Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.     

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.     

Localization and imaging of gangliosides in mouse brain tissue sections by laserspray ionization inlet

A new ionization method for the analysis of fragile gangliosides without undesired fragmentation or salt adduction is presented. In laserspray ionization inlet (LSII), the matrix/analyte sample is ablated at atmospheric pressure, and ionization takes place in the ion transfer capillary of the mass spectrometer inlet by a process that is independent of a laser wavelength or voltage. The softness of LSII allows the identification of gangliosides up to GQ1 with negligible sialic acid loss. This is of importance to the field of MS imaging, as undesired fragmentation has made it difficult to accurately map the spatial distribution of fragile ganglioside lipids in tissue. Proof-of-principle structural characterization of endogenous gangliosides using MS(n) fragmentation of multiply charged negative ions on a LTQ Velos and subsequent imaging of the GD1 ganglioside is demonstrated. This is the first report of multiply charged negative ions using inlet ionization. We find that GD1 is detected at higher levels in the mouse cortex and hippocampus compared with the thalamus. In LSII with the laser aligned in transmission geometry relative to the inlet, images were obtained in approximately 60 min using an inexpensive nitrogen laser.     

Metabolism of cartilage proteins in cultured tissue sections.

The asparagine-linked oligosaccharides of the complex acidic-type from [3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined with extensive digestion with endo- and exo-glycosidases from bacterial and eukaryotic sources. The neutral products from the digestion with a mixture of exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral oligosaccharides that were all converted by digestion with eukaryotic beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small oligomannosyl core containing two alpha-linked mannose residues and a third mannose residue beta-linked to N-acetylglucosamine. These studies suggested that the complex acidic-type oligosaccharides from cellular and viral membrane glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of outer branch N-acetylglucosamine residues resulting in partial resistance to beta-N-acetylglucosaminidase from a bacterial source.     

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.     

The biosynthesis of gangliosides. Labelling of rat brain gangliosides in vivo

1. After injection of [6-(3)H]glucosamine into 8-day-old rats it was found that all the major brain gangliosides and their sialyl groups were labelled at essentially the same rate, except the hematoside, which was the least labelled. In 18-day-old rats it was found that the two major gangliosides with the sialyl (2-->8)-sialyl linkage, and their sialyl groups were more labelled than the hematoside, the Tay-Sachs ganglioside, the other two major gangliosides and their respective sialyl groups. 2. No difference was found in any of the cases studied between the specific radioactivities of the neuraminidase-resistant and -labile sialyl groups belonging to the same ganglioside. The same was found for the specific radioactivities of the galactosyl groups proximal and distal to the ceramide moiety of total brain gangliosides from rats injected with [U-(14)C]glucose. From this it was concluded that partial turnover of the ganglioside molecule does not occur. 3. A model for the synthesis of gangliosides is presented that accounts for results from previous experiments in vitro and the lack of precursor-product relationships observed in experiments in vivo.     

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature- dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine- labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.     

GPI-anchored proteins at the node of Ranvier

Contactin and TAG-1 are glycan phosphatidyl inositol (GPI)-anchored cell adhesion molecules that play a crucial role in the organization of axonal subdomains at the node of Ranvier of myelinating fibers. Contactin and TAG-1 mediate axo-glial selective interactions in association with Caspr-family molecules at paranodes and juxtaparanodes, respectively. How membrane proteins can be confined in these neighbouring domains along the axon has been the subject of intense investigations. This review will specifically examine the properties conferred by the lipid microenvironment to regulate trafficking and selective association of these axo-glial complexes. Increasing evidences from genetic and neuropathological models point to a role of lipid rafts in the formation or stabilization of the paranodal junctions.     

Mechanism of activation of acetylcholinesterase in brain tissue homogenates by detergents]

The mechanisms of activation of acetylcholinesterase (AChE) in the homogenates of rat and frog forebrains and medullae oblongatae under effects of non--ionic detergents (Triton X-100 and Tw-en 80) were studied. Titration of the active centers of enzyme by the highly selective phosphoorganic inhibitor HD-42 showed that the inhibitor-induced activation is due to the appearance of previously masked active sites of the enzyme. Activation of AChE in the homogenates of rat brain medulla oblongata was caused by lysis of the structural elements of nervous tissue which were not destroyed by homogenization. Another mechanism of AChE activation by the detergents (frog brain homogenate) is the destruction of the vesicular membrane structures present in the homogenate with AChE centers located on the inner surface of the vesicles.     

