Small cell lung cancer is not associated with the presence of anti-fucosyl-GM1 ganglioside autoantibodies reactive in immunoenzymatic test.

The characteristic feature of small cell lung cancer carcinoma (SCLC) is the aberrant expression and abundant presentation of fucosyl-GM1 ganglioside (FucGM1). In the present study we searched for the presence of anti-FucGM1 ganglioside, as well as anti-GM1, GM2 and GD3 ganglioside autoantibodies in the sera of patients with SCLC and as a control, in sera of patients with renal cell cancer (RC) and healthy blood donors. The autoantibodies against FucGM1 were present at low titer in only three of 36 SCLC patients, and with similar titer in two of 36 RC patients and four of 36 healthy controls. Likewise, the autoantibodies against GM2 and GM3 gangliosides were found only sporadically and with the same titer and frequency in cancer patients as in healthy persons. Anti-GD3 autoantibodies could not be detected in any of the screened sera.

[The use of monoclonal antibodies in the detection of small cell lung cancer metastases in bone marrow]. / Próba zastosowania przeciwcial monoklonalnych do wykrywania przerzutów drobnokomórkowego raka pluca w szpiku.

Expression of a number of antigens associated with small cell lung cancer (SCLC) have been proposed as a marker of malignancy and the diagnostic tool for the staging procedures and important prognostic factor. Since the bone marrow (BM) was described as a frequent site for SCLC metastases, we have decided to assess clinical importance of cancer cells detection in BM, using immunofluorescence with MAC-1, MAC-31, NSE and anti-Fucosyl-GM1 (PF3) antibodies. The group of 32 patients with SCLC was assessed using our panel of antibodies. Control group consisted of 5 patients with other malignancies (3 patients with malignant lymphoma, 1 with chronic lymphocytic leukaemia and 1 with non-SCLC). The study revealed no correlation between the expression of SCLC markers in patients BM and the cancer treatment outcome measured as a response for treatment, time to progression, and survival time, and no significant difference was found between the patients and control group.

Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides.

To detect HL-60 human promyelocytic leukemia cell proteins involved in the uptake of gangliosides from the culture medium we used photoreactive, 4-azidosalicylic acid (ASA) acylated and radioiodinated (200 Ci/mmole) derivatives of GM3, GD3, GM1, and FucGM1 gangliosides. Gangliosides-ASA, added to the medium at 15-20 nM concentration, followed a similar time course of uptake. After 1 min incubation cell bound gangliosides-ASA could not be removed with trypsin, but only 5-10% remained after incubation with BSA. The proportion of cell bound gangliosides-ASA resistant to BSA treatment increased with time of incubation up to 76% after 20 h. As shown on TLC, GM3- and GD3-ASA were catabolized to LacSph-ASA and ceramide-ASA, while GM1-ASA was hydrolyzed to GM2-ASA. FucGM1-ASA was converted to GM1-ASA very slowly. Upon irradiation with UV lamp, cell bound gangliosides-ASA crosslinked to and photolabeled many proteins but the distribution of radioactivity after SDS/PAGE was very uneven and did not correlate with Coomassie staining. In all experiments the 42 kDa protein bands were most intensely photolabeled. Photolabeling of 42 kDa proteins decreased with time of incubation as compared to lower molecular mass pro teins. With all gangliosides-ASA used similar but not identical protein photolabeling patterns were obtained. Photolabeling patterns with GM3- and GD3-ASA differed from those with GM1- and FucGM1-ASA.

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides.

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.

Anti-fucosyl-GM1 ganglioside IgG and IgM autoantibodies in human serum: no link to pathology.

Anti-fucosyl-GM1 ganglioside antibodies were detected in sera of five persons: four patients with autoimmune neuropathies and more recently, IgG antibodies in one with Graves' disease (Adler et al., Autoimmunity 18, 149-152, 1994) [1]. In the latter case, we were unable to find any relation between the occurrence of antibodies and thyroid disease. Now we report a detailed study on the anti-glycolipid antibodies in this patient. We found that her serum contained not only IgG but also a high level of anti-FucGM1 IgM antibodies, with a titer stable over a period of 5 years of treatment and follow-up. The carbohydrate structure of the epitope recognized by IgG and some of IgM antibodies seems to consist of Fuc-Gal-GalNAc-Gal- or a part of this sequence. Moreover, this patient's serum contained other IgM antibodies active against FucGM1 and also asialo GM1 glycolipids. Our results indicate that anti-FucGM1 ganglioside antibodies of G and M classes occur in serum of this patient with no apparent adverse health effects.

Photochemical labeling of human erythrocyte membranes with radioiodinatable azidosalicylic acid derivative of globoside.

In an attempt to define glycolipid functions we have prepared photoactivatable, iodinatable derivative of globoside and used it for photoaffinity labeling of human erythrocyte membranes. Lysogloboside (Gb4Sph) was prepared from globoside through deacylation in methanolic KOH followed by re-N-acetylation of galactosaminyl residue. The NH2 group of sphingosine residue in Gb4 Sph reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid resulting in the formation of Gb4Sph-ASA which was purified by preparative tlc and column chromatography. It migrated on tlc as a single spot in two solvent systems, was susceptible to leech ceramide glycanase and could be radioiodinated to a specific radioactivity of about 200 Ci/mmol. Gb4Sph-[125I]ASA was incorporated into human erythrocytes in a time and concentration-dependent manner. Before photolysis 96% of the Gb4Sph-ASA could be removed with albumin but not with trypsin. After photolysis about 50% of the label was firmly bound to erythrocytes being resistant to albumin and trypsin treatment. The label was distributed between membrane proteins and lipids in about 1:2.3 ratio. Photolabeled proteins were analyzed by SDS-PAGE followed by autoradiography and immunostaining. Most of the radioactivity was detected in band 3 and its proteolytic fragments irrespective of the duration of photolysis. Photolabeling of erythrocyte lipids was demonstrated by Sephadex LH-20 column chromatography.

