Membrane glycoprotein transferases: Time courses of activity changes in a synchronized cell population and enzyme activity half-lives in an asynchronous population

The time course of activity changes of five membrane glycoprotein transferases: the glycoprotein:galactosyl, the fetuin:fucosyl, the PSM:fucosyl, the fetuin:N-acetylglucosaminyl, and the polypeptide: N-acetylgalactosaminyl was determined using defined acceptors and conditions in synchronized L5178Y cells. In addition, time course of activity changes of these transferases was determined for the endogenous acceptors present in the cell extract. Each of the membrane glycoprotein transferases was an S peak enzyme in the L5178Y cell. Activity with endogenous acceptors, in general, was constant throughout the cell cycle indicating that acceptor availability may, in part, control glycoprotein synthesis in vivo.     

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 mm; N-glycolylneuraminic acid, 0.90 mm; d-glucosamine, 4.9 mm; d-galactosamine, 7.6 mm; N-acetyl-d-glucosamine, 23 mm; and N-acetyl-d-galactosamine, 24 mm. d-Galactose, d-glucose, d-mannose, α-methyl-d-glucoside, α-methyl-d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.     

D-aspartate oxidase in mammalian brain and choroid plexus

Abstract— Synaptosomes from guinea-pig cerebral cortex contain a fetuin: sialyl glyco-protein: glycosyl transferase; evidence is presented which indicates that both a sialyl transferase; evidence is presented which indicates that both a sialyl transferase and endogenous acceptors were located in the synaptosome ‘ghost’ fractions. Following solubilization of synaptosomes with Triton X-100 and the use of fetuin minus NANA as acceptor, 25 per cent of the transferase was recovered after centrifugation and column chromatography on Sephadex G-100 and G-200 with a 64·0-fold purification. The enzyme had a pH optimum of 6·3, required no divalent metal cation for activity, and exhibited high activity with either fetuin minus sialic acid, prothrombin minus sialic acid, Tamm-Horsfall glycoprotein minus sialic acid, or orosomucoid minus sialic acid as acceptor; neither BSM nor PSM minus NANA functioned as an effective acceptor. The fetuin:sialyl transferase using fetuin minus sialic acid and CMP-sialic acid as substrates a and b, respectively, gave the following kinetic constants when using the Cleland bisubstrate model: K_a= 35μM; K_b= 3 μM; K_ia, = 25 μM; K_ib= 25μM; and V₁= 92 pmoles. min^?1.mg^?1 of protein. The following divalent cations inhibited the reaction: Ba²⁺ > Hg²⁺ > Pb²⁺ > Cu²⁺.     

GLYCOSYL TRANSFERASE ACTIVITY IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN

—The conditions required for the optimal activity of several glycosyl transferases were determined in normal and scrapie-affected mouse brain. Particulate preparations were analysed for fucosyl, galactosyl, mannosyl and N-acetyl glucosaminyl transferase activity using endogenous acceptors. Solubilized preparations were analysed for fucosyl, galactosyl and sialyl transferase activity using defined exogenous acceptors. The activity of each of these enzymes was followed at intervals throughout the development of scrapie in the mouse. In the endogenous system no change was found in the fucosyl and mannosyl transferase activities of the scrapie material, but the levels of galactosyl and N-acetyl glucosaminyl transferase began to rise as clinical signs of scrapie developed i.e. at 15–16 weeks post-inoculation. In the exogenous system the levels of galactosyl and fucosyl transferase began to rise in the scrapie material at 11–12 weeks post-inoculation, rising to twice the normal value at 18–20 weeks. Sialyl transferase showed no change in activity.     

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

A lectin from the Asian horseshoe crabTachypleus tridentatus: purification,specificity and interaction with tumour cells

A lectin from the haemolymph of the Asian horseshoe crabTachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-boundN-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues,N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin theN-acetyl group and the glyceryl side chain ofN-acetylneuraminic acid are important, while presence of an aglycon, specially an -glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin andCollocalia mucin, all beingO-chain glycoproteins but carrying completely different carbohydrate chains. The majority ofN-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the Tachypleus tridentatus agglutinin-receptor which is present on red cells andO-chain glycoproteins.Abbreviations TTA Tachypleus tridentatus agglutinin - SDS sodium dodecyl sulfate - BSM bovine sub-maxillary mucin - VCS Vibrio cholerae sialidase - OSM ovine submaxillary mucin - WGA Wheat germ agglutinin - NeuAc N-acetylneuraminic acid.     

UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.GalNAc transferaseHuman intestinal cancerous tissueBovine submaxillary gland mucin O-Glycosidically linked sugar chain     

Platelet adhesiveness and aggregation: the collagen:glycosyl, polypeptide:N-acetylgalactosaminyl and glycoprotein:galactosyl transferases of human platelets

Four glycoprotein:glycosyl transferases were identified, purified, and characterized from human platelets. The enzymes present were collagen:glc, collagen:gal, polypeptide:galNAc, and glycoprotein:gal; the fetuin:glcNAc transferase was absent. Each of the 4 transferases was found to be almost exclusively bound to the platelet plasma membrane. Incubation of platelet homogenates without exogenous acceptors yielded no transfer of monosaccharide, indicating the complete lack of endogenous acceptors. These results lead to the interesting speculation that the transferases may not be responsible in the mature platelet for glycoprotein synthesis at all, but rather that they may function for intercellular adhesion and the primary step in hemostasis, the adhesion of collagen to platelets.     

SUBCELLULAR AND SUBMICROSOMAL DISTRIBUTION OF GLYCOLIPID-SYNTHESIZING TRANSFERASES IN YOUNG RAT BRAIN

The subcellular and submicrosomal distributions of four glycolipid-synthesizing transferases were studied in young rat brains. (1) Two galactosyl transferases involved in the synthesis of cerebrosides, the cerebroside sulphotransferase which catalyses the synthesis of sulphatides, and the glucosyl transferase which plays an important role in the ganglioside biosynthesis were localized essentially in the microsomal fraction. Only low activities were detected in the crude mitochondrial and synaptosome-enriched fractions. (2) A comparison of the activities of these enzymes in the crude myelin and two myelin subfractions showed that the galactosyl transferases and the cerebroside sulphotransferase had similar activities in the crude myelin and myelin-like fractions. A considerable galactosyl transferase activity was found in purified myelin. In this respect these two enzymes were different from cerebroside sulphotransferase, whose activity was much lower in purified myelin. On the other hand, glucosyl transferase had a relatively low specific activity in all three myelin fractions. Analysis of different markers showed that the activities were considerably higher than those expected from the maximum microsomal contamination calculated. (3) Subfractionation of the microsomes demonstrated that the galactosyl transferases were more concentrated in the lower parts of the gradient, containing vesicles with attached ribosomes. Cerebroside sulphotransferase and glucosyl transferase were found predominantly in the upper and intermediate parts of the gradient, which were composed essentially of smooth-surfaced vesicles and membrane fragments. Chemical analysis of submicrosomal fractions confirmed the morphological observations.     

Glycoprotein biosynthesis: Folic acid effects on glycoprotein:glycosyl transferase activities of rat kidney and liver

Folic acid at 14 μM to 1.4 mM increased the activity of the collagen:glc and fetuin:gal and decreased the activity of the fetuin:NANA glycoprotein:glycosyl transferases of rat liver and kidney in vitro; highest effects were found with 1.4 mM folic acid. 1.4 mM folic acid increased kidney fetuin:gal activity 5-fold and decreased fetuin:NANA activity 3-fold. At 1.4 mM, folinic acid and p-methylaminobenzoic acid were totally inactive toward the transferases, methasquin was moderately active, and homofolic, tetrahydrohomofolic and methotrexate were very active toward the transferases. In all instances, however, the fetuin:gal and collagen:glc transferases were activated while the fetuin: NANA transferase was inhibited. From the data presented, folic acid is viewed as a possible control molecule in the synthesis of glycoprotein.     

Glycosyltransferases with endogenous acceptor activity in plasma membranes isolated from rat liver

Plasma membrane fractions from rat liver exhibited glycosyltransferase activity with endogenous membrane-associated acceptors and either UDP-galactose, UDPglucose, UDP-N-acetylglucosamine, or GDPmannose donors. Of these, incorporation into non-lipid acceptors was most active with UDP-galactose and only with UDPgalactose and UDPmannose was there incorporation into endogenous lipid acceptors. CMP-N-acetylneuraminic acid was inactive as a donor with the isolated plasma membranes. In order to demonstrate transferase activity, low concentrations of substrate sugar nucleotides and short incubation times were used as well as sulfhydryl protectants and a phosphatase inhibitor (NaF) in the reaction mixtures. The findings support the concept of surface localization of at least a galactosyl transferase in cells of rat liver.     

