Faculty Bibliography

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans

In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development

Three monosialogangliosides are highly labeled when PC12 pheochromocytoma cells are cultured in the presence of L-[3H]fucose, and two additional monosialogangliosides are labeled to a lesser extent. In contrast, neither of the two disialogangliosides of PC12 cells contains fucose residues. Removal of sialic acid and fucose by formic acid hydrolysis demonstrated the presence of 3 major 'core' structures in the monosialogangliosides, and a single asialo derivative of the disialogangliosides which has the same chromatographic mobility as one of the monosialoganglioside hydrolysis products. None of the major formic acid hydrolysis products of the PC12 cell gangliosides corresponds to asialo-GM1, supporting our previous conclusion that PC12 cells do not contain significant amounts of brain-type gangliosides

Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix

We have examined the oligosaccharide structure of a major Mr = 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri-and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77-90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N-acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris, together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the alpha-linked mannose residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Large polysialosyl glycopeptides can be derived from the glycoproteins of both mature as well as early postnatal brain, although their concentration (in terms of sialic acid content) decreases by approximately 75 percent during the first month in rat brain. These glycopeptides were found to contain O-sulfate residues, and subcellular fractionation studies indicated that most of the glycoproteins having polysialosyl units are present in non-synaptic plasma membranes. Large polysialosyl glycopeptides can also be obtained from the membranes of PC12 pheochromocytoma cells, demonstrating that this type of structure is not unique to brain

Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans

The composition and biosynthesis of glycoproteins, proteoglycans, and gangliosides have been studied in a clonal line of rat pheochromocytoma (PC12) cells. Glycoproteins account for approximately 78% of the glucosamine-labeled complex carbohydrates found in the culture medium, together with 17% chondroitin sulfate and 5% heparan sulfate. 10% of the glycoproteins but less than 1% of the proteoglycans are released by trypsin treatment of the cells, whose complex carbohydrates are composed of 93% glycoproteins, 1.3% chondroitin sulfate, 3.4% heparan sulfate, and 2.6% of mono- and disialogangliosides. Sequential lectin affinity chromatography and alkali treatment of glycopeptides prepared from the medium, trypsin-releasable, membrane, and cell-soluble glycoproteins demonstrated that in all of the subfractions large tri- and tetraantennary complex oligosaccharides account for 82 to 97% of those present in PC12 cell glycoproteins. Biantennary oligosaccharides account for approximately 2-6% of those in medium and trypsinate, as compared to 10-13% in the membrane and cell soluble glycoproteins, and there were large differences (ranging from 7 to 60%) in the proportions of biantennary oligosaccharides which are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. High mannose oligosaccharides are present predominantly in the cell membrane and soluble glycoproteins, where they account for 4 to 5% of the total glycoprotein labeling. In response to nerve growth factor (NGF), the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. Significant alterations were also observed in the complex carbohydrates of NGF-treated cells, the most striking of which were an almost 3-fold increase in labeled gangliosides and a 75% increase in trypsin-releasable glycoproteins. Cellular heparan sulfate decreased by 70% in response to NGF and increased by an equivalent amount in the culture medium, whereas an NGF-induced increase in chondroitin sulfate labeling occurred specifically in the cell membranes

The chromogranins, a family of related acidic glycoproteins, and two chondroitin sulfate/dermatan sulfate proteoglycans were isolated from the soluble contents of bovine adrenal chromaffin granules by chromatography on DEAE-cellulose. These chromaffin granule matrix glycoconjugates were treated with trypsin, and the resulting peptides were fractionated by HPLC. The two proteoglycans, which differ in their concentration of glycosaminoglycans and glycoprotein oligosaccharides, yielded almost identical peptide patterns and would both appear to have the same protein moiety. The peptide profile of the proteoglycans differs, however, from that of the chromogranins, which they closely resemble in terms of amino acid composition. The various chromogranin fractions obtained by gel filtration were also found to have significant differences in the chromatographic patterns of their tryptic peptides