Incidental presence of antibodies against gangliosides in Graves' disease.

In Graves' disease an increased immunological activity against glycoconjugate antigens is observed. Since gangliosides seem to be important for thyroid stimulation we searched for the presence of antiganglioside antibodies in Graves' patients sera by the immunoenzymatic method. Antibodies against some of 12 different gangliosides used in this study were found in 22% of the patients. However, in most cases the titer of antiganglioside antibodies was very low. Only the serum from one patient reacted with FucGM1 ganglioside at the dilution up to 1:1300. Anti-FucGM1 antibodies were present in serum samples of this patient taken before and during the treatment when she was hyper-hypo- and euthyroid. The lack of relation of the anti-FucGM1 antibody titer to the thyroid status of this patient, and the very low titer of antiganglioside antibodies in sera of some other patients, suggest that the presence of these antibodies has no close connection to the thyroid disease.

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.

Metabolism of cholesterol, phosphatidylethanolamine and stearylamine analogues of GM1 ganglioside by rat glioma C6 cells.

Tritium-labeled neoglycolipids consisting of the oligosaccharide of ganglioside GM1 attached to cholesterol (GM1OSNH-X-CHOL), phosphatidylethanolamine (GM1OS-PE) and stearylamine (GM1OSNHC18) were synthesized and their uptake and metabolism by GM1-deficient rat glioma C6 cells were determined. When the neoglycolipids were added to serum-free culture medium, all three were rapidly taken up by the cells and initially inserted into the plasma membrane based on their resistance to trypsin and their ability to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity to cell surface toxin binding increased; this process was slow for GM1OSNH-X-CHOL and GM1OS-PE and rapid for GM1OSNHC18. Analysis of lipids extracted from the cells indicated that the neoglycolipids also underwent metabolism to GD1aOS-based analogues. In addition, GM1OSNH-X-CHOL and GM1OSNHC18 were degraded to their GM2OS-based analogues, whereas GM2OS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GM1OS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglycolipid. In the presence of monensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, GM1OS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to GD1aOS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin.

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.

A novel form of GD1c ganglioside IV3 (NeuAc alpha 2-8 NeuGc)Gg4Cer from murine X-ray induced thymoma.

Four disialoganglioside fractions were isolated from X-ray induced thymoma in C57Bl/6 mice. On the basis of compositional and methylation analyses as well as degradation with specific exoglycosidases a novel form of GD1c ganglioside NeuAc alpha 2-8 NeuGc alpha 2-3 Gal beta 1-3 GalNAc beta 1-4 Gal beta 1-4 GlcCer was identified.

Glycolipids and glycopeptides of red cell membranes in congenital dyserythropoietic anaemia type II (CDA II).

The composition and structure of neutral and acidic oligoglycosylceramides, polyglycosylceramides and polyglycosylpeptides were determined in erythrocyte membranes of two patients with congenital dyserythropoietic anaemia type II. In keeping with previous studies we found an elevated accumulation in CDA II erythrocytes of LacCer, Lc3Cer and nLc4Cer. Gb4Cer was elevated in erythrocytes of only one of the two patients tested. In addition we found a significant increase of 6IVNeuAcnLc4Cer ganglioside. Polyglycosylceramides were elevated 6-fold but they resembled those of cord erythrocytes with respect to complexity and the number of side chains. Polyglycosylpeptides of CDA II erythrocytes were decreased 7-fold. These glycopeptides were, however, heterogeneous with respect to branching pattern; the minor fraction was highly branched whereas the major one was more linear in structure. Both polyglycosylceramides and polyglycosylpeptides exhibited high I and i antigenicity. We postulate that the accumulation of glycolipids and underglycosylation of glycoproteins in CDA II membranes results from the prolongation of G1 and possibly M phases of the mitotic cycle of the erythroid cells in which glycolipids are preferentially synthesized.

Structure of a new disialoganglioside GD1c from spontaneous murine thymoma.

A major mono- and a di-sialoganglioside were isolated and purified to homogeneity from a spontaneous thymoma that occurs in AKR mice. Compositional and methylation analyses and the use of exoglycosidases established the monosialoganglioside to be alpha Neu(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----4)Cer and the disialoganglioside to be alpha NeuAc(2----8)alpha NeuAc(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----1)Cer (GD1c). A possible pathway for the biosynthesis of this disialoganglioside is presented.

Glycosphingolipids in murine lymphocytes from thymus and spontaneous and X-ray induced thymomas.

Lymphocytes from spontaneous thymoma in AKR mice and from X-ray induced thymoma in C57B1/6 mice showed elevated levels (by 50% and 100%, respectively) of lipid-bound sialic acid as compared with lymphocytes from normal thymuses used as controls. Some ganglioside fractions in thymomas were elevated 4-6-fold over those in normal thymuses while other fractions decreased or disappeared. Neutral glycosphingolipid (NGSL) content in lymphocytes from thymomas was also changed. Thin-layer chromatography of NGSLs showed that the fractions migrating as ceramide monohexoside (CMH), dihexoside (CDH) and below globoside standards were increased, respectively, 2-3-fold, 3-6-fold and 2-fold in both types of thymomas. Methylation and gas-liquid chromatography analysis confirmed the presence of CMH, CDH and globoside in NGSLs isolated from X-ray induced thymoma.