A brain sialyltransferase having a narrow specificity for O-glycosyl-linked oligosaccharide chains

The existence of a brain sialyltransferase catalyzing the specific transfer of NeuAc on native fetuin was demonstrated. This enzyme was not able to sialylate either asialofetuin or desialylated and nondesialylated orosomucoid, transferrin, and bovine submaxillary mucin. It required the presence of Mn2+ for optimal activity. Moreover, in fetuin, this activity was closely related to the proportion of NeuAc residues, but in liver tissue sialylation occurred only onto asialofetuin. In native fetuin, sialylation took place on O-glycan chains to give an O-disialyltetrasaccharidic structure. The Gal----GalNAc----protein was not an acceptor, but alpha-NeuAc-(2----3)-Gal----GalNAc----protein was, suggesting a specific transfer alpha-(2----6) to the GalNAc residue.     

Trypsin Inhibitory Activity of Bovine Fetuin De-O-glycosylated by Endo-α-N-acetylgalactosaminidase

The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-α-N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl β1,3 N-acetylgalactosamine (Galβ1→3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.     

Isolation and properties of a sialidase fromTrypanosoma rangeli

In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-(2–3)-lactose> N-glycoloylneuraminy-(2–3)-lactose> N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg²⁺ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca²⁺ Mn²⁺ or Mg²⁺ ions.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - CMP cytidine monophosphate - EDIA ethylenediaminetetraacetic acid - ESM equine submandibular gland mucin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - Lac lactose - MU-Neu5Ac 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4Ac5Gc N-glycoloyl-4-O-acetylneuraminic acid - Neu2en 2-deoxy-2,3-didehydroneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - PMSF phenylmethylsulfonyl fluoride - PSM pig submandibular gland mucin - SDS sodium dodecyl sulfate - Tris tris-(hydroxymethyl)aminomethane Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday.     

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species.

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.     

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×10⁵ Da and aggregates in excess of 10⁶ Da, while the minor mucin ranged from 3.0 × 10⁵ to 10⁶ Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.Abbreviations PBS 0.01m sodium phosphate buffer, pH 7.3, containing 0.15m NaCl - Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - GalNAc-ol N-acetylgalactosaminitol     

Some biochemical properties of human breast tumor sialyltransferase

Sialyltransferase activity in normal human breast tissue and tumors was investigated with lactose, desialylated fetuin, and bovine submaxillary mucin as the acceptors. While microsomal preparations from the normal tissue showed little or no sialyltransferase activity toward these acceptors, tumors showed elevated enzymic activities. Tween-20 at 0.5% concentrations stimulated sialic acid transfer to all three acceptors. Another nonionic detergent, Triton X-100, stimulated asialo fetuin sialyltransferase activity while inhibiting activity toward asialo BSM and lactose. Interestingly, lysolecithin, a normal cellular constituent which possesses detergent properties also had an effect similar to that of Triton X-100. Thermal denaturation curves of enzymic activity toward asialo BSM, however, resembled those seen with asialo fetuin as the acceptor. Kinetic studies showed that at acceptor concentrations of 500 micrograms each, sialyl transfers to asialo fetuin, asialo BSM, and lactose showed apparent Km values of 50, 60, and 300 microM, respectively. At CMP-sialic acid concentrations of 300 microM, the Km values for the above acceptors were 25, 15, and 5000 microM.     

Studies on glycosyltransferases in fusion-defective conA-resistant L6 rat myoblast cell lines

1. Sialyl- and galactosyl-transferase activities were determined in wild type and conA-resistant L6 rat myoblasts with substrates derived from fetuin, alpha 1-acid glycoprotein and bovine submaxillary mucin; fetuin was the best acceptor for both enzyme activities, whereas the mucin did not act as an acceptor. 2. The optimum pH for sialyltransferase was 6.6 in both cell lines. 3. The optimum pH for galactosyltransferase in the wild type cell line was 6.2 which was slightly higher than the value of 5.8 found for the conA-resistant cells. 4. Values for Km for both enzyme activities increased five to ten-fold in the variant cell line with both acceptors. 5. The main sialyltransferase activity was the Gal beta 1----4GlcNAc alpha 2----3sialyltransferase for N-linked chains. The galactosyltransferase was most likely the enzyme that is responsible for the synthesis of the Gal beta 1----4GlcNAc structure.