Novel fucolipids of human adenocarcinoma: disialosyl Lea antigen (III4FucIII6NeuAcIV3NeuAcLc4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure

A new fucoganglioside, disialosyl Lea, has been found in the disialoganglioside fraction of human colonic adenocarcinoma. The ganglioside has been isolated from four other disialogangliosides by high-performance liquid chromatography followed by preparative high-performance thin-layer chromatography. The structure of the antigen was characterized by its conversion to lactofucopentaosyl(II)ceramide (Lea-active ceramide pentasaccharide), methylation analysis, and high-mass range electron-impact as well as field-desorption mass spectrometry of the permethylated derivative, as shown below: (Formula; see text) Specific removal of alpha 2----3-linked sialosyl residue by influenza virus A2 sialidase, or preferential hydrolysis of the same residue by Clostridium perfringens sialidase in the absence of detergent, resulted in the formation of an intermediate product, monosialosyl LeaII (III4FucIII6NeuAcLc4), which reacted with anti-Lea antibody and with monoclonal antibody FH7 and may have a sialic acid linked at the 6 position of GlcNAc. The IgG3 monoclonal antibody FH7 was established, which reacts specifically with disialosyl Lea and monosialosyl LeaII as above, but does not react with disialosyllactotetraosylceramide (IV3NeuAcIII6Neu-AcLc4), monosialosyl LeaI (IV3NeuAcIII4FucLc4), and other mono- and disialogangliosides isolated from the same cancer tissue. The antibody FH7 may be useful in the detection of human cancer.     

III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression

Monospecific rabbit antibodies directed against the human milk sialyloligosaccharides III6NeuAcLcOse4 (sialyltetrasaccharide b), IV3NeuAcLcOse4 (sialyltetrasaccharide a), and IV6NeuAcnLc4Ose (sialyltetrasaccharide c) were used to detect their homologous haptens as gangliosides or ganglioside-derived sialyloligosaccharides from the human colorectal carcinoma cell line SW1116. III6NeuAcLc4Cer was first detected in human meconium [P. A. Prieto and D. F. Smith (1985) Arch. Biochem. Biophys. 241, 281-289], and its presence in a total ganglioside fraction of SW1116 cells together with its absence from a total lipid extract of normal human intestinal mucosa are consistent with III6NeuAcLc4Cer being a tumor-associated oncofetal antigen. IV3NeuAcLc4Cer, a ganglioside in human meconium [P. A. Prieto and D. F. Smith (1986) Arch. Biochem. Biophys. 249, 243-253], was also detected in SW1116 cells; an observation that is consistent with its being the immediate precursor to the sialyl-Lea ganglioside in SW1116 cells. Specific antisera against sialylated type 1 oligosaccharide chains whose expression is independent of the Lewis gene fucosyltransferase may be useful diagnostic reagents for oncofetal, carbohydrate antigens.     

A novel ganglioside isolated from renal cell carcinoma

In renal cell carcinoma (RCC), the level of higher gangliosides is correlated with degree of metastatic potential, and cell lines derived from metastatic deposits of RCC are characterized by high expression of disialogangliosides (Saito, S., Orikasa, S., Ohyama, C., Satoh, M., and Fukushi, Y. (1991) Int. J. Cancer 49, 329-334 and Saito, S., Orikasa, S., Satoh, M., Ohyama, C., Ito, A., and Takahashi, T. (1997) Jpn. J. Cancer Res. (Gann) 88, 652-659). We now report two disialogangliosides, G1 and G2, found in the RCC cell line TOS-1. G1 from TOS-1 cells was characterized as having a novel hybrid structure between ganglio-series (region I as in Structure; same as the terminal structure of ganglioside GM2), and the lacto-series type 1 (region II). The characterization was based on reactivity with various monoclonal antibodies (mAbs) with defined epitope specificity, as well as monosaccharide and fatty acid component analysis, (1)H NMR spectroscopy, and electrospray ionization mass spectrometry of the intact compound. G1 showed strong reactivity with mAb RM2, raised originally against TOS-1 cells, and weak cross-reactivity with anti-GM2 mAb MK-1-8. The antigen is hereby termed GalNAc disialosyl Lc4Cer (IV4GalNAcIV3NeuAcIII6NeuAcLc4; abbreviated GalNAcDSLc4). G2 was identified by 1H NMR and mass spectrometry as having a structure similar to Structure but without the GalNAcbeta1-->4 substitution and showed strong reactivity with mAb FH9 reported previously to be specific for disialosyl lacto-series type 1 (disialosyl Lc(4)) having vicinal alpha2-->3 and alpha2-->6 sialosyl residues, an antigen associated with human colonic cancer. Clinicopathological studies indicate that expression of these disialoganglioside antigens in RCC tissue is correlated with the metastatic potential of RCC.     

