Baiba K. Gillard

A solvent partition method for microscale ganglioside purification

A simple and rapid method for the purification of gangliosides from the total lipid extract of plasma, cells, or tissue is described. The novel component of the method is the partition of the dried total lipid extract in the three-component solvent system consisting of diisopropyl ether, 1-butanol, and 50 mM aqueous NaCl (6/4/5, v/v/v). Gangliosides partition nearly quantitatively into the lower aqueous phase, and other lipids into the upper organic phase, resulting from the mixture of these three solvents. The ganglioside-containing aqueous phase is then freed of salts and other low-molecular-weight impurities by gel filtration. The thin-layer chromatographic patterns of total gangliosides thus obtained are clear and distinct, even when small samples with very low ganglioside concentrations (e.g., 1-ml samples of plasma) are processed by this method. Thus, this new ganglioside purification method is especially applicable to comparative qualitative studies of gangliosides requiring the analysis of multiple small samples.

Modulation of the immune response by gangliosides. Inhibition of adherent monocyte accessory function in vitro.

Gangliosides are potent inhibitors of lymphoproliferative responses. Selectively greater inhibitory effects of gangliosides on antigen-induced (vs. mitogen-induced) proliferation have been documented; e.g., 50 nmol of highly purified bovine brain gangliosides (BBG)/ml caused greater than or equal to 87% inhibition of proliferative responses of human peripheral blood mononuclear cells (PBMC) to three soluble specific antigens (Candida, streptokinase-streptodornase, and tetanus toxoid) vs. less than or equal to 37% inhibition of responses to three nonspecific mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen). The possibility that BBG interfere with adherent monocyte accessory function, upon which responses to soluble specific antigens are strictly dependent, was therefore considered. PBMC were separated into the adherent and nonadherent subpopulations, exposed to BBG, recombined, and their proliferative responses were measured. Unseparated PBMC preincubated for 48-72 h with 100 nmol BBG/ml and then washed to remove unbound BBG exhibited 73-76% inhibition of subsequent antigen-induced lymphoproliferation. Separate pretreatment of both adherent and nonadherent cell subpopulations in BBG under the same conditions resulted in similar (72-82%) inhibition, which was reproduced by preincubation of only the adherent cells in BBG. Preincubation of only the nonadherent cells in BBG was not inhibitory. Inhibition (a) was independent of whether gangliosides were added in solution or incorporated into liposomes, (b) was abrogated by adding untreated monocytes to cultures containing adherent cells that were preexposed to BBG (excluding the possibility that BBG was inducing suppression mediated by adherent cells), and (c) was reversible by further incubation of BBG-pretreated adherent cells in control medium. Together, these results delineate a mechanism by which gangliosides modulate lymphoproliferative responses--direct, noncytotoxic, and ultimately reversible inhibition of the accessory function of adherent monocytes.

Measurement and significance of antibodies against GM1 ganglioside Report of a workshop, 18 April 1989, Chicago, IL, U.S.A.

Twelve laboratories from the United States, Canada, France, Italy and Switzerland participated in a workshop to compare assays used to measure anti-GM1 antibodies, and to discuss the clinical significance of these antibodies. A panel of test samples containing varying amounts of anti-GM1 antibody was prepared by mixing varied proportions of normal serum with a serum containing a monoclonal IgM antibody that bound GM1 ganglioside. Enzyme-linked immunosorbent assay (ELISA) data were supplied by eight laboratories and ten laboratories classified the sera as negative, weakly or strongly positive. Most laboratories correctly identified the two samples that contained the highest quantities of antibody, but there was considerable disagreement on the classification of the three samples with moderate or small amounts of antibody. The sensitivity of the assays varied considerably. The more sensitive assays did not use detergent in the washing buffers, and incubated the human serum with the antigen at 4 degrees C overnight. Several investigators have identified a subset of patients with lower motor neuron disease or multifocal neuropathy who have high titers of anti-GM1 antibodies. Many patients with neurological and non-neurological diseases have low to moderate levels of anti-GM1 antibodies, and the significance of these antibodies is unclear. There was general agreement that standardization of the ELISA assays is urgently required, and that distribution of a reference high-titered antiserum would facilitate this process.

Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside GM3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and GM3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and GM3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-gamma decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb3, nLc4, IV2FucnLc4, and IV3NeuAcnLc4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.

Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A2

Small, dense, electronegative low density lipoprotein [LDL(−)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(−) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(−) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(−) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(−). In contrast, lipoprotein-associated phospholipase A2 (LpPLA2) is highly enriched only in small, dense LDL(−). The association of LpPLA2 with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core.

