Robert K. Yu

[10] Gangliosides: Structure, Isolation, and Analysis

Publisher Summary This chapter discusses structure and procedure for isolation, and analysis of gangliosides. Most widely used procedure for isolation is based on the ability of gangliosides to partition from the chloroform-methanol phase in which they are extracted into the upper water-enriched phase, leaving behind the bulk of other lipids. There is also a procedure based on DEAE-Sephadex. This method employs an anion-exchange resin to separate gangliosides first, along with other acidic lipids, from the much larger quantity of neutral and zwitterionic lipids that do not bind to the resin. The subsequent steps of base treatment, dialysis, and silicic acid chromatography are designed to remove the other acidic lipids and any acidic low molecular weight contaminants that accompany the gangliosides. Other types of chromatography is used for resolution. Quantitative estimation of gangliosides is usually based on the sialic acid moiety. Various procedures are outlined, along with a gas-liquid chromatographic method that has the attributes, of specificity and sensitivity.

High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species

Abstract Improved resolution of complex brain ganglioside mixtures was achieved by high-performance thin-layer chromatography. The percentage distribution of individual gangliosides was then determined by direct densitometric seanning, employing a transmittance mode, of the resorcinol-positive spots on the plate. As little as 90 pmol (29 ng) of lipid-bound sialic acid could be detected with a good signal-to-noise ratio. A linear detector response was observed up to 3.0 μg of lipid-bound sialic acid. The brain white matter ganglioside patterns of eight animal species, including human, chimpanzee, monkey, chicken, bovine, sheep, and pig, were examined in detail. In addition, human brain gray matter, rat cerebral, rat brain gray matter, and rat cerebellar ganglioside patterns were also studied. Ganglioside G M4 (G 7 ) was found to be one of the major components in primate and chicken brain white matter, but it represented only a minor ganglioside in other species. Other major gangliosides in all brain samples studied were G M1 , G D1a , G D1b , and G T1b . G M1 was more abundant in white matter than in gray matter. G T1a , a recently discovered ganglioside species, was found in all species examined, but was most abundant in the rat cerebellum. The latter source also contained high proportions of G T1b and G Q1b .

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Abstract: Developmental changes in ganglioside composition and biosynthesis were 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‐acetylgalacto‐saminyltransferase 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.

Role of tumor-associated gangliosides in cancer progression

Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted.

Gangliosides GM1 and GD1b are antigens for IgM M-protein in a patient with motor neuron disease

We studied a patient with an IgM M-protein and lower motor neuron disease to identify the antigens to which the M-protein bound. Gangliosides from peripheral nerve and spinal cord were separated by high-performance thin-layer chromatography and immunostained with the patients serum. The serum IgM immunostained two gangliosides identified as GM1 and GD1b, and immunostaining was specific for the M- protein light chain type. IgM-binding to the two gangliosides was detectable by ELISA at serum dilutions of greater than 1:10,000, and the M-protein was selectively immunoabsorbed by liposomes containing GM1 or GD1b. The IgM M-protein also bound to asialo-GM1, indicating reactivity to the galactosyl(beta 1-3)N-acetylgalactosaminyl moiety shared by GM1, GD1b, and asialo-GM1.

GD3 ganglioside is a glycolipid characteristic of immature neuroectodermal cells

Biochemical studies have indicated that the disialoganglioside, GD3, is a major glycolipid component of the immature vertebrate CNS, but a minor element within the mature CNS. We have investigated its cellular localization in rat CNS by immunofluorescence using a mouse monoclonal antibody that recognizes GD3. In tissue sections of postnatal CNS, the antibody bound to cells in several areas known to contain immature neuroectodermal populations: the subventricular zone beneath the lateral ventricle, the external germinative layer of the cerebellar cortex, and the dentate gyrus of the hippocampus. GD3-containing cells were also found in developing white matter of the forebrain and cerebellar folia. Using a double-label immunofluorescence method, we found that the GD3-positive white matter cells did not express the astrocytic marker, glial fibrillary acidic protein. In adult rat CNS, we could not detect antibody binding to neurons or glia. Scattered GD3-positive cells were apparent in the adult subventricular zone. Our results indicate that GD3 ganglioside is a membrane component characteristically expressed in the rat CNS by neuroectodermal stem cells, both neuronal and glial precursors.

Monoclonal IgM with unique specificity to gangliosides GM1 and GD1b and to lacto‐N ‐tetraose associated with human motor neuron disease

IgM lambda monoclonal antibodies in two patients with motor neuron disease showed the same unique antigenic specificity. They bound to gangliosides GM1 and GD1b and to lacto-N-tetraose-BSA. By immunofluorescence microscopy they bound to central and peripheral nerve tissue and to motor end-plates at the neuromuscular junction. Sera from control subjects did not contain antibodies of similar specificity. Monoclonal IgMs with the same unique specificity could be responsible for motor neuron disease in some patients with monoclonal gammopathies.

Calcium/Ganglioside‐Dependent Protein Kinase Activity in Rat Brain Membrane

Abstract: The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high‐molecular‐weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low‐molecular‐weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+ ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1. GD1a was the most potent activator tested. Asialo‐GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+ ‐dependent, ganglioside‐stimulated phosphorylation was qualitatively similar to the pattern for calmodulin‐dependent phosphorylation. However, while calmodulin‐dependent kinase activity was inhibited with an IC50 of 10 μM trifluoperazine, ganglioside‐stimulated kinase was inhibited with an IC50 of 200 μM trifluoperazine. These results indicate that gangliosides have complex effects on membrane‐associated kinase activities and suggest that Ca2+ ‐ganglioside complexes are potent stimulators of membrane kinase activity.

Development regulation of ganglioside metabolism

Publisher Summary Various reports have appeared indicating that the expression of gangliosides may be largely regulated at the genetic level. Evidence from analysis of the gene for this enzyme revealed binding sequences for a number of liver restricted transcription factors such as hepatocyte nuclear factor l a, liver specific factors D-binding protein (DBP), and liver enriched transcription activator protein (LAP), as well as the more general transcription factors AP-1 and AP-2. Presumably, the expression of the various glycosyltransferases for ganglioside synthesis may be regulated in an analogous manner. Factors involved in the regulation of ganglioside expression include the proper translocation and sorting of the glycolipid products in multi-glycosyltransferase systems. Disruption of the flow of gangliosides along their biosynthetic pathways also can cause alterations in ganglioside expression. It is demonstrated that polysialogangliosides exerted inhibitory effects feedback control. Another intriguing mechanism for the regulation of ganglioside synthesis states that in cultured cerebellar cells a shift in the biosynthesis from a- to b-series gangliosides occurred when the pH of the medium was lowered from 7.4 to 6.2, with the effect being reversible. Whether changes in pH occur in vivo to effect the shift in biosynthesis of gangliosides during development remains to be substantiated.

STALOSYLGALACTOSYL CERAMTDE AS A SPECIFIC MARKER FOR HUMAN MYELIN AND OLIGODENDROGLIAL PERIKARYA: GANGLIOSIDES OF HUMAN MYELIN, OLIGODENDROGLIA AND NEURONS

Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with GM4 and GM1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of GM4 was found in oligodendroglial fractions in addition to GM1, GD1a, GD1b, and GT1b. The usually‐ minor brain gangliosides GM3, GM2, and GM3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of GM4. These results indicate that GM4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter GM1 and GM4.