Characterization of Sialyltransferase-IV Activity and Its Involvement in the c-Pathway of Brain Ganglioside Metabolism

Abstract: To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg²⁺ or Mn²⁺ and Triton CF-54 for its full activity. The V_max value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent K_m values for CMP-sialic acid and GM1 were 172 and 78 µM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b-synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [³H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GD1a-synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1b, whereas the endogenous GT1b-synthase activity was suppressed by GA1 or GM1. GA1, GM1, and GD1b did not affect the endogenous GM3- and GD3-synthase activities. These results clearly demonstrate that sialyltransferase-IV in brain tissues catalyzes the reaction for GQ1c synthesis in the c-pathway as well as the corresponding steps in the asialo-, a-, and b-pathway in ganglioside biosynthesis.     

Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.     

Activities of Five Different Sialyltransferases in Fish and Rat Brains

Abstract: To investigate the role of Sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different Sialyltransferases (GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase) using total membrane preparations from cichlid fish and Sprague-Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglloside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3-synthase activity was lower than GD3-synthase activity, whereas the opposite relationship was observed in rat brain. The GT3-synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3-synthase activity was detected in rat brain. The GD1a-and GT1a-synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a-synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c-series gangliosides including GT3 and polysialo-species, whereas a-and b-se-ries gangliosides were major components in rat brain. These results suggest that the species-specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous GD1a

Previous reports indicated the presence of both gangliosides and sialidase in the nuclear envelope (NE) of primary neurons and the NG108-15 neural cell line. GM1, one of the major gangliosides of this membrane, was shown to be tightly associated with a sodium-calcium exchanger in the inner membrane of the NE and to potentiate exchanger activity. GD1a was the other major ganglioside detected in the NE and, like GM1, occurs in both inner and outer membranes. A subsequent report indicated the presence of sialidase activity in the NE without specification as to which of the two membranes express it. The present study was undertaken to determine the nature and locus of this activity within the NE of two cell lines: NG108-15 and SH-SY5Y. Western blot analysis of the separated membranes revealed occurrence of Neu3 in the inner membrane and Neu1 in the outer membrane of the NE. Moreover, sialidase activity at both sites was shown capable of catalyzing conversion of endogenous GD1a to GM1.     

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.     

The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane-bound sialidase

Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidase. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5-7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes.     

Peroxidase-amplified assay of sialidase activity toward gangliosides.

Sialidase assays were carried out with the substrate, ganglioside GD1a, coated onto enzyme immunoassay plate wells. Following the incubation of GD1a with sialidase from V. cholerae, the amount of ganglioside GM1 produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to GM1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of GM1 was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.     

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli

Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Secretary sialidase activity and GM3 ganglioside

The sialidase activities with GM3 ganglioside and sialyllactitol were demonstrated in the conditioned medium of human fibroblasts. pH versus activity profiles of conditioned medium with GM3 as substrate suggested the presence of two sialidases with optimal activities at pH 4.5 and pH 6.5. The GM3 sialidase activity at pH 6.5 was suppressed in the medium of contact-inhibited cells. This sialidase may function in the metabolism of cell surface GM3 since there was a selective loss of labeled sialic acid from GM3 at different times of incubation after pulse-labeling with a radioactive sialic acid precursor ([3H]N-acetyl-mannosamine) and a radioactive ceramide precursor ([14C]serine). In addition, a sialidase inhibitor, 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (NeuAc-2-en) resulted in a reversible growth inhibitory effect and the suppression of the sialidase activity in the medium. We have speculated that GM3 hydrolysis on the cell surface by the sialidase may be coordinated with the cell cycle and may be at its maximum during early in the G1 phase.     

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.     

Desialylation of extracellular GD1a-neoganglioprotein suggests cell surface orientation of the plasma membrane-bound ganglioside sialidase activity in human neuroblastoma cells

The orientation of the catalytic site of a ganglioside-specific sialidase in the plasma membrane of SK-N-MC neuroblastoma cells was probed using water-soluble GD1a-neoganglioprotein substrate on intact cells and GM1-product detection by cholera toxin B. Desialylation of substrate was readily observed, whereas specific sialidase inhibitors prevented the reaction, and conditioned medium was inactive. Inhibitors of endocytosis and acidification had no effect on substrate degradation, and lowering temperature to 18 degrees C reduced activity but did not abolish it. We conclude that the ganglioside sialidase activity is cell surface-orientated and displays an in situ specificity that mirrors enzyme preparations in vitro.     

Compartmental Organization of the Synthesis of GM3, GD3, and GM2 in Golgi Membranes from Neural Retina Cells

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [³H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[³H]Gal. Conversion of the synthesized [³H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [³H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.     

Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules

Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.     

Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver

Synthesis of ganglioside GD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer----GA3----GD3----GT3.