Characterization of one neutral and two acidic glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU).

One neutral and two acidic glycoasparagines were isolated from the urine of patients with aspartylglycosylaminuria (AGU). The neutral one was identified as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn. The acidic ones were composed of 1 mole of sialic acid and 2 moles each of galactose and N-acetylglucosamine, attached to asparagine, and were isomeric with respect to the position of sialic acid attachment since they produced the same glycoasparagine on incubation with the neuraminidase [EC 3.2.1.18] from Clostridium perfringens. The structure of the resulting sialic acid-free glycoasparagine was determined to be beta-Gal-beta-GlcNAc-beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based mainly on the results of sequential enzymatic degradations.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Some studies on the composition of bovine cortical-bone sialoprotein

1. An analysis of bovine bone sialoprotein, a homogeneous glycoprotein isolated from cortical bone, is presented. 2. Analytical results agree with earlier physical measurements indicating a molecular weight of about 23000. 3. Mild acid hydrolysis and treatment with neuraminidase showed that fucose and sialic acid occupy terminal positions on oligosaccharide chains. 4. Treatment of the sialic acid-free glycoprotein with beta-galactosidase showed that much of the galactose occupies a sub-terminal location in the intact glycoprotein. 5. The polypeptide chain is rich in aspartic acid, glutamic acid, serine, threonine and glycine, and has no detectable free terminal amino group. 6. Glycopeptides were studied after proteolytic digestion. 7. It is considered that the carbohydrate moiety is highly branched and is probably linked by an acid- and alkali-stable glycosylamine bond involving aspartic acid.     

Glycoproteins from ascitic fluid of Ehrlich ascites tumor. Isolation and chemical characterization.

Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7-37.3% of the total weights of these glycoproteins.     

Studies on the glycopeptides of canine prothrombin]

After degradation of canine prothrombin by the complex of Streptomyces griseus proteases four glycopeptides were obtained. Each of them contained aspartic acid, hexosamines, mannose, galactose and sialic acids. Canine prothrombin contains two or three carbohydrate chanins, which are bound to aspartic (asparagine) residues. Microheterogenity of the carbohydrate chains of canine prothrombin was found.     

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [(35)S]sulphate, heparin is the major radioactive product. However, [1-(14)C]glucosamine and d-[(14)C]glucose are incorporated largely into the glycoprotein. 4. The fate of [(35)S]sulphate-labelled and [1-(14)C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t((1/2)) of [(35)S]sulphate-labelled material in cells is 70hr. and that of [1-(14)C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [(35)S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1-(14)C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [(14)C]sialic acid being concentrated in the microsomal fraction.     

Glycoproteins from ascitic fluid of Ehrlich ascites tumor: Isolation and chemical characterization

Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7–37.3% of the total weights of these glycoproteins.     

Structure of the carbohydrate chain of free secretory component from human milk.

Secretory component from human milk was found to contain 23.4% carbohydrate, which includes galactose, mannose, fucose, glucosamine, and sialic acid. Secretory component could be degraded by pronase or base-borohydride to yield the same, single type of carbohydrate chain. In the glycopeptide produced by pronase digestion, aspartic acid was the only amino acid present in molar quantities after amino acid analysis, which suggests that the carbohydrate moiety is linked to the polypeptide chain at asparagine residues. The positions of links between the various sugar units were studied by methylation analyses of: secretory component, periodate-oxidized and reduced secretory component, the fragment produced by base-borohydride treatment, and the pronase glycopeptide after treatment with specific glycosidases. Sugars released from the glycopeptide by various glycosidases were also quantitated. From the results of these studies a branched chain structure was assigned to the carbohydrate chain of secretory component.     

Evidence for sialic acid on the nicotinic acetylcholine receptor from Torpedo marmorata

The nicotinic acetylcholine receptor from Torpedo marmorata was treated with neuraminidase. Direct determination of sialic acid released gave about 1 mole sialic acid per mole receptor. Lectin binding studies of the sugars accessible on the receptor molecule were performed after sialic acid hydrolysis. They indicated that the terminal sialic acid is linked to a galactose residue.The present findings confirm the presence of one terminal sialic acid residue per receptor molecule.     

