with growing neurites.

The activity of neurotoxin-responsive Na+ channels in mouse neuroblastoma cells, N-18, was examined after treating the cells with compounds that are reported to perturb intracellular traffic. The compounds used have been shown to either alter glycoprotein synthesis and processing, (swainsonine, castanospermine, monensin, and retinoic acid) or receptor mediated endocytosis (mevinolin, 7-ketocholesterol, and chloroquine), or both. All of these compounds inhibited the activity of the neurotoxin-responsive Na+ channel with the exception of retinoic acid which increased the activity. Na+ channel activity was measured by two methods: (a) In vivo, the efflux of 86Rb was measured by use of the cells in monolayer culture, and (b) in vitro, the flux of 86Rb was measured from artificial phospholipid vesicles containing the partially purified Na+ channel. In both cases, 86Rb flux responded to stimulating neurotoxins, veratridine and scorpion venom, and was inhibited by tetrodotoxin as characteristic of excitable membranes. One of the perturbing compounds, swainsonine, was examined in detail. Treatment of N-18 cells with 10 microM swainsonine for 24 h markedly reduced the activity of the neurotoxin-responsive Na+ channel, as shown by the neurotoxin-stimulated efflux of 86Rb in vivo. In addition, after reconstitution into phospholipid vesicles of the partially purified Na+ channel from swainsonine-treated cells, reduced 86Rb flux was observed when compared with that of nontreated cells. Furthermore, the activity was not recovered in other less purified fractions. A comparison of the glycopeptides from the treated and nontreated cells by size, charge, and lectin-binding affinities was consistent with the formation of hybrid oligosaccharides after swainsonine treatment. It is concluded that the oligosaccharide residues of the Na+ channel glycoprotein must be processed to the mature complex-type for full activity. The stimulation of channel activity by treatment with retinoic acid supported this conclusion.

A membrane glycoprotein, Mr = 20,000, has been purified from human neuroblastoma cells (IMR-5) with the use of monoclonal antibody selected for binding capacity to human neuroblastoma cell lines. The antigen was extracted with 0.5% Nonidet P-40 from cells metabolically labeled with L-[3H]fucose or D-[3H]glucosamine. A double antibody affinity column was used to purify the membrane glycoprotein. Goat anti-mouse IgM was coupled to cyanogen bromide-activated Sepharose 4B. The absorption of the monoclonal antibody contained in ascites fluid completed the affinity column. Appropriate controls of similar material from other cell types and another monoclonal antibody demonstrated the specificity of the affinity column. Glycopeptides from the surface of human neuroblastoma cells, IMR-5 and CHP-134, had antigenic activity, as radioactive pronase-digested material bound to the affinity column and inhibited complement-mediated cytolysis. Glycolipids extracted from the cells had no antigenic activity. It was concluded that the carbohydrate residues of the glycoprotein conferred the antigenic specificity. Three methods were devised to aid in detection and purification of the antigen. These were: 1) an assay for the detection of complement-mediated cytolysis by measuring the enzyme creatine phosphokinase in the nonlysed target cells; 2) precipitation of the antigen . antibody complex with 4% polyethylene glycol; and 3) removal of the antibody by a wheat germ agglutinin-agarose column.

L-Fucose and D-galactose in low concentrations (0.27 or 2.7 mM) inhibited the induction of active Na+ channels in mouse and human neuroblastoma cells when the monosaccharides were added to the culture medium for 4 days with the inducing agent dimethyl sulfoxide. Active Na+ ionophores were determined by measurement of the toxin-stimulated efflux of 86Rb from the cells. At the same time, the amount of a radioactive glycoprotein (Mr 200,000), which was shown previously to be associated with neurite and membrane preparations from cells with active Na+ channels, was decreased. Cell growth and viability were not affected. The nonphysiological isomer D-fucose or the addition of D-glucose in the same concentration did not inhibit differentiation. Vibrio cholerae neuraminidase, added to the cells prior to the stimulation of 86Rb efflux by veratridine and scorpion venom, was inhibitory. The implications of these findings, which suggest a key role for glycoproteins in at least a portion of the excitability process, are discussed.

Human tumor cells of neurectoderm origin contain a high proportion of alpha-L-fucosyl linkages were determined by high-resolution, 500-MHz, 1H-n.m.r. spectroscopy which gave signals characteristic for alpha-L-Fucp-(1 leads to 3)-D-GlcNAc residues these L-fucosyl residues. This was shown by use of a specific alpha-L-fucosidase from almond emulsin and a broad-spectrum alpha-L-fucosidase from rat testes. The exact alpha-L-fucosyl linkages were determined by high-resolution, 500-MHz, 1H-n.m.r. spectroscopy which gave signals characteristic for alpha-L-Fucp-(1 leads to 3)-D-GlcNAc residues linked to branches and for alpha-L-Fucp-(1 leads to 6)-D-GlcNAc residues linked to the core. More than 95% of the asparagine-linked GlcNAc residues were substituted with (1 leads to 6)-alpha-L-fucosyl groups. Further definition of the range of neutral glycopeptides was obtained with immobilized lectins. Binding to E-PHA-agarose suggested the presence of a beta-D-mannopyranosyl residue substituted at O-4 by a 2-acetamido-2-deoxy-D-glucopyranosyl group. alpha-L-Fucp-(1 leads to 3)-GlcNAc interfered with this binding since removal of alpha-L-fucosyl groups by almond emulsin alpha-L-fucosidase increased the binding by 100%. These studies demonstrate the ability of a combination of high-resolution 1H-n.m.r., enzyme degradation, and lectin-binding affinities to delineate structural elements of small amounts of oligosaccharide residues.

