PubMed Health⌕ Search

Biomedical subjects

M C Glick

Publications and source records attributed to M C Glick.

At least 37 records · Page 2Linked to original sources
PubMed1991

Purification and characterization of GDP-L-fucose-N-acetyl beta-D-glucosaminide alpha 1----6fucosyltransferase from cultured human skin fibroblasts. Requirement of a specific biantennary oligosaccharide as substrate.

GDP-L-fucose-N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase which catalyzes the transfer of fucose from GDP-L-fucose to the asparagine-linked N-acetyl-beta-D-glucosamine of N-linked glycoproteins has been purified 37,000-fold from cultured human skin fibroblasts. The Km values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2 microM, respectively. The Vmax was 1.4 mumols/mg/min. The key step in enzyme purification was affinity chromatography using the immobilized substrate asialoagalactotransferrin glycopeptide-CH-Sepharose. The affinity-purified enzyme had a minimum substrate requirement for a biantennary oligosaccharide with GlcNAc in terminal position, having a Km value of 55 microM. It was heretofore unexpected that the oligosaccharide would serve as substrate, since the site of enzyme activity is GlcNAc-1-linked to Asn. Although the presence of amino acids on this oligosaccharide enhanced the activity 3-fold, it is proposed that this may be the result of an alpha/beta anomeric mixture (2:1) of oligosaccharide used in these studies with only the beta anomer active as substrate. The implication is that the amino acid is required only to retain the beta anomeric position of the substrate. Removal of GlcNAc or addition of Gal to either the oligosaccharide or glycopeptide destroyed the ability to serve as substrates. In addition, di-N-acetylchitobiose, tri-N-acetylchitotriose and GlcNAc beta 1----Asn were nonpermissible substrates. This rigid substrate requirement is unique among fucosyltransferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.

Carbohydrate Sequence↗
PubMed1991

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme.

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.

Chromatography, Affinity↗
PubMed1990

Additional fucosyl residues on membrane glycoproteins but not a secreted glycoprotein from cystic fibrosis fibroblasts.

Glycopeptides derived from peripheral membrane glycoproteins of skin fibroblasts of seven patients with cystic fibrosis (CF) had an increase in fucosyl residues when compared with those of seven age, race and sex matched controls (Pediatr Res 1985;19:368-374). To further define these results, the membrane glycopeptides which bound to immobilized lentil lectin and thereby enriched in fucosyl residues linked alpha 1----6 to N-acetylglucosamine attached to asparagine, were Pronase digested, partially purified and examined by 500-MHz 1H-NMR spectroscopy. The CF derived glycopeptides had two different features when compared to those from Controls (1) an increased number of fucosyl residues linked alpha 1----6 to the N-acetylglucosamine attached to asparagine and (2) fucosyl residues linked alpha 1----3 to a branch N-acetylglucosamine. The glycopeptides from both sources were of the di and triantennary type containing sialic acid linked alpha 2----3 and alpha 2----6 to galactose in an approximate molar ratio of 3:2 and 2:1, from CF and Control, respectively. Glycopeptides derived from a glycoprotein, fibronectin, secreted from CF fibroblasts were also examined by 1H-NMR spectroscopy and showed no evidence of fucosyl residues linked alpha 1----3 to branch N-acetylglucosamine and a lesser percentage of core fucose than found in the peripheral membrane glycopeptides. These results define further the altered fucosylation of the CF peripheral membrane glycoproteins.

Carbohydrate Sequence↗
PubMed1989

N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae.

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Membrane excitability expressed in human neuroblastoma cell hybrids.

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.

Action Potentials↗
PubMed1988

Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells.

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗
PubMed1988

A quantitative method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase activity with lectin affinity chromatography.

A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosylation of the asparagine-linked N-acetylglucosamine. The enzyme was extracted from human skin fibroblasts and incubated with GDP-[14C]fucose and a specific substrate, asialo-agalactotransferrin glycopeptide. The product of the enzyme reaction, [14C]fucose alpha 1----6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 M methyl alpha-D mannopyranoside. The method was shown to be specific after characterization of the lentil lectin-bound glycopeptides by enzyme degradation and affinity chromatography. Quantitation of the method was shown by several parameters, including the linearity of product formed with respect to time, GDP-[14C]fucose concentration and enzyme concentration.

Chromatography, Affinity↗
PubMed1988

Lack of proteolytic processing of alpha-L-fucosidase in human skin fibroblasts.

Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of alpha-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of alpha-L-fucosidase, Mr 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-beta-D-glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L-[3H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L-leucine. alpha-L-Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N-Acetyl-beta-D-glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra- and extracellular enzyme was observed, although of smaller size, Mr 52,000-53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that alpha-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.

Acetylglucosaminidase↗
PubMed1987

NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine.

Phenotypic expression of transformation was inhibited by swainsonine at concentrations which affect the late stages of glycoprotein processing but not growth of cells. In the presence of swainsonine, NIH 3T3 fibroblasts transfected with human tumor DNA (al-l) no longer grew in soft agar or expressed complex type oligosaccharides characteristic of transformed cells. Thus, it appears that glycoproteins with fully processed oligosaccharides are necessary for the maintenance of the transformed phenotype in these cells.

Alkaloids↗
PubMed1986

Characterization of human fibronectin glycopeptides from cystic fibrosis and control skin fibroblasts.

Fibronectins isolated from different species and tissue sources are glycosylated differently. We report here a characterization of the glycopeptides of fibronectin isolated from the culture medium of skin fibroblasts from patients with cystic fibrosis together with age-, race-, and sex-matched control subjects. The characterization of this fibronectin is of special interest because it is derived from: a non-fetal, cellular source; eight different individuals; and cystic fibrosis and control individuals. The fibronectin glycopeptides were purified by gel-permeation chromatography and Con A-Sepharose and were analyzed by anion-exchange chromatography and affinity columns of immobilized 5-hydroxytryptamine and lectins. One half of the glycopeptides of skin fibroblast fibronectin were shown to contain biantennary oligosaccharides which were core-fucosylated and partially sialylated. Although the remaining half was a complex mixture of glycopeptides, there was remarkably little inter-individual variation. No difference between cystic fibrosis and control subjects was discernible by the techniques employed here. Unlike the biantennary glycopeptides of human plasma fibronectin, those from skin fibroblast fibronectin were core-fucosylated and less highly sialylated. However, compared to human cellular fibronectin glycopeptides from fetal sources, those from skin fibroblast fibronectin were both more highly fucosylated and sialylated.

Adolescent↗
PubMed1986

Perturbation of glycoprotein processing affects the neurotoxin-responsive Na+ channel in neuroblastoma cells.

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.

Animals↗
PubMed1986

Similarities in glycosylation of human neuroblastoma tumors and cell lines.

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.

Acetylglucosamine↗