Novel fucolipids accumulating in human adenocarcinoma. III. A hybridoma antibody (FH6) defining a human cancer-associated difucoganglioside (VI3NeuAcV3III3Fuc2nLc6)

A new fucoganglioside, 6B, which accumulates in human colonic adenocarcinoma but is absent in normal colonic mucosa, was isolated from a monosialoganglioside fraction of colonic adenocarcinomas. The structure of this ganglioside was identified as shown below by methylation analysis, direct probe mass spectrometry, and enzymatic degradation followed by examination of the degradation products with specific monoclonal antibodies. (formula; see text) The hybridoma (FH6) secreting a monoclonal IgM antibody directed to this glycolipid was selected by reactivity of the antibody with this ganglioside and lack of reactivity with other glycolipids having a closely related structure, such as sialosyllactoneotetraosylceramide (IV3NeuAcnLc4), sialosyllactofucopentaosy(III)ceramide (IV3NeuAcIII3FucnLc4), sialosyllactofucopentaosy(II)ceramide (sialosyl-Lea glycolipid; IV3NeuAcIII4FucLc4), and 6C fucoganglioside (sialosyl 2 leads to 6 fucoganglioside; VI6NeuAcIII3FucnLc6). The antibody was highly reactive with a large variety of human cancer cells, but was less reactive or did not react with a variety of normal cells.     

A ganglioside of rat ascites hepatoma AH 7974F cells. Occurrence of a novel disialoganglioside (GD1 alpha) with a unique N-acetylneuraminosyl (alpha 2-6)-N-acetylgalactosamine structure

A novel disialoganglioside has been isolated from rat ascites hepatoma AH 7974F cells. Based on the results of sequential enzymatic hydrolysis and gas chromatography-mass spectrometry analysis of the methylated sugars, the structure was concluded to be (Formula: see text) Proton magnetic resonance spectra of the ganglioside have been obtained and peaks of protons were assigned based on the analytical results. This is the first report on the occurrence in mammalian cells of an example of this new series of gangliosides which has NeuAc linked to the C6 position of GalNAc of the gangliotetraosyl backbone. The present ganglioside was named GD1 alpha.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

Differential expression of fucosyl GM1 and a disialoganglioside with a NeuAc alpha 2-6GalNAc linkage (GD1e) in various rat ascites hepatoma cells

A distinct difference in ganglioside composition among various rat ascites hepatomas and Yoshida sarcoma was observed on TLC-immunostaining with anti-fucosyl GM1 antibody, and chemical and enzymatic analyses. Yoshida sarcoma and ascites hepatomas, AH13, AH66F and AH66, but not the other 9 tumor cell lines investigated, specifically contained a disialoganglioside, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1ceramide (GD1e), whereas the 9 ascites hepatoma cells without GD1e contained fucosyl GM1. The differential expression of fucosyl GM1 and GD1e in various tumor cell lines indicates that different cell lineages express distinct metabolic pathways for gangliosides, and that the gangliosides are useful markers for distinguishing tumor cell lines.     