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb3, and Gb4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB3H4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were GM3 and IV3NeuAcnLc4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.

Isolation and purification of gangliosides from plasma.

Publisher Summary The high degree of structural diversity of this class of lipids and alterations in the cellular ganglioside complement associated with differentiation and with the neoplastic state have suggested that particular gangliosides may be markers for certain tumors. The degree of purification necessary to resolve individual plasma gangliosides by thin-layer chromatography has, using available methodology, required larger volumes of plasma and sometimes is associated with significant losses during purification. The method described in this chapter was originally developed to address the specific problem of the purification of gangliosides from small volumes of biological fluids in which the ganglioside concentration is low. The procedure results in highly resolved ganglioside thin-layer chromatographic patterns using small (l.0-ml) samples of human plasma. The first step of the overall three-step-procedure is total lipid extraction of the plasma sample. Next, gangliosides are separated from most other lipids, and then the lower aqueous phase that results from this partition and which contains the gangliosides is purified by gel filtration to remove low molecular weight contaminants, both inorganic (salts) and organic (polypeptides). The gangliosides then can be quantitated and qualitatively analyzed by thin-layer chromatography. The chapter discusses the applications of the partition method for ganglioside isolation and purification.

Apolipoproteins A-I, A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.

Carbohydrate tolerance in cystic fibrosis is closely linked to pancreatic exocrine function.

ABSTRACT. We evaluated carbohydrate tolerance in nine thin cystic fibrosis (CF) patients and in six controls, measuring responsiveness to the following insulinotropic secretagogues: oral glucose, IV glucose, and IV tolbutamide. Glucose responses segregated patients into two groups: Group I with normal carbohydrate tolerance associated with normal to slightly increased insulin responses, and Group II with impaired carbohydrate tolerance associated with insulinopenia. This latter group included one patient with frank diabetes. The CF patients demonstrated a significant positive correlation between insulin secretion, in response to each secretagogue, and pancreatic exocrine function as measured by serum pancreatic amylase isoenzyme concentration. Pancreatic a-cell function, as reflected by basal plasma glucagon concentrations, also correlated well with exocrine function in the CF patients, excluding the diabetic individual. The enteroinsular axis of the CF group was intact as reflected by normal plasma gastric inhibitory polypeptide concentrations in Group I and by elevated levels, basally and in response to oral glucose, in the insulinopenic Group II patients. Furthermore, those patients with impaired tolerance demonstrated a greater magnitude of insulinopenia compared to controls following IV glucose and possibly IV tolbutamide, than following oral glucose.Thus, these data suggest that loss of carbohydrate tolerance in patients with CF, like that seen with classical chronic pancreatitis, 1) parallels the loss of exocrine function, 2) is associated with appropriate enteroinsular signaling, and 3) can be detected earlier or more easily following testing with direct IV secretagogues than following oral glucose stimulation.

Interferon-γ alters expression of endothelial cell-surface glycosphingolipids☆

Abstract Our previous work on human endothelial cell (EC) glycosphingolipids (GSLs) demonstrated that these cells contain a large diversity of GSLs, predominantly with lacto core structures. In order to evaluate the role of GSLs as EC antigens and receptors, we investigated their cell-surface expression on confluent EC monolayers and ECs activated by interferon-γ (IFN-γ) and interleukin-1 (IL-1). IFN-γ activation of endothelial cells resulted in a small change in GSL composition, but greatly increased surface expression of gangliosides and decreased surface expression of neutral GSLs. In particular, surface expression of the major neutral GSL, globoside, decreased three- to fourfold as measured both by galactose oxidase labeling and by binding of the anti-globoside monoclonal antibody 9G7. IFN-γ did not significantly alter the total cell content of globoside, as measured by metabolic labeling, but rather altered the ratio of accessible cell surface to intracellular globoside. Two mechanisms appear to contribute to the decreased cell-surface globoside expression. IFN-γ treatment increased the relative proportion of intracellular globoside which is associated with the cell cytoskeleton. IFN-γ treatment also caused more of the cell-surface globoside to be inaccessible to antibody, and both neuraminidase and trypsin treatment of the cells increased globoside accessibility. IL-1 treatment increased total cell GSL content, but did not alter GSL composition or cell-surface binding by six anti-carbohydrate antibodies. The specific modulation of cell-surface GSLs by IFN-γ suggests that GSLs may play a role in the altered adhesive and receptor activities of IFN-γ-activated ECS.