Richard U. Margolis

Neurobiology of glycoconjugates

1 Structure and Localization of Gangliosides.- 2 Biosynthesis, Metabolism, and Biological Effects of Gangliosides.- 3 Structure and Localization of Glycoproteins and Proteoglycans.- 4 Biosynthesis of Glycoproteins.- 5 Biosynthesis of Glycosaminoglycans and Proteoglycans.- 6 Lysosomal Degradation of Glycoproteins and Glycosaminoglycans.- 7 Glycoproteins of the Synapse.- 8 Glycoproteins of Myelin and Myelin-Forming Cells.- 9 Axonal Transport and Intracellular Sorting of Glycoconjugates.- 10 Synaptic Vesicle Glycoproteins and Proteoglycans.- 11 Carbohydrate Recognition, Cell Interactions, and Vertebrate Neural Development.- 12 Polysialic Acid as a Regulator of Cell Interactions.- 13 Extracellular Matrix Adhesive Glycoproteins and Their Receptors in the Nervous System.- 14 Hyaluronate and Hyaluronate-Binding Proteins of Brain.- 15 Inborn Errors of Complex Carbohydrate Catabolism.

Disposition of fucose in brain.

Abstract— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium‐labelled fucose into brain glyco‐proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half‐time for the turnover of glycoprotein‐fucose in young rats was approximately 2 weeks.

CARBOHYDRATE-PEPTIDE LINKAGES IN GLYCOPROTEINS AND MUCOPOLYSACCHARIDES FROM BRAIN

Abstract— Treatment of glycopeptides, prepared from glycoproteins of rat and rabbit brain, with NaOH‐NaBH4 leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, α‐aminobutyric acid and galactosaminitol. These results indicate that N‐acetylgalactosamine at the reducing end of oligosaccharide chains in brain glycoproteins is linked O‐glycosidically to the hydroxyl groups of serine and threonine residues. 2‐acetamido‐1‐(L‐β‐aspartamido)‐l,2‐dideoxy‐β‐D‐glucose was also detected after partial acid hydrolysis of the alkali‐stable glycopeptides, and most of the carbohydrate in brain glycoproteins appears to be linked by N‐acetylglucosaminylasparagine linkages. The results of the treatment of the sulphated mucopolysaccharides from bovine brain with alkaline‐borohydride indicate that the polysaccharide chains in chondroitin sulphate and heparan sulphate are linked exclusively to serine.

The hyaluronidase of brain.

Abstract— Hyaluronidase (hyaluronate glycanohydrolase, EC 3.2.1.35), with a pH optimum of 3.7, was detected in rat and bovine brain. It degraded hyaluronic acid and, at a slower rate, chondroitin sulphate to a mixture of higher oligosaccharides with N‐acetylhexosamine at the reducing end. The enzyme was enriched 5‐ and 6‐fold in a crude lysosomal fraction of rat brain or bovine cerebral cortex, and was further purified to a total enrichment of 9‐fold by ammonium sulphate fractionation. The enzyme activity in grey matter was more than twice that found in white matter, and there was no significant change in enzyme activity as a function of increasing age from the neonatal to the adult rat brain. The level of hyaluronidase activity in rat brain is considerably greaterthan that required to account for the rate of catabolism of hyaluronic acid and chondroitin sulphate measured in vivo.

Sulfate turnover in mucopolysaccharides and glycoproteins of brain

Abstract The turnover of sulfate in chondroitin sulfate, heparan sulfate, and the sulfated glycopeptides obtained from brain glycoproteins has been meaured in adult rat brain in vivo . The calculated half-time for the turnover of heparan sulfate was 3 days, and that of chondroitin sulfate was 1 week. The disappearance of radioactivity from both mucopolysaccharides followed a linear course for the entire time period of the experiment (18 days), indicating that only one major pool of each of these mucopolysaccharides was present in brain. 90% of the chondroitin sulfate in rat brain was identified as chondroitin 4-sulfate, and both chondroitin 4-sulfate and chondroitin 6-sulfate turned over at approximately the same rate. There was no difference in the turnover of the N - and O -sulfate groups in heparan sulfate. Sulfate-labeled glycoproteins demonstrated a biphasic disappearance of radioactivity, with half-times of 2.5 and 14 days. Evidence is presented which indicates that these two components represent pools of sulfated glycoproteins in brarin having distinctly different rates of metablic activity.

