Reduced inflammatory potential of peritoneal macrophages recruited in mice pretreated with a glycolipid synthesis inhibitor.

Integral components of mammalian cell membranes, glycosphingolipids (GSL) reside in specialized plasma membrane microdomains critical for cell signaling. N-alkylated nojirimycins are compounds developed for GSL substrate deprivation therapy, blocking GSL synthesis by specifically inhibiting an essential enzyme, ceramide glucosyltransferase. Peritoneal macrophages recruited in mice pretreated with an inhibitory N-alkylnojirimycin displayed a reduced capacity to release either TNFalpha or interleukin-6 when re-exposed to whole killed E. coli in vitro. Cell viability and protein content were not affected. A nojirimycin analogue without GSL inhibitory capacity had no effect. The results show inhibition of GSL synthesis in vivo by an N-alkylnojirimycin can reduce the response to an inflammatory stimulus and indicate N-alkylnojirimycins have experimental and potential clinical value for modulating innate immune responses in vivo.

The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures.

Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods. Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses. Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation. All gangliosides were resistant to beta-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties. Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set. In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid. One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an alpha(2-6)-linked sialic acid residue with the same mass as another doublet. All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II(3)NeuAcLacCer (sialolactosyl ceramide, GM3), IV(3)- and IV(6)NeuAcnLcOse(4)Cer (sialoparagloboside, nLM1), and IV(3)NeuAcnLcOse(6)Cer (a sialohexosylceramide).

Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice.

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine B lymphocytes purified by adherence to anti-immunoglobulin ("antibody panning") possess identical gangliosides but different ganglioside surface accessibilities. We investigated the distribution and surface accessibility of gangliosides of B lymphocytes purified by adherence to plastic ("plastic panning") or by subtraction of non-B-lymphocyte components. As with antibody panning, there were no entirely new or absent gangliosides in plastic-panned or subtraction-purified B lymphocytes of each strain. However, striking changes in relative expression of five gangliosides were detected with each purification protocol. Moreover, five gangliosides of antibody-panned and plastic-panned B lymphocytes but only two gangliosides of subtraction-purified B lymphocytes were inaccessible to surface labeling. Unlike the situation for antibody-panned B lymphocytes, no interstrain (HeN vs. HeJ) surface accessibility differences existed in gangliosides of plastic-panned or subtraction-purified cells. Exposure of subtraction-purified B lymphocytes to anti-immunoglobulin failed to elicit changes in ganglioside expression. Murine B lymphocytes have distinct protocol-dependent differences in glycolipid phenotype which likely denote individual subpopulations.

Expression of mouse sialic acid on gangliosides of a human glioma grown as a xenograft in SCID mice.

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.

Tumor-infiltrating macrophages influence the glycosphingolipid composition of murine brain tumors.

A procedure was developed to analyze glycosphingolipids (GSLs) in tumor-infiltrating macrophages (TIMs). The procedure entailed dissociating tumors into single cell suspensions with a concurrent metabolic labeling of GSLs using [14C]galactose. TIMs were then separated from tumor cells and other host cells by magnetic activated cell sorting and CD11b (Mac-1) microbeads. Gangliosides and neutral glycosphingolipids were analyzed in the TIM-enriched and TIM-depleted fractions in two different murine brain tumors (EPEN and CT-2A). The TIM gangliosides consisted of over 30 structures as assessed by two-dimensional high performance thin-layer chromatography. GSLs enriched in TIMs, relative to the tumors, included Gg4Cer (asialo GM1), GM1b, and GD1alpha. TIM GSLs were similar in EPEN and CT-2A despite their differences in growth and morphology. TIM GSLs were similar whether TIMs were isolated from tumors grown intracranially or subcutaneously. TIM GSLs were also similar to activated peritoneal macrophage GSLs, although differences in the ceramide structure were observed. Knowledge of TIM GSLs will be important in determining the function of these molecules in macrophage-tumor interactions. In addition, these methods will be helpful in determining the cellular origin of human brain tumor GSLs and in identifying tumor-associated GSLs for immunotherapy.

Ganglioside composition of a mouse brain tumor grown in the severe combined immunodeficiency (SCID) mouse.

The content and composition of gangliosides were examined in an experimental mouse brain tumor, EPEN, that was grown subcutaneously in the flank of the syngeneic C57BL/6J (B6) host and in the B6 severe combined immunodeficiency (SCID) host. SCID mice lack functional T- and B-lymphocytes, but have a normal complement of macrophages. The content and distribution of the brain tumor gangliosides were similar whether the tumor was grown in the immunocompetent B6 host or in the B6-SCID host. N-acetylneuraminic acid- (NeuAc) containing GM3 was the major ganglioside in the subcutaneous tumors and in the cultured EPEN cells. Significant amounts of N-glycolylneuraminic acid- (NeuGc) containing gangliosides were found in the tumor grown in both mouse hosts. NeuGc-containing gangliosides are not expressed in normal mouse brain, but are present in macrophages and serum. An extremely complex pattern of minor gangliosides was found in the subcutaneous tumors on two-dimensional, high-performance thin-layer chromatograms. Most of the minor gangliosides comigrated with those found in mouse macrophages. The results show that the absence of functional T- and B-lymphocytes does not markedly affect brain tumor ganglioside composition and suggest that NeuGc-containing gangliosides in the EPEN can be derived from tumor infiltrating host cells (mostly macrophages) and from the extracellular milieu (serum).

