Expression of alpha 2-glycoprotein by glial precursor cells: an immunocytochemical study with glial cultures.

Studies on the presence of the brain-specific alpha 2-glycoprotein in cultures of newborn rat brain cells revealed that a population of glial precursor cells expressed this antigen at an early stage of development. This cell population consisted of small, phase-dark cells that proliferated in culture and occupied the surface of a layer of flat epithelial-like astrocytes. The latter cell type did not react with the antibodies. The number of alpha 2-glycoprotein positive cells gradually decreased from a high concentration of 88% of the total overlying cells at 6 days of culture to 44% at 23 days. The morphological heterogeneity of the overlying cells was noticeable after 10 days in culture as clusters of cells with elaborate processes started to develop. alpha 2-Glycoprotein was found to be concentrated in these structures. A glioma cell line (C-6 glia) which represents a unique in vitro model for the glial progenitor cells, was also found to express this glycoprotein antigen.

Synthesis of sulfated glycoproteins by glial precursor cells.

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [3H]glucosamine and [35S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [3H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [35S]sulfate into glycoproteins.

Preparation of astrocyte-deficient cultures containing neurons and oligodendroglia from embryonic rat cerebral hemispheres.

Complement-dependent anti-glial fibrillary acidic protein (GFAP) antibody-mediated cytotoxicity was utilized in order to prepare astrocyte-deficient cultures from mechanically dissociated cells from 18-day-old embryonic rat cerebral hemispheres. Neurons and a lesser number of oligodendroglia were the major cell components in such cultures.

Changes in glycoprotein carbohydrate content in the aging human brain.

There is no significant change in the concentration, per gram of fresh tissue, of the total carbohydrate associated with brain glycoproteins as the human brain ages from 25 to 85 years. Nevertheless, notable shifts in the concentration of varied types of oligosaccharides do occur. The concentration and percentage of total glycopeptide-carbohydrate recovered from the whole brain represented by the N-glycosidically linked complex sialooligosaccharides of the tri- and tetraantennary type show a marked decline with age. On the other hand, the N-linked mannose high oligosaccharides of the ovalbumin type, the N-linked biantennary sialoglycopeptides of the transferrin type, and the O-linked oligosaccharides show an increase as the brain ages. Other age related changes that were noted include an increase in the number of sulfate ester groups associated with the N-linked sialoglycopeptides and a decrease in the number of phosphorylated high mannose oligosaccharides as the brain ages.

Expression of hyaluronectin by C-6 glial cells at high density.

Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C-6 glial cells grown to high density. The density-dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2',3'-cyclic nucleotide-3'-phosphohydrolase, glutamine synthetase, S-100 protein, and glial fibrillary acidic protein.

Synthesis and release of sulfated glycoproteins by cultured glial cells.

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.

Failure of plasma membranes of astrocytes in primary culture to insert exogenous ganglioside GM1.

Primary cultures of astrocytes derived from newborn rat brain cerebral hemispheres did not incorporate ganglioside GM1 into their plasma membranes when cultured in a medium containing the added ganglioside. This finding suggests that the function and/or structure of the astrocyte cell membrane differs from that of the neuron and oligodendroglia cell.

Absence of ganglioside GM1 in astroglial cells from newborn rat brain.

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the "pure" astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of N-[ (3)H]acetylmannosammine appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.

Cellular localization of GM1-ganglioside with biotinylated choleragen and avidin peroxidase in primary cultured cells from rat brain.

A new technique capable of demonstrating the presence and cellular localization of the ganglioside GM1 in primary cultured cells from the brains of newborn rats is described. The method is based on the highly specific binding of biotinylated choleragen to ganglioside GM1, and takes advantage of the high affinity of avidin for biotin. Thus, the biotinylated choleragen-ganglioside GM1 complex can be visualized by the use of avidin peroxidase. The results of this nonimmunologic method indicate that the concentration of ganglioside GM1 is much lower in culture astroglial cells than in neurons and oligodendroglial cells.