Release of Chromaffin Granule Glycoproteins and Proteoglycans from Potassium‐Stimulated PC12 Pheochromocytoma Cells

Abstract: Cultured PC12 pheochromocytoma cells were labeled with [3H]gIucosamine, 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 β‐hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(β l ± 3)N‐acetylgalactosamine, as well as several mono‐ and disialyl O‐glycosidically‐linked oligosaccharides, and the tetra‐saccharide AcNeu(α2 ± 3)Gal(β l ± 3)[AcNeu(α2 ± (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 triand 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.

Extractability of glycoproteins and mucopolysaccharides of brain.

Very little is known about the localization and functions of the glycoproteins and mucopolysaccharides of nervous tissue. There have been two major approaches to the study of these substances in brain. The first involves the isolation of glycopeptides and mucopolysaccharides after digestion of the lipid‐free protein residue from whole brain with proteolytic enzymes (Margolis, 1967; Di Benedettaet al., 1969; Margolis and Margolis, 1970; Katzman, 1972). This approach has the advantage that sufficient tissue is used to permit analysis of the structure and metabolism of the carbohydrate components of these macromolecules. However, any differentiation of the various glycoproteins and mucopolysaccharides based on such features as their anatomical location, association with proteins, lipids or other membrane components, and the properties conferred by their non‐carbohydrate portion, is unavoidably lost as a consequence of the procedures used for their isolation.

Dissociative extraction of brain proteoglycans

IT HAS been demonstrated that sulfated glycosaminoglycans (chondroitin sulfate and heparan sulfate) are present in brain as proteoglycans, in which the polysaccharide chains are covalently linked to serine residur; in the protein moiety (MARGOLIS er a/.. 1972). In an attempt to characterize these proteoglycans. BRANFORD WHITE & HUDSON (1977) have recently reported the isolation of a chondroitin sulfate proteoglycan from sheep brain using a dissociative extraction method. Briefly, the method involves extraction of a lipid-free residue of whole brain with 0.1 M-citrate buffer (pH 3.l), followed by extraction with 4 M-guanidine hydrochloride in 50 mM-Tris buffer (pH 7.5). Guanidine was then removed from this second extract by dialysis against distilled water, and a chondroitin sulfate proteoglycan was isolated from the precipitated protein by extraction with 0.1 M-citrate buffer, pH 5.0. The authors did not provide any quantitative data concerning the yield of proteoglycan in the final citrate extract (Fraction Bm). However, in the course of our own studies on the isolation and characterization of proteoglycans from brain (MARWLIS et at., 1976) we had evaluated a similar type of dissociative extraction procedure and found it to be unsatisfactory from the standpoints of both the yield and purity of the resulting product. On the basis of an encouraging report on their results by BRANFORD WHITE et al. (1976) and their later more detailed description of the isolated proteoglycans (BRANFORD WHITE & HUDSON, 1977) we have re-evaluated the possible utility of dissociative extraction techniques using the procedure described by these authors.

Metabolism of the protein moiety of brain glycoproteins

There were also rapid and slow components for the turnover of hexosamine in hyaluronic acid and heparan sulphate, but chondroitin sulphategavecvidencc on only a single metabolic pool having a half-time of 3 weeks (MARGOLIS & MARGOLIS, 1972b, 1973~). Other data indicated that the sulphate groups on chondroitin sulphate and in one fraction of heparan sulphate turn over an average of 3 times as rapidly as hexosamine in the polysaccharidc backbone, and that thc glycoproteins with a rapid turnovcr are prcsent mainly in the soluble fraction of brain (MARC~LIS & MARGOLIS, 1973~; MARGOLIS & GOMEZ, 1973). In order to obtain more information concerning the biochemical basis and possible physiological significance of this metabolic heterogeneity of complex carbohydrates in nervous tissue, we have studied the metabolism of the protein moiety of brain glycoproteins with the aid of labelled threonine. MARGOLIS, 1972b, 1973~; MARGOLIS & GOMEZ, 1973).