Glycopeptides suggesting a complex oligosaccharide composition are present on the surface of cells from human neuroblastoma tumors and several cell lines derived from the tumors. The glycopeptides, labeled with radioactive L-fucose, were removed from the cell surface with trypsin, digested with Pronase, and examined by chromatography on Sephadex G-50. Human skin fibroblasts, brain cells, and a fibroblast line derived from neuroblastoma tumor tissue show less complex glycopeptides. Although some differences exist between the cell lines and the primary tumor cells, the similarities between these human tumors and animal tumors examined previously are striking.

Glycoproteins were purified from a clonal cell line of mouse neuroblastoma, N-18, labeled metabolically with L-[3H]fucose. The purified radioactive glycoproteins were reconstituted into artificial phosphatidylcholine vesicles. When the vesicles were preloaded with cesium acetate and treated with neurotoxins to activate the Na+ channel, a shift in intravesicular density was observed to a less dense position after centrifugation on sucrose gradients. This shift was partially inhibited by tetrodotoxin, which prevents the activation of the Na+ channel. A similarly derived fraction of [14C]fucose-containing glycoproteins from a neuroblastoma cell line that does not possess excitable membranes, N1A-103, was reconstituted into phospholipid vesicles, and, after preloading with cesium ions, the fraction was combined with those of the 3H-labeled glycoproteins of the differentiated cells, N-18, which have excitable membranes. Only the 3H-labeled glycoprotein-containing vesicles were responsive to the neurotoxins, as shown by a shift in intravesicular density on sucrose gradients. These results are interpreted as a demonstration of the reconstitution of glycoproteins to form the activated Na+ channel. Comparison of the radioactive glycoprotein profiles after polyacrylamide gel electrophoresis showed that glycoproteins of Mr 200,000, Mr 165,000, and Mr 65,000 were common to the reconstituted fractions that were biologically active.

It is suggested that a correlation exists between the proportions of specific groups of glycopeptides that are found in the surface membranes and the ability of neuroblastoma clones to differentiate morphologically. The evidence for this is examined and a comparison is made with other properties of the differentiated cells.

The presence of fucosyl residues linked alpha 1----3(4) to N-acetylglucosamine was demonstrated on the oligosaccharides from glycoproteins of 11 human neuroblastoma tumors from ten different patients. This finding is in complete agreement with the previous report that human neuroblastoma cell lines contained an unusually large proportion of metabolically incorporated L-[3H]fucose in this specific linkage (U. V. Santer and M. C. Glick, Cancer Res., 43:4159-4166, 1983). Furthermore, the glycopeptides derived from the neuroblastoma tumors had a low percentage of fucose-containing biantennary oligosaccharides as determined by affinity to concanavalin A-Sepharose and in this characteristic were similar to glycopeptides from virus transformed and other tumor cells. To obtain these results, the tumor cells were labeled metabolically for 48 h with L-[3H]fucose. The cells were harvested and digested with Pronase, and the glycopeptides were isolated and treated with alpha-L-fucosidase from almonds, specific for the release of fucose linked alpha 1----3(4) to N-acetylglucosamine. A portion of the glycopeptides was characterized by serial affinity chromatography on immobilized concanavalin A and lentil lectin. The phenotypic similarity of the tumor cells to the cell lines, particularly CHP-134, included the paucity of biantennary oligosaccharides and the presence of fucosyl residues on the multiantennae of the glycopeptides.

Three neuroblastoma systems were used to define fucose-containing glycopeptides on the cell surface and to relate them to the phenotypic expressions of neuronal functions. These systems were a) clonal lines of mouse neuroblastoma C-1300, b) cell hybrids of mouse neuroblastoma and rat glioma, and c) human neuroblastomas, primary cells from the tumor, and cell lines. The results suggest that similarities exist in the membrane glycopeptides available at the surface of the mouse and human cells. It is proposed that these similarities correspond to the ability of the cells to perform the differentiated functions of neuronal cells or to exist as tumors. Based on analogy with other cell membranes, a schema is given for the structure of the membrane glycopeptides on the neuroblastoma cell.

Sequential removal of surface glycopeptides was achieved by subjection of mouse neuroblastoma cells to a two-step trypsin treatment under different conditions. The glycopetides released by each trypsinization step were digested by Pronase and examined on columns of Sephadex G-50. Different chromatographic patterns were found for the two digests. Thus, several groups of glycopeptides can be distinguished by the trypsinization procedure. One group is readily removed and appears to be at a more accessible location on the cell surface. Among the four neuroblastoma clones examined, the glycopeptide patterns from axon-forming cells differed from those of axon-minus cells.