苯污染对大鼠脑组织环核苷酸与相关蛋白水平的影响

为了探索苯污染对人类健康损伤作用的因素,选用SPF级Wistar大鼠为实验对象,分为4组:低剂量组灌胃苯0.19 g·kg-1,中剂量组灌胃苯0.38 g·kg-1,高剂量组灌胃苯0.76 g·kg-1,对照组灌胃菜籽油2 mL·kg-1;用酶联免疫吸附法(ELISA)检测脑组织中环核苷酸和相关蛋白的水平.结果发现,连...     

Phylogenetic and regional variations in brain gangliosides of tetrapods.

1. Ganglioside patterns were analyzed from four neural tissues (medulla, midbrain, forebrain and retina) in a representative from each of the four tetrapod classes. 2. Regional variations in ganglioside patterns were noted within some species, but differences were greater across phylogenetic lines. 3. These results suggest that evolutionary history plays a greater role than neural differentiation in the expression of brain ganglioside patterns.     

Lipid remodeling of GPI-anchored proteins and its function

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.     

Studies on gangliosides of goldfish brain

Abstract— Purified gangliosides from goldfish brain have been separated on thin layer chromatography. Incorporation of radioactivity into gangliosides from intracranially- injected [³H]galactose was not affected by concentrations of puromycin (170 μg intra- cranially) known to exert behavioural effects. Puromycin (500 μg intracranially) inhibited the incorporation of radiolabelled galactose into infant rat brain gangliosides, a result which confirms a previous report in the literature.     

Differential sorting and fate of endocytosed GPI-anchored proteins

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI- APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway.     

Molecular profiling of experimental Parkinson's disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry

Direct molecular profiling of biological samples using matrix-assisted laser desorption ionization mass spectrometry is a powerful tool for identifying phenotypic markers. In this report, protein profiling was used for the first time to generate peptide and protein profiles of brain tissue sections obtained from experimental Parkinson's disease (unilaterally 6-hydroxydopamine treated rats). The mass spectrometer was used to map the peptide and protein expression directly on 12 microm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein expression profile differences were found in the dopamine depleted side of the brain when compared to the corresponding intact side, for example, calmodulin, cytochrome c, and cytochrome c oxidase. An increased ratio of post-translational modifications such as acetylations were found in the striatum of proteins in the dopamine depleted side of the brain. These modifications were decreased after subchronic administration of L-Dopa. The present study shows that unique protein profiles can be obtained in specific brain regions (and subregions) directly on brain tissue sections and allows for the study of complex biochemical processes such as those occurring in experimental Parkinson's disease.     

Human brain gangliosides in development, aging and disease.

In this study, brain gangliosides in prenatal and postnatal human life and Alzheimer's disease were analyzed. Immunohistochemically, the presence of the "c"-series of gangliosides (GQ1c) was only registered in the embryonic brain at 5 weeks of gestation. Biochemical results indicated a two-fold increase in ganglioside concentration in the human cortex between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except in the cerebellar cortex, which was characterized by increasing GT1b. During prenatal human development, regional differences in ganglioside composition could only be detected between the cerebrum ("a"-pathway) and the cerebellum ("b"-pathway). Between birth and 20-30 years of age, a cerebral neocortical difference of ganglioside composition occurred, characterized by the lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In the frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in the occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In the cerebellar cortex, GD1b and GT1b fractions decreased with aging. In Alzheimer's disease, we found all ganglio-series gangliosides (GM1, GD1a, GD1b, GT1b) to be decreased in regions (temporal and frontal cortex and nucleus basalis of Meynert) involved in pathogenesis of disease. In addition, in Alzheimer's disease we found simple gangliosides (GN2, GM3) to be elevated in the frontal and parietal cortex, which might correlate accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.