Herbert C. Yohe

Incorporation of N-acetylmannosamine into rat brain subcellular gangliosides: Effect of pentylenetetrazol-induced convulsions on brain gangliosides

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N‐acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides GT1b and GQ1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, GT1b and GQ1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane‐bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water‐soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin‐layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin‐layer chromatogram.

Ganglioside alterations in stimulated murine macrophages

A two-dimensional thin-layer chromatographic technique has been used to separate and display gangliosides from murine peritoneal macrophages in different functional states. Resident macrophages have a relatively simple ganglioside pattern with about 15 resorcinol-positive spots. Gangliosides from resident cells contained mostly (90%) N-glycolylneuraminic acid. Thioglycolate-elicited and Corynebacterium parvum-activated macrophages have much more complex patterns with about 40 resorcinol-positive spots. Although ganglioside sialic acid content of stimulated macrophages was only slightly higher than that of resident cells, it consisted of nearly equal amounts of N-acetyl- and N-glycolylneuraminic acid. The shift in the ganglioside sialic acid type and the expression of different gangliosides in macrophages upon stimulation may help explain some of the differences in function and responsiveness noted in these macrophage populations.

Further Evidence for an Intrinsic Neuraminidase in CNS Myelin

Abstract: 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‐associ‐ated 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°C, the myelin neuraminidase activity was more than sevenfold of that observed at 37°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. Developmentally, the myelin‐associated neuraminidase activity in the cerebral hemispheres was also the highest at 20–30 days of age and declined to adult levels soon after. The combined evidence strongly indicates the presence of an intrinsic neuraminidase for gangliosides in CNS myelin and this neuraminidase may play an important role in determining the unique ganglioside pattern in the myelin sheath.

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.

Characterization of some minor gangliosides in Tay—Sachs brains

Abstract The ganglioside distribution of Tay—Sachs brain was re-examined in detail. In both the gray and white matter, the levels of lipid-bound sialic acid were increased 6- and 10-fold, respectively, over normal infant brain, and approximately 90% of the total ganglioside was G M2 . The level of G M2 was increased about 90 times in gray matter and 220 times in white matter in comparison with that in normal controls. The level of G D1a -GaINAc was increased 19 times and 10 times in gray and white matter, respectively. The concentration of G D2 was increased about 4-fold in Tay-S-Sachs white matter. In addition, the G M3 level was increased 2.7 and 3.5 times and the G D3 level 2 and 2.4 times over normal gray and white matter, respectively. However, the levels of other complex gangliosides such as G M1 , G D1a , G D1b , G T1b and G Q1b decreased remarkably. Since G M2 , G D2 , G D1a -GalNAc and a recently characterized ganglioside G M1b -GalNAc possess a common N-acetylgalactosaminyl terminal structure, their accumulation in Tay—Sachs brains is therefore consistent with the known hexosaminidase A deficiency. However, the accumulation of hexosamine-free G M3 and G D3 is not. The in vitro incorporation of N-acetylgalactosamine into G M3 to form G M2 was examined in a rat brain microsomal fraction in the presence of large amounts of other glycolipids. Acidic glycolipids were slightly stimulating and then became increasingly inhibitory when the molar ratio of lipid to substrate G M3 exceeded 10 to 1. Neutral glycolipids and the phospholipid, phosphatidylcholine, were inhibitory at all levels tested. The data suggest that the accumulation of G M3 and G D3 in Tay—Sachs brains could be due to an inhibition of N-acetylgalactosaminyl-transferas by high levels of glycolipids, and the inhibition is not due to chelation of the obligate divalent cation necessary for the activity of this enzyme. The inhibition of this enzyme may also be responsible for the decreased levels of other complex gangliosides.

Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I).

LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAcalpha2-3Galbeta1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAcalpha2-3Galbeta1-3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a, respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside-protein interactions.

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 μg/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 γ (IFN‐γ)‐primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN‐γ‐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‐γ 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.

Identification of the sialosyl-sialosyl linkage in biosynthesized gangliosides

Abstract A technique was developed to examine the sialosyl-sialosyl linkage in the minute quantities of complex gangliosides biosynthesized in vitro. The disialoganglioside, GD3 1 , and the trisialoganglioside, GT1a, were biosynthesized as secondary products by the reaction of CMP-(4-14C)sialic acid with the glycolipid substrates, lactosyl ceramide and GM1 ganglioside, respectively. Following periodate oxidation-borohydride reduction, the intact sialic acid residue, and the susceptible sialic acid residue converted into an N-acetylheptulosaminic acid residue, were released by hydrolysis, esterified, separated, and analyzed for radioactivity. Analysis of the biosynthesized GD3 and GT1a gangliosides indicated radioactivity in both the susceptible and the nonsusceptible sialic acid residues, demonstrating that a sialosyl-(2→8)-sialosyl linkage had been synthesized. When the primary products (GM3 and GD1a, respectively) of the synthesis just described were examined, radioactivity was detected, as expected, only in the susceptible sialic acid residue. Because the N-acetylneuraminic acid and N-acetylheptulosaminic acid derivatives formed in the reaction were identified by chromatography, the standards used for comparison were verified by g.l.c.-m.s.

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.