Stereo- and regio-selectivity of diethylaminosulfur trifluoride as a fluorinating reagent for methyl glycosides.

Methyl glycopyranosides reacted with diethylaminosulfur trifluoride (DAST) in the absence of solvent to yield methyl dideoxy-difluoro and deoxy-fluoro glycopyranosides. Methyl alpha-D-glycopyranosides produced 6-deoxy-6-fluoro- and 4,6-dideoxy-4,6-difluoro derivatives with Walden inversion at C-4. Methyl beta-D-glucopyranoside also produced a 3,6-dideoxy-3,6-difluoro derivative, with Walden inversion at C-3. Methyl 6-O-trityl-alpha-D-glucopyranoside, reacted with DAST to yield the corresponding 4-deoxy-4-fluorogalactopyranoside derivative.

Structure of the mannose-rich oligosaccharide chains of concanavalin A-binding glycopeptides derived from beef brain glycoproteins.

A neutral, mannose-rich, concanavalin A (Con A)-binding glycopeptide fraction was obtained by proteolytic digestion of defatted beef brain tissue. Hydrazinolysis followed by gel filtration of the reaction products provided three oligosaccharides. A portion of each oligosaccharide was treated by exhaustive digestion with alpha-mannosidase. Another portion was subjected to selective acetolysis of Man alpha 1 leads to 6Man linkages, providing two fragments that were recovered by gel filtration. The structure of the intact oligosaccharides, as well as the fragments obtained by selective acetolysis and enzymatic treatment, were resolved by gas-liquid chromatographic-mass spectrometric analysis. The structures of the three oligosaccharides were: (a) Man alpha 1 leads to 2Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4-N-acetylglucosamine (GlcNAc)beta 1 leads to 4N-acetylglucosaminitol (GlcOLNAc); (b) Man alpha 1 leads to 2Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcOLNAc; and (c) Man alpha 1 leads to 6(Man alpha 1 leads to 3) Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc-beta 1 leads to 4GlcOLNAc. These structures account for 15-20% of the glycoprotein-carbohydrate of whole beef brain and most of the oligosaccharides that demonstrate a high affinity for Con A. In view of the large number of Con A-binding glycoproteins in brain tissue, it appears that many of these different glycoproteins must contain structurally identical oligosaccharides.

Absence of ganglioside GM1 in astroglial cells from 21-day old rat brain: immunohistochemical, histochemical, and biochemical studies.

A procedure was developed for the cultivation of cells derived from the cerebral hemispheres of the 21-day old rat. Approximately 98 percent of the cells in a 10 day culture are astrocytes that contain glial fibrillary acidic protein. Analysis of the extracted gangliosides by thin layer chromatography revealed that ganglioside GM1 was absent and that the predominant ganglioside was GM3. Very small amounts of the polysialogangliosides GD1a, GD1b, and GT1b were detected. The concentration of gangliosidic NeuNAc per mg protein in these astrocytes was only 3 percent that observed in the 5 day culture of a mixed cell preparation from newborn rat brain. Immunohistochemical and histochemical studies were performed on the mixed cell population of the minced tissue of 21-day old rat brain prior to cultivation. Astrocytes did not stain for hyaluronectin. These cells also did not provide a positive staining reaction for ganglioside GM1 utilizing the antiganglioside GM1 peroxidase-antiperoxidase procedure and the biotinylated choleragen-avidin-peroxidase procedure. These two histochemical methods for ganglioside GM1 also did not stain astrocytes that had been cultured for 5 days. Oligodendroglial cells, which were also present in the uncultured 21-day-old minced brain tissue, stained positively for ganglioside GM1 and hyaluronectin. Hyaluronectin had previously been shown to be a marker for oligodendroglia. Oligodendroglial cells which were present in the 5 day cultures of 21-day old brain tissue also provided a positive reaction for ganglioside GM1. It is concluded that ganglioside GM1 is absent in astroglia. The presence of small amounts of polysialogangliosides in the "pure" astrocyte preparation is discussed.

