Faculty Bibliography

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain

The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of 'hybrid' brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core

PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain

Hyaluronate is actively synthesized by cultured epidermis and dermis, but no direct histological data have been available about its localization in normal human skin. A hyaluronate-specific biotinylated probe, prepared from the hyaluronate binding region of cartilage proteoglycan, was applied to human skin sections and visualized using the biotin-avidin-peroxidase system. The specificity of this staining was confirmed by hyaluronidase predigestion and by hyaluronate-derived oligosaccharides added to the staining solution. All dermis showed diffuse binding of the probe, but the highest staining intensity was observed in the epidermal intercellular spaces. The stainability extended from basal cells to the middle layers of the epidermis, whereas the granular layer and stratum corneum were completely negative. Also, the basal side of basal cells (basement membrane) did not bind the hyaluronate probe. The abundance of hyaluronate on surfaces and intercellular spaces of the spinous cells is suggested to have an important role in the physiology of human epidermis

Poly(N-acetyllactosaminyl) oligosaccharides have been identified, on the basis of their susceptibility to endo-beta-galactosidase, in a large-molecular-size glycopeptide fraction derived from chromaffin granule membrane glycoproteins. The glycoproteins containing poly(N-acetyl-lactosaminyl) oligosaccharides were selectively labeled by treatment of chromaffin granule membranes with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography demonstrated specific labeling in the 41-47 kilodalton (kD) region and in a distinct band at 90 kDa. Two-dimensional SDS-PAGE revealed that the poly(N-acetyllactosaminyl) oligosaccharides are predominantly present in glycoprotein IV, together with lesser labeling of glycoproteins II and III, whereas they are absent from dopamine beta-hydroxylase and carboxypeptidase H, which are the major glycoproteins of chromaffin granule membranes

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)