Ganglioside biosynthetic gene expression in experimental mouse brain tumors.

The genes for cytidine monophospho-N-acetylneuraminic acid hydroxylase (NeuAc-H) and beta-1,4-N-acetylgalactosaminyl transferase (GalNAc-T) were examined using reverse transcription-PCR in two experimental mouse brain tumors, EPEN and CT-2A. NeuAc-H is required for the synthesis of gangliosides containing N-glycolylneuraminic acid, whereas GalNAc-T is required for the synthesis of ganglioside GM2. The genes were analyzed in solid tumors grown in vivo and in tumor cells grown in vitro. NeuGc-containing gangliosides are abundant in cells of the mouse immune system, including macrophages, but are undetectable in normal mouse brain. GM2 is expressed in both neural and nonneural mouse cells and tissues. The EPEN tumor cells synthesize only ganglioside GM3, whereas the CT-2A tumor cells synthesize GM3, GM2, GM1, and GD1a. NeuAc-H gene expression was detected in both solid tumors grown in vivo but was undetectable in either tumor cell line. The presence or absence of NeuAc-H gene expression in the tumor tissues and cells correlates with the presence or absence, respectively, of NeuGc-containing gangliosides. Differences in GalNAc-T gene expression between the solid tumors and the cultured tumor cells correlate with the expression of ganglioside GM2. The findings suggest that the differences in ganglioside biosynthetic gene expression between brain tumors grown in vivo and in vitro are associated with the presence or absence, respectively, of tumor-infiltrating host cells.

Structural characterization of the disialogangliosides of murine peritoneal macrophages.

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.

Influence of host cell infiltration on the glycolipid content of mouse brain tumors.

Previous studies showed that levels of some glycosphingolipids (GSLs) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT-2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro. The markers included ganglioside N-glycolylneuraminic acid (NeuGc), GA1 (asialo-GM1), and Fc receptor-bearing cells. NeuGc-containing gangliosides, GA1, and Fc receptors are expressed by macrophages and lymphoid-type cells of the mouse host immune system but are not normally expressed by mouse neural cells. Differences in the relative content of Fc receptor-bearing cells in ependymoblastoma and CT-2A tumors grown in vivo (8.3 and 16.8%, respectively) were proportional to differences in the relative content of NeuGc-containing gangliosides (25.5 and 45.1%) and GA1 (8.5 and 13.8%), respectively. Neither cultured tumor cell line expressed Fc receptors, GA1, or NeuGc-containing gangliosides. These findings suggest that non-neoplastic host infiltrating cells (macrophages) contribute significantly to the GSL composition of solid tumors growing in vivo.

In situ accessibility of murine macrophage gangliosides.

Gangliosides are implicated in cell signal transduction. Prior to investigating this phenomenon in macrophages, the in situ accessibility of gangliosides to macromolecules was assessed for peritoneal macrophages isolated from normal C3H/HeN and endotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident and thioglycolate-elicited macrophage ganglioside patterns are the same as normal strains, and no strain differences in galactose oxidase accessibility for resident or thioglycolate-elicited macrophage gangliosides were found. The only gangliosides accessible to galactose oxidase in resident macrophages are GM1a structures. In thioglycolate-elicited macrophages, an additional ganglioside is accessible. For Escherichia coli-activated macrophages, where ganglioside distribution differs between strains, a difference in galactose oxidase-accessible gangliosides also exists. Escherichia coli-activated C3H/HeN patterns show three triplets absent in C3H/HeJ patterns. There were no differences in ganglioside accessibility to Vibrio cholerae sialidase between the thioglycolate-elicited C3H/HeJ and C3H/HeN macrophages. However, despite differences in sialidase-sensitive ganglioside content between E.coli-activated macrophages of these strains, sialidase accessibility for E.coli-activated macrophages was also similar. Sialidase-susceptible GM3 was cryptic in either strain under all conditions examined. The accessibility of murine macrophage gangliosides to galactose oxidase or sialidase was independent of their sialic acid species and chain length of the ceramide fatty acid. With the exception of GM3, major murine macrophage gangliosides are accessible in situ to macromolecules, especially to exogenous pathogenic bacterial sialidase which can alter macrophage cell surface characteristics. Altered macrophage ganglioside accessibility appears sometimes as a consequence, but not a cause, of C3H/HeJ endotoxin hyporesponsiveness.

Removal of borate from tritiated gangliosides via the mannitoborate complex.

