A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.     

Monomer-micelle transition of the ganglioside GM1 and the hydrolysis by Clostridium perfringens neuraminidase.

The action of Clostridium perfringens neuraminidase on the ganglioside Gm1 tritiated in the ceramide moiety was studied. The rates of hydrolysis of the Gm1 ganglioside were determined from radioactivity in the neutral glycolipid product, which was separated from the substrate on DEAE-Sephadex columns. In order to study the physical state of the substrate in the conditions used in the neuraminidase treatment, the critical micelle concentrations of the Gm1 ganglioside were determined using formation of the triiodide anion in aqueous iodine solution as an indicator. The critical micelle concentrations were also obtained by determining the non-sedimenting radioactivity at different concentrations of the labeled ganglioside per total volume used in ultracentrifugation experiments. In addition, the concentrations of the monomeric ganglioside were concluded from the results of the ultra-centrifugation studies. The increase in the reaction rate of the Gm1 hydrolysis as the function of the substrate concentration was leveled off at 25-28 microM ganglioside. The abrupt change at this concentration is interpreted as reflecting the monomer-micelle transition of the ganglioside in the conditions used (50mM sodium acetate buffer, pH 4.6). The critical micelle concentration was 29 microM on the basis of the triiodide test, and ultracentrifugation revealed the critical micelle concentration 28 microM. The reaction velocity of the hydrolysis was decreased immediately above the critical micelle concentration, and became constant at higher concentrations of the ganglioside. A close correlation to these changes in the reaction rate is suggested to exist in the concentrations of the monomeric Gm1 ganglioside. Saturation of the buffer used in the neuraminidase assays with butanol effected a striking change in the plot of reaction rate versus ganglioside concentration. The reaction rate increased up to 100-110 microM Gm1 ganglioside. The shift of the inflexion point in the rate plot from 25-28 microM to 100-110 microM ganglioside concentration is suggested to be due to a respective change in the critical micelle concentration effected by butanol. N-Acetylneuraminyllactosyl ceramide, lactosyl ceramide and asialo-Gm1 ganglioside had an inhibitory effect on the reaction. In contrast, N-acetylneuraminyllactose, lactose and some other free saccharides were not inhibitory. The results demonstrate that factors other than the saccharide structure must be taken into account when substrate specificity of a glycosidase is studied using competition experiments. It is suggested that the inhibition effected by the glycolipids is due to an increase in the micellar state of the Gm1 ganglioside.     

Immobilized Clostridium perfringens neuraminidase. Substrate cleavage and enzyme release during incubation.

Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures. The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme. The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay. The activity of the immobilized enzyme was dependent on the nature of the support and the substrate. Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties. Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates.     

Decreased ganglioside neuraminidase activity in fibroblasts from mucopolysaccharidosis patients. Inhibition of the activity in vitro by sulfated glycosaminoglycans and other compounds

The neuraminidase activities towards the ganglioside substrates GD1a, GD3 and GM3 were found to be markedly diminished in homogenates of fibroblasts cultured from patients with various genetic mucopolysaccharidoses. Mixing normal and patients' fibroblast homogenates revealed this effect to be due to the presence of diffusible inhibitors. The neuraminidase acting on the trisaccharide sialyllactose, on the other hand, showed normal activity in all the cell lines tested. Experiments in vitro revealed the sulfated glycosaminoglycans chondroitin 4-sulfate and heparin, the polysaccharide dextran sulfate, and the trypanocidal drug suramin to be strongly inhibitory on the ganglioside GD1a neuraminidase activity of normal fibroblast homogenates. Regarding chondroitin 4-sulfate, this inhibition was of the non-competitive type. A disulfated tetrasaccharide prepared from chondroitin 4-sulfate, on the other hand, was not at all inhibitory. These and additional findings led us to propose a model for the interaction between enzyme and inhibitor, involving a 'clamping' mechanism by the polysulfated compounds. We conclude that the decreased ganglioside neuraminidase activities of mucopolysaccharidosis fibroblasts are due to an inhibition by the accumulated sulfated glycosaminoglycans and that such inhibition is responsible for the storage of certain gangliosides in the tissues of the patients.     

The human GM2 activator protein. A substrate specific cofactor of beta-hexosaminidase A.

Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

Action of ortho- and paramyxovirus neuraminidase on gangliosides. Hydrolysis of ganglioside GM1 by Sendai virus neuraminidase

The action of neuraminidase of influenza A virus, Sendai virus and Newcastle disease virus particles on bovine brain ganglioside GM1 and the properties of Sendai virus neuraminidase for GM1 were studied. With Sendai virus, GM1 was hydrolyzed to asialo-GM1 (GA1) and N-acetylneuraminic acid even in the absence of surfactant or other additives, while the hydrolysis of GM1 by Newcastle disease virus or influenza A virus was very low or undetectable under the same conditions. The formation of GA1 by Sendai virus neuraminidase was confirmed by thin-layer chromatography and immunodiffusion test using anti-GA1 antiserum. The apparent Km of Sendai virus neuraminidase for GM1 hydrolysis was found to be 2.67 x 10(-4) M and the optimum pH was 5.6. GM3, GM2 and oligosaccharide of GM1 were hydrolyzed more effectively than GM1 in the absence of surfactant (GM3 greater than GM2 greater than oligosaccharide of GM1 greater than GM1). The hydrolysis of GM1 by the Sendai virus enzyme was stimulated by the addition of sodium cholate or sodium taurocholate, but was inhibited by divalent cations (10 mM), Ca2+, Mg2+, ZN2+, Fe2+ and CU2+. In the absence of the surfactant, Sendai virus neuraminidase hydrolyzed GM1 more efficiently than Arthobacter ureafaciens neuraminidase which has been reported recently as being an adequate enzyme to hydrolyze ganglioside GM1 as a substrate.     