Localization of hyaluronectin in oligodendroglial cells.

Astroglia and oligodendroglia in primary cell cultures were identified by immunohistochemical staining with antiglial fibrillary acidic protein and anticerebroside antisera, respectively. The antiserum to hyaluronectin (HN) was utilized to show that this hyaluronic acid-binding glycoprotein was localized in the oligodendroglial cels. This is consistent with the recent finding that HN is localized in the node of Ranvier. Astroglia did not react with antihyaluronectin.

Changes in the concentration and composition of human brain gangliosides with aging.

Ganglioside content and composition were studied in whole brains from 9 neurologically normal male individuals ranging from 25 to 85 years in age. The content of ganglioside-bound sialic acid decreased from 1,070 to 380 micrograms/g fresh tissue at 85 years. Ten individual ganglioside fractions were identified on high-performance thin-layer chromatography, seven of which were quantified. With age, ganglioside composition shifted to a more polar pattern due to an increase in the relative concentration of the more polar fractions GQ1b, GT1b and GD1b and a decrease in GD1a and GM1. Except for GQ1b, the absolute concentration of all gangliosides decreased with age. All changes were more pronounced in younger ages. Results are discussed in relation to structural changes occurring in the aging brain, and the involvement of gangliosides is suggested.

The metabolism and structure of phosphoglycoproteins in rat brain.

Glycoproteins that contain phosphohexosyl groups were found to be present in the myelin- and synaptosomal-enriched fractions as well as in the microsomes of rat brain. The kinetics of flow of intraperitoneally injected [32P]phosphate suggests that the phosphate is enzymatically added in structures found in the microsomal fraction. The newly synthesized phosphoglycoproteins then appear in the soluble fraction of the synaptosomes and in the cytosol, prior to incorporation into the membranes of the synaptosomes and myelin. Phosphoglycopeptides recovered from the phosphoglycoprotein contain 3 Mannose units per N-acetylglucosamine residue; one of the mannose residues is phosphorylated. [13C]NMR studies indicate that the phosphoglycopeptides contain a chitobiose group and more than four sugar residues. Thus, the phosphomannoglycopeptides from rat brain contain an average of 2 N-acetylglucosamine, 6 mannose, and two phosphate moieties per oligosaccharide chain. Enzymatic treatment with alpha-mannosidase failed to remove the phosphomannose, although some mannose residues were released. Thus, the phosphorylated mannose is not removed by the glycosidase and terminal nonphosphorylated mannose residues are present in the oligosaccharide. The phosphate residues are removed by treatment with alkaline phosphatase.

Urinary glycoconjugates in schizophrenic patients.

The level of glycoconjugates excreted in the urine of five schizophrenic patients was compared to that excreted by six controls. Urinary samples were fractionated by means of gel filtration and anion exchange chromatography to yield (i) fraction I consisting of basic, neutral, or slightly acidic glycopeptides and/or oligosaccharides, (ii) fraction II consisting of acidic glycopeptides and/or oligosaccharides, and (iii) fraction III consisting of glycosaminoglycans (acidic mucopolysaccharides). The hexose levels of fraction II (p less than 0.05) and uronate levels of fraction III (p less than 0.025) were significantly reduced in schizophrenic patients. The ratio of galactose/mannose in the glycoconjugates of fraction II was lower than normal in the urine from schizophrenic patients. Significantly (p less than 0.05) lower levels of rhamnose and higher levels of fucose were found in the glycoconjugates of fraction I from schizophrenic patients. Contrary to a previous report, we found no evidence for the presence of an abnormally elevated rhamnose-containing glycoprotein or glycoconjugate in fraction II. I appears that the pattern of metabolism of glycoproteins and glycosaminoglycans in schizophrenic patients deviates from the normal.