Content and accessibility of sialic acid on the surface of rat hepatocytes during primary culture

The content and accessibility of terminal sialic acid and galactose residues of rat hepatocytes in primary culture were determined by in situ labeling using either periodate or sialidase/galactose oxidase treatment followed by sodium borotritiide reduction. Rat erythrocytes which were used for comparison showed a strongly enhanced tritium incorporation into galactose after sialidase treatment. In contrast, with freshly prepared rat hepatocytes only a small amount of galactose labeling was achieved after sialidase treatment. The amount of galactose labeled following sialidase treatment increased with time in culture up to day 6 and roughly paralleled the increase of the total sialic acid content. Major changes of sialic acid-containing glycoconjugates were restricted to the gangliosides. There was a transient drop in surface labeling of ganglioside-associated sialic acid on the first day in culture. The specific radioactivity of the in situ-tritiated ganglioside-sialic acid also fell by 50% in this period. Between day 2 and 4, there was an increase in gangliosidesialic acid labeling but the specific radioactivity of the sialic acid remained constant. This indicates that newly synthesized gangliosides but not the preexisting ones were accessible to periodate oxidation. The data allow conclusions about turnover and topology of the sialic acid-containing glycolipids.     

Purification and chemical characterization of the major glycoprotein of avian myeloblastosis virus.

The major glycoprotein of avian myeloblastosis virus (AMV) has been purified to an apparent state of homogeneity by gel filtration on a Sepharose 4B column in the presence of 6 m guanidine hydrochloride followed by dialysis against distilled water and then extraction with chloroform-methanol. The AMV glycoprotein remains soluble in the aqueous phase whereas contaminating proteins precipitate, either upon dialysis against distilled water or after treatment with chloroform-methanol.Carbohydrate, represented by glucosamine, mannose, galactose, fucose, and sialic acid, constitutes 40% of the weight of AMV glycoprotein. Glucosamine is the major carbohydrate component whereas fucose and sialic acid are present in relatively low amount. Amino acid analysis indicates a relatively high content of aspartic and glutamic acid, serine, threonine, and glycine. Based on SDS-polyacrylamide gel electrophoresis, a molecular weight value of 77,500 ± 500 was determined for AMV glycoprotein.     

Structure of glycoproteins of human erythrocytes. Alkali-stable oligosaccharides

Studies have been made on the oligosaccharide residues of the alkali-stable carbohydrate-protein linkage of sialoglycopeptides derived from human erythrocytes. Four glycopeptides were isolated after alkaline borohydride treatment and Pronase digestion of MN-active sialoglycopeptides. The structure of one of these glycopeptides (GPIV) has been studied by sequential hydrolysis with specific glycosidases. Glycopeptide GPIV contained (per mol): 1mol of fucose, 1mol of sialic acid, 3mol of galactose, 3mol of mannose, 4mol of acetylglucosamine, 1mol of aspartic acid and fractional amounts of threonine, serine and glycine. The molecular weight of the glycopeptide was estimated to be 2330 by gel filtration. On the basis of glycosidase-digestion results, a tentative structure is proposed for the oligosaccharide moiety of glycopeptide GPIV.     

Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.     

Correlation of gastric mucosal damage with sialic acid profile in rats: effect of hydrochloric acid, pepsin and hypertonic saline

Sialic acids occupy terminal positions on gastric mucus glycoprotein where they contribute to the high viscosity of mucin. Desialylation of mucus may lead to degradation of the mucus and eventually to the breakdown of the gastric mucus barrier. The effect of a variety of damaging agents (0.1 M HCl, 2 mg ml(-1) pepsin and 2 M NaCl) on sialic acid profile was determined in pylorus-ligated rats. The relationship between sialic acid, galactose, pyruvate and the extent of gastric mucosal damage were studied. Instillation of pepsin significantly increased total sialic acid, galactose and macroscopic mucosal lesions in the stomach. Instillation of 0.1 M HCl reduced the total sialic acid but this decrease was not significant. Acidity led to a significant increase in the amount of free sialic acid in the gastric instillates and the macroscopic lesions induced by acid was not significantly different from the control animals (0.15 M NaCl). 2 M NaCl induced the macroscopic lesions in the stomach and also free sialic acid in the instillates. Pepsin potentiates the action of 2 M NaCl. In all the agents examined with the exception of acid, it was observed that an increase in free sialic acid and galactose was accompanied by gastric mucosal erosion and elevation of pyruvate concentration. It is concluded that gastric acidity alone is not inherently damaging and that resistance of gastric mucosa to destructive agents may be dependent on the integrity of the sialic acids.     

Carbohydrate composition of serum low and high density lipoproteins of nonhuman primate species.

