Megumi Saito

Structure and Localization of Gangliosides

Gangliosides are sialic acid-containing glycosphingolipids (GSLs) found primarily in the plasma membrane of virtually all vertebrate tissues and are particularly abundant in the nervous system. They constitute part of the glycocalyx network surrounding the cell surface and are crucial in determining the properties and functions of cells. Structurally, they contain a hydrophobic ceramide chain to which a hydrophilic oligosaccharide is glycosidically linked. Some representative brain gangliosides are shown in Figure 1.

Ganglioside GD2 specificity of monoclonal antibodies to human neuroblastoma cell

Four monoclonal antibodies (3F8, 3A7, 3G6, and 2F7) against human neuroblastoma cells have been suggested to react with surface glycolipids of these cells. In this report these monoclonal antibodies were shown to be specific to the disialoganglioside GD2 using a thin-layer chromatography (TLC)-immunostaining method. When mixed human brain gangliosides were developed by TLC in two different solvent systems and incubated with each of the monoclonal antibodies, only GD2 was stained. These antibodies also reacted with highly purified GD2 on the plate. These findings suggest that GD2 provides an antigenic site on the surface of human neuroblastoma cells.

In situ immunological determination of basic carbohydrate structures of gangliosides on thin-layer plates

An immunological method for the determination of the basic carbohydrate structure of gangliosides by using a thin-layer chromatographic immunostaining technique was developed. After high-performance thin-layer chromatography of gangliosides, the chromatogram is treated with a 0.4% polyisobutylmethacrylate solution. Arthrobacter ureafaciens neuraminidase is then applied to the separated gangliosides in situ on the chromatographic plate. This procedure will remove both external and internal sialic acid residues from the core oligosaccharide backbone. The resulting glycolipid products are then incubated with anti-Gg4 serum and 125I-staphylococcal protein A, successively, and exposed to an X-ray film. Through a highly specific binding, the anti-Gg4 antibody detects only those gangliosides having the oligosaccharide backbone of Gg4.

The neuropathy of plasma cell dyscrasia Binding of IgM M‐proteins to peripheral nerve glycolipids

In some patients with neuropathy and plasma cell dyscrasia, the M-proteins bind to peripheral nerves. Binding of M-proteins to peripheral nerve glycolipids was examined by immunostaining after thin-layer chromatography. The IgM from 16 patients with anti-MAG M-proteins bound to the same two glycolipid bands in peripheral nerve. The IgM that bound to the glycolipids had the same idiotype as the anti-MAG M-protein, indicating that it was the M-protein that bound to both glycolipids. The reactive glycolipids did not contain sialic acid and were not gangliosides. No immunostaining of peripheral nerve glycolipids was observed with IgM from patients with neuropathy and IgM M-proteins that did not bind to MAG, and the anti-MAG antibodies did not bind to brain glycolipids. Anti-MAG M-proteins probably bind to the same or closely related carbohydrate determinants that are shared by a number of glycoproteins and glycolipids of peripheral nerve.

Biochemistry and Function of Sialidases

Sialidases (EC 3.2.1.18; N-acylneuraminosyl glycohydrolase) are a family of exoglycosidases that catalyze the cleavage of nonreducing sialic acid residues ketosidically linked to mono-or oligosaccharide chains of glycoconjugates. They are widely distributed in viruses, bacteria, fungi, mycoplasma, and protozoa as well as avian and mammalian species (Rosenberg and Schengrund, 1976; Corfield et al., 1981a; Corfield and Schauer, 1982; Conzelmann and Sandhoff, 1987; Corfield, 1992). Among the various sialidase species, viral and bacterial enzymes have been studied extensively; a number of them have been purified to homogeneity and characterized for their properties and structures. Mammalian sialidases are more labile and often are bound tightly to membranes, hindering successful purification of these enzymes. Much attention, however, has been directed toward these enzymes as interest in the metabolism and biological function of sialoglycoconjugates in mammalian cells has grown in recent years (Schauer, 1982, 1985, 1991; Ledeen, 1989; Schengrund, 1990; Varki, 1992). The term “sialidase” was first proposed by Heimer and Meyer (1956), and “neuraminidase” was introduced a year later (Gottschalk, 1957). Both names have been used interchangeably in the literature. Since the enzyme does not usually apply to neuraminic acid itself, but to its derivatives, sialic acid, the term “sialidase” is deemed more appropriate (Rosenberg and Schengrund, 1976).

Sialidase Activity in Nuclear Membranes of Rat Brain

Abstract: A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N‐acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N‐acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane‐bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain.

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.

Glycosphingolipids in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Glycosphingolipids in cerebrospinal fluid (CSF) of individual patients with multiple sclerosis (MS) were analyzed using a glycolipid-overlay technique. The ganglioside composition of CSF of non-MS patients was characterized by an abundance of polysialo species, including GT1b and GQ1b. This pattern is completely different from that of human white or gray matter, in which mono- and disialogangliosides predominate. Increased levels of GM1, either associated with or without increases of other gangliosides, such as GD1a, were observed in 16% of the patients with MS (6 of 37 cases: 1 of 15 progressive progressive stage, 4 of 16 progressive stationary stage, and 1 of 6 relapsing stage). The concentration of GD3 was increased in 23% (3 of 13 cases), whereas 1 of 13 cases (8%) showed a dramatic increase of sulfoglucuronyl paragloboside (SGPG) associated with a high level of GD3. These changes may reflect the cellular changes associated with the known pathological lesions in MS, which are characterized by demyelination, gliosis, and/or remyelination with oligodendrocytic proliferation.

Thin-layer chromatography-densitometry of minor acidic phospholipids: Application to lipids from erythrocytes, liver, and kidney

A method for quantitative determination of acidic phospholipids by thin-layer chromatography (TLC) followed by densitometry is described. The total lipids were separated into neutral and acidic fractions by DEAE-Sephadex column chromatography. A clear-cut separation of acidic phospholipids was achieved by high-performance TLC with a solvent system of chloroform/acetone/acetic acid/formic acid/water (60/60/4/10/3). Each phospholipid band was quantitated by densitometry with the use of an internal standard. The lipid compositions of sheep and mouse erythrocytes and of rat liver and kidney were determined by the present method.

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

Abstract: 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(α2 → 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°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 × 10−6M versus 6.3 × 10−6M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 × 10−4M and 4.3 × 10−4M, 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°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.