The biosynthesis of gangliosides. The incorporation of galactose, N-acetylgalactosamine and N-acetylneuraminic acid into endogenous acceptors of subcellular particles from rat brain in vitro

Gangliosides bound to subcellular particles from rat brain were labelled by incubation of the particles (i) with CMP-N[(3)H]-acetylneuraminic acid and (ii) simultaneously, with CMP-N[(3)H]-acetylneuraminic acid and UDP-N-acetyl-[(14)C(1)]galactosamine or with CMP-N[(3)H]-acetylneuraminic acid and UDP-[U-(14)C]-galactose. Analysis of the labelled gangliosides showed that in (i), (a) the labelling was mostly in the neuraminidase-labile sialyl groups, (b) rigid relationships exist between the enzymes and the sialyl acceptors; the enzymes are not free to interact with all the specific substrates present in the preparation and (c) the precursor of the trisialoganglioside was the major disialoganglioside with a sialyl 2-->8 sialyl group. In (ii), (a) precursor-product relationships between the main pools of each ganglioside apparently do not exist, (b) for the labelling of Tay-Sachs ganglioside the amount formed from hematoside was at least 2.5 times that from aminoglycolipid and (c) the major monosialoganglioside was the precursor for the major disialoganglioside with a sialyl 2-->8 sialyl group.     

Appearance of a novel type of ganglioside (GD1 alpha) in a differentiation-resistant clone of mouse myeloid leukemia cells, M1-R1

Glycolipid compositions of three mouse myeloid leukemia cell clones, two that are sensitive to differentiation inducers (M1-T22 and M1-S1) and one that is differentiation-resistant (M1-R1), have been compared. The T22 and S1 clones contained glucosylceramide (GlcCer), lactosylceramide (LacCer) and gangliotriaosylceramide (Gg3Cer) as the major neutral glycolipids. The differentiation resistant clone, R1, was characterized by the appearance of globotriaosylceramide (Gb3Cer) and a decrease of Gg3Cer. There was a distinct difference in the ganglioside profile between the differentiation-inducible and -resistant clones: T22 and S1 cells contained no detectable amounts of ganglioside, whereas six different gangliosides were detected in the R1 clone. These gangliosides were isolated and identified as GM3, GM2, GM1a, GD1a, GM1b, and a unique disialoganglioside, GD1 alpha, having the following structure: (formula; see text) Based on these comparative studies, the relationship between the glycolipid composition and the differentiation potential of leukemia cells is discussed.     

Disialoganglioside GDla of rat brain subcellular particles during development.

The increase observed in the amount of the disialoganglioside GDlof the rat cerebrum during development between 21 and 81 days of age accounted for nearly 40% of the overall increase in total ganglioside in the tissue during the same period. Subcellular fractionation showed the microsomal fraction to contribute by far the most towards this increase in Cerebral ganglioside GDla. It is suggested that microsomal ganglioside GDla may serve as a marker for dendritic arborization in the rat cerebrum.     

Myelin Gangliosides of Human Peripheral Nervous System: An Enrichment of GM1 in the Motor Nerve Myelin Isolated from Cauda Equina

Myelins of the PNS were isolated from human motor and sensory nerves of cauda equina, and their ganglioside compositions were compared. The predominant ganglioside in the human PNS myelins, both from motor and sensory nerves, was LM1 (sialosylneolactotetraosylceramide). Sialosyl-nLc6Cer and disialosyl-nLc4Cer, GD3, GM3, and GD1b were detected as common components of the two nerve myelins. Furthermore, it was revealed that the motor nerve myelin contained GM1 (about 15% of total gangliosides), whereas sensory nerve myelin contained only a trace amount of GM1 (less than 5%), by TLC analyses together with TLC immunostaining using anti-GM1 antibody. As for the disialoganglioside fraction, the content of GD1a, as well as that of GM1, differed in motor and sensory nerves. Thus, the different contents of the ganglioseries gangliosides in human motor and sensory nerve myelins were demonstrated.     

Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.     

A novel disialoganglioside in rat spleen lymphocytes.