A procedure for the preparation of GM3 ganglioside from GM1-lactone

A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.     

Role of Myelin-Associated Neuraminidase in the Ganglioside Metabolism of Rat Brain Myelin

The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.     

On the presence of a "particle-bound" neuraminidase in retina: a developmental study.

Neuraminidase activity was detected in chicken retina. A “particle-bound” neuraminidase was demonstrated in retina; however no “soluble” neuraminidase was present. For the endogenous “particle-bound” neuraminidase the pH optimum was 4.0 and the enzyme was stimulated by 0.15 % Triton X-100. Total activity measured in the presence of both endogenous and G_Dla (0.085 mM) substrates, reached a maximum at the same pH (4.0) in the presence of lower amounts of Triton X-100 (0.075–0.1 %). An excess of G_Dla inhibited the activity. Total and endogenous activity profiles of the “particle-bound” neuraminidase behaved similarly during chick retina ontogenesis; the activities were low during the early embryo period, reached maximum levels near hatching and remained stable throughout adulthood. In chicken retina, neuraminidase, which may take part in ganglioside catabolism, reached the maximum of activity when the retina was morphologically mature, while glycosyltransferases, implicated in the first steps of retinal ganglioside synthesis, are known to attain maximum levels of activity at an early stage of brain development.     

Quantification of gangliotetraose gangliosides with cholera toxin

A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level. The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase. Color is developed and quantified spectrophotometrically. Omission of neuraminidase gives a measure of GM1 alone. Linearity was obtained between 0.5 and 3 pmol. This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells. For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides. Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides.     

Characterization of two gangliosides from human leukemic polymorphonuclear leukocytes.

Eight bands of gangliosides, from human polymorphonuclear leukocytes were demonstrated by thin-layer chromatography. Bands 4 and 5 were isolated and purified in sufficient amounts to allow their biochemical identification by thin-layer chromatography, gas chromatography and sequential action of glycosidases and neuraminidase. The major ganglioside was characterised as N-acetylneuraminylgalactosyl-beta-N-acetylglucosaminyl-beta-galactosyl-beta-glucosylceramide. A second ganglioside was tentatively identified as N-acetylneuraminyl-galactosyl-beta-N-acetylglucosaminyl-beta-(N-acetylneuraminyl)galactosyl-beta-glucosylceramide. Both gangliosides isolated were hydrolysed by neuraminidase. However, treatment of the intact cells with neuraminidase did not alter the ganglioside pattern.     

Antibodies against gangliosides and ganglioside complexes in Guillain-Barré syndrome: new aspects of research

Guillain-Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.     

Antibodies against gangliosides and ganglioside complexes in Guillain–Barré syndrome: New aspects of research

Guillain–Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.     

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.     

Gangliosides of murine T lymphocyte subpopulations

Gangliosides from murine T lymphoblasts were analyzed by high-performance thin-layer chromatography followed by in situ neuraminidase treatment and immunostaining of the resulting asialogangliosides and compared with those from thymocytes and cloned T lymphocytes with defined functions. The ganglioside IVNeuGc/Ac-GgOse5Cer (GalNAc-GM1b), a marker for T lymphoblasts [Müthing, J., Egge, H., Kniep, B., & Mühlradt, P. F. (1987) Eur. J. Biochem. 163, 407-416], was found only in small amounts as the N-acetylated species in gangliosides from thymocytes and a cytolytic T cell clone. Two helper clones expressed this ganglioside like T blasts. The structures of the two major disialogangliosides from T blasts, IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha type) with C24:0/24:1 and C16:0 fatty acids, were elucidated by neuraminidase treatment and immunostaining and by fast atom bombardment mass spectrometry. Gangliosides of this type were detected in thymocytes only in minor amounts, whereas GM1b-type gangliosides prevailed in cells from this organ. Analysis of the T lymphoblast gangliosides from six genetically unrelated mouse strains showed that terminally sialylated GgOse4Cer (GM1b), IVNeuAc-GgOse5Cer (GalNAc-GM1b), and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha) were conserved structures in all strains examined. We conclude that maturation or stimulation of T cells may be correlated with elongation of a common GM1b-type precursor structure resulting in GalNAc-GM1b or GD1 alpha-type gangliosides.     

Antibodies against ganglioside complexes in Guillain-Barré syndrome and related disorders

Guillain-Barré syndrome (GBS) is acute autoimmune neuropathy, often subsequent to an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful diagnostic markers and possible pathogenetic factors. Recent data demonstrated that sera from some patients with GBS react with ganglioside complexes (GSCs) consisting of two different gangliosides, but not with each constituent ganglioside. Those antibodies may specifically recognize a new conformational epitope formed by two gangliosides. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. The antibodies to GM1/GalNAc-GD1a and those to GSCs containing GQ1b or GT1a are associated with pure motor GBS and Fisher syndrome, respectively. In contrast, the binding activities of the antibodies highly specific to GD1b are strongly inhibited by the addition of GD1a to GD1b. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which two different gangliosides may form a new conformational epitope. Future investigation is necessary to elucidate the roles of GSCs in the plasma membrane and of the clinical relevance of the anti-GSCs antibodies.     

Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

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.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.