Purified gangliosides were radiolabeled by exposure to sodium boro [3H]hydride in the presence of palladium on barium sulfate. After centrifugation to remove the catalyst, borate was complexed with mannitol and the resulting mannitoborate complex and salts were removed by reversed phase chromatography. The labeled gangliosides were repurified by ion exchange and silica gel chromatographies. Radiopurity was determined by autoradiography of two-dimensional thin-layer chromatograms of the gangliosides. The method eliminates the need for extensive dialysis or repeated methanol evaporations to remove borate thus reducing time and the volume of labeled waste.

GM1b and GM1b-GalNAc: major gangliosides of murine-derived macrophage-like WEHI-3 cells.

WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)GM1b and (NeuAc)GM1b-GalNAc and the minor component as (NeuAc)GM1b-GalNAc-Gal. The presence of GM1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. GM1b-GalNAc and GM1b-GalNAc-Gal have been reported in T-lineage cells but not in resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoGM1 synthetic pathway. Preliminary results indicate a disialo member of this pathway, GDlc, may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.

The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages. Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse.

The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.

Altered B-lymphocyte membrane architecture indicated by ganglioside accessibility in C3H/HeJ mice.

We have analyzed both the total ganglioside composition and the surface accessibility of C3H/HeN B lymphocytes and C3H/HeJ B lymphocytes. Seventeen individual resorcinol-positive moieties were visualized by two-dimensional thin-layer chromatography of the purified gangliosides from both strains. Complete homology between strains was seen in the patterns of total gangliosides purified from the endotoxin-responsive and -hyporesponsive strains, with only minor differences in the relative concentrations of four gangliosides. In comparison, only 12 individual gangliosides were accessible to surface labeling following galactose oxidase treatment in these same strains, suggesting that some gangliosides are masked at the cell surface in both strains. However, labeling of the more polar components was greatly reduced in the endotoxin-hyporesponsive (C3H/HeJ) strain, suggesting that these gangliosides have decreased accessibility to galactose oxidase at the cell surface. Therefore, while the total ganglioside compositions of the two strains were nearly equivalent, there were dramatic differences in ganglioside surface accessibility. These findings indicate that an alteration in membrane structure that is associated with the endotoxin hyporesponsiveness observed in C3H/HeJ B lymphocytes exists.

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages.

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.

Comparison of thymocyte and T lymphocyte gangliosides from C3H/HeN and C3H/HeJ mice.

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28-30 discrete gangliosides of which eight represented major gangliosides. Splenic T lymphocytes from both strains had much simpler patterns, with six to seven major gangliosides and 12-13 minor gangliosides. Computerized analysis of the thymocyte ganglioside patterns between LPS-responder C3H/HeN mice and lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice revealed no significant difference in the major gangliosides. However, with splenic T cell gangliosides, there is a striking difference in the relative proportion of three homologous gangliosides between the two strains. Consistent with previous observations on macrophage gangliosides, the ratio of N-acetylneuraminic acid-containing ganglioside to N-glycolylneuraminic acid-containing ganglioside was higher in both thymocytes and T-cells from the LPS-responder strain. These results show that sialic acid-containing glycolipids from thymocytes and T lymphocytes between endotoxin responder and hyporesponder strains manifest small but significant changes. These differences are present in unstimulated cell populations and may represent a manifestation of the Lps gene.

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice.

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.

Further evidence for an intrinsic neuraminidase in CNS myelin.

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF-54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60 degrees C, the myelin neuraminidase activity was more than sevenfold of that observed at 37 degrees C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin-associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution proton NMR studies of gangliosides. III. Elucidation of the structure of ganglioside GM3 lactone.

Ganglioside GM3 lactone (1) was prepared in 95% yield from the parent ganglioside by incubation at 25 degrees C for 4 days in glacial acetic acid. Inspection of the 500 MHz proton NMR spectra of 1 and its precursor in dimethylsulfoxide-d6-deuterium oxide at 30 degrees C revealed a large deshielding (+1.42 ppm) of the H-2 resonance of the galactosyl residue. This suggests that 1 must be the lactone formed by esterification of the sialic acid carboxyl group with the C-2 hydroxyl of the galactosyl residue. Consideration of all the NMR data leads to a specific structure proposal in which 1 has a highly rigid structure. Interesting features of the structure include a hydrophobic inner surface and a semicircular outer edge of seven-oxygen atoms, which may have physiological importance.

Lack of specificity of brain gangliosides in the modulation of lymphocyte activation.

A potential role for glycolipid gangliosides to act as immunomodulating agents has been suggested. Most studies have employed brain gangliosides. We have systematically investigated highly purified murine brain gangliosides for their ability to modulate lymphocyte activation. All sialic acid classes of ganglioside inhibited lipopolysaccharide (LPS)-induced antibody secretion and all polysialated gangliosides inhibited LPS-induced DNA synthesis. Monosialated gangliosides had no effect on DNA synthesis induced by LPS. 8-BrcGMP-induced DNA synthesis was also inhibited, suggesting that a negative signal was delivered to B lymphocytes by co-cultivation with exogenous gangliosides. The lack of specificity with respect to sialic acid class observed in these studies suggests that further investigation of an immunomodulatory role for gangliosides focus on endogenous lymphocyte gangliosides.