A novel ganglioside has been identified as the predominant disialoganglioside of the lymphocytes prepared from rat spleen. The ganglioside was isolated from rat spleen and characterized by compositional analysis, methylation analysis, sialidase hydrolysis, proton NMR spectroscopy, and negative ion fast atom bombardment mass spectrometry. The structure was determined as follows. [formula: see text] This ganglioside is a unique derivative of N-acetyllactosaminyl-GM1. The three monosialogangliosides containing N-acetyllactosaminyl-GM1 structure, which had been originally isolated from rat spleen (Nohara, K., Suzuki, M., Inagaki, F., Ito, H., and Kaya, K. (1990) J. Biol. Chem. 265, 14335-14339), were also found in the lymphocytes and were hardly detected in the spleen remnant tissue depleted of single cells. On the other hand, GD1c(NeuGc,NeuGc) (IV3(NeuGc alpha 2-8NeuGc)-Gg4Cer), the overwhelmingly predominant ganglioside of rat thymocytes (Nohara, K., Suzuki, M., Inagaki, F., and Kaya, K. (1991) J. Biochem. (Tokyo) 110, 274-278), was demonstrated to be only a minor component of the gangliosides of the spleen lymphocytes. These results suggested that GD1c is characteristic for the immature T lineage lymphoid cells and the gangliosides having lactosaminyl-GM1 structure are specific for other populations of the lymphocytes in rat.     

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Chemical structure of carcinoma ganglioside antigens defined by monoclonal antibody C-50 and some allied gangliosides of human pancreatic adenocarcinoma

A hybridoma, C-50, obtained by fusion of mouse myeloma cells with spleen cells from a mouse immunized with cells from the colorectal carcinoma cell line COLO 205, produced antibodies that detected ganglioside antigen in human adenocarcinomas in many organs. The major ganglioside antigen fraction isolated from liver metastases of a pancreatic adenocarcinoma, behaving as a homogenous band on thin-layer chromatography, consisted of three different gangliosides. One of them, A (25%), had the same carbohydrate structure as the ganglioside antigen defined by monoclonal antibody 19-9, NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4Glc-Cer(Fuc-3'-isoLM1) Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369). The major ganglioside, B (60%), was the isomeric hexasaccharide ganglioside (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3-Gal beta 1-4Glc-Cer(Fuc-3'-LM1) and the third ganglioside, C, was 6'-LM1, NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer (15%). Ganglioside B, isolated from human kidney, did not react with the C-50 MAb. Based on this result and on studies of COLO 205 cell induced tumours where the ganglioside antigen fraction only consisted of A, it is suggested that the C-50 MAb defines an antigen determinant present in A.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Frog brain uridine diphosphate galactose–N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase

1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1-->4)-N-acetylneuraminyl-(2-->3)-galactosyl-(1-->4)-glucosylceramide (G(M2)) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose-G(M2) galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11(1/2)) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2-7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn(2+) for maximal activity and the K(m) for Mn(2+) was determined as 2.2mm. The half-maximal velocity was obtained at a G(M2) concentration of 0.18mm. Inhibition of the enzymic reaction was found when the G(M2) concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

A Ganglioside Species (GD1α) Migrates at a Slow Rate and CMP-Sialic Acid Severalfold Faster in Xenopus Sciatic Nerve: Fluorographic Demonstration

The ninth dorsal root ganglion of adult Xenopus laevis was labeled with N-acetyl-D-[6-3H]mannosamine, and intraaxonal migration of gangliosides was examined by analysis of the chloroform/methanol extract of each of 5-mm consecutive nerve segments by TLC coupled with fluorography. A unique disialoganglioside (GD1 alpha), which amounted to up to 83% of the total ganglioside in this nerve, migrated at 1-2 mm/day at 15 degrees C. This contrasts with the rapid transport of other ganglioside species previously reported in the optic systems of goldfish, rabbits, chickens, and rats. Fluorographic analysis also revealed a trichloroacetic acid-soluble substance migrating at a velocity of approximately 8 mm/day at 15 degrees C. The substance was considered to be CMP-sialic acid on the basis of observations that it comigrates with authentic CMP-N-acetylneuraminic acid in TLC developed with two different solvent systems, it is very labile to weak acid but resistant to neuraminidase from Vibrio cholerae, it is converted to N-acetylmannosamine when treated first with weak acid and subsequently with N-acetylneuraminic acid aldolase, and it has a beta-sialosyl group in its structure. Because CMP-sialic acid is believed to be the sole sialosyl donor in the cells, its migration in axons toward terminals, together with the previous demonstration of sialyltransferase activity in the synaptosomal plasma membrane, strongly supports the possibility that sialosylation of gangliosides and probably of other sialoglycoproteins is not confined to the Golgi apparatus, but can also occur after the compounds are committed to axonal transport.