1. Carbohydrate composition of serum low and high density lipoproteins obtained from 5 nonhuman primate species (chimpanzee, patas, baboon, rhesus, and spider) and humans was studied. 2. Individual lipoproteins were isolated from pooled sera of each species by ultracentrifugal flotation between the densities 1.019-1.063 for LDL-2; 1.063-1.12 for HDL-2; and 1.12-1.21 for HDL-3. After delipidation, sialic acid, fucose, glucosamine, mannose, galactose, and glucose were determined on apo LDL-2, apo HDL-2, and apo HDL-3. 3. Glucosamine, galactose, and mannose constituted a major component of the sugars in apo LDL-2, with similar relative proportions in all species. Sialic acid, fucose, and glucose formed a minor component, the proportions of which varied greatly among the species. 4. Unlike apo LDL-2, sialic acid, fucose, and glucosamine constituted the bulk of the sugars in apo HDL-2 and apo HDL-3. Mannose, galactose, and glucose were minor components, with galactose predominating. 5. Qualitative differences were observed in electrophoretic mobilities of apo HDL-2 and apo HDL-3 on polyacrylamide gel. One faster moving band was unique to chimpanzee. 6. Intraspecies differences in the content of sialic acid and fucose of apolipoproteins may be related to lipoprotein metabolism and species susceptibility (or resistance) to either spontaneous or diet-induced atherosclerosis.     

Characterization of ganglioside GM4 lactones isolated from the whale brain

Two novel ganglioside lactones were isolated from the brain of Bryde's whale (Balaenoptera edeni) and characterized. These gangliosides migrated above GM4 and were designated M4-1 and M4-2 in the order of location from the bottom of the TLC plate. These gangliosides were converted to GM4 by mild alkali treatment. Although M4-1 and M4-2 contained 1 mol each of N-acetylneuraminic acid and galactose, they behaved as neutral lipids on ion-exchange chromatography. Inner ester linkages were found in these gangliosides by secondary ion mass spectrometry and infrared spectroscopy. Two-dimensional J-correlated proton NMR spectra revealed that an inner ester linkage was formed in M4-1 between the sialic acid carboxyl group and the C-4 hydroxyl group of the galactosyl residue and another inner ester linkage was formed in M4-2 between the sialic acid carboxyl group and the C-2 hydroxyl group of galactose.     

Biosynthesis of the major brain gangliosides GD1a and GT1b

Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.     

The acid mucopolysaccharides of cattle retina

1. Two polysaccharides were isolated from the interstitial matrix surrounding the photoreceptor cells of cattle retina. They were liberated from this region of the tissue in a soluble form after agitation of whole retinas in 0.9% sodium chloride. One, which comprises two-thirds of the polysaccharides present, is a hyaluronidase-sensitive ;half-sulphated' chondroitin sulphate containing uronic acid, galactosamine and sulphate in the molar proportions 1.27:1.0:0.54. The other is a hyaluronidase-resistant non-sulphated heteropolysaccharide for which the name sialoglycan is proposed. It contains galactose, glucosamine and sialic acid in the molar proportions 2.4:1.0:0.4. Both polysaccharides contain only small amounts of nitrogen in excess of the amount calculated from their amino sugar and sialic acid content. 2. A similar combination of mucopolysaccharides is associated with the pigment epithelial-cell layer but in quantities only one-fifth of those present in the adjacent matrix area. 3. The ease with which they are released into aqueous media is consistent with the assumption that they are present in the extracellular spaces in both of these tissue layers. 4. The retinal residue left after removal of the two soluble polysaccharides is rich in amino sugar- and sialic acid-containing polymers, which appear to be firmly bound to the tissue fragments. 5. About one-third of the sialic acid and one-tenth of the amino sugar could be extracted with chloroform-methanol. The components in this fraction were tentatively identified as gangliosides. 6. Digestion of the chloroform-methanol-insoluble residue with Pronase yielded as the principal product a heteropolysaccharide containing 16.5% of glucosamine, 24.3% of neutral sugar (galactose plus fucose) and 18.1% of sialic acid. This substance has been classified as a sialoglycan of composition similar to (but not identical with) that of the soluble one isolated from the matrix area of the tissue.     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130.

Plasma membranes were isolated from an ascites hepatoma, AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared by digesting the membranes with pronase, then by fractionating the digest chromatographically and electrophoretically. Isolated fractions were analyzed for their amino acid and carbohydrate compositions. Results were compared with those for corresponding fractions from AH 66 (J. Biochem. 76, 319-333 (1974)). Mucopolysaccharides and a series of glycopeptides were isolated from the fraction excluded from Sephadex G-50. The mucopolysaccharides were identified as a family of heparan sulfates with different electrophoretic mobilities. The glycopeptides contained serine, threonine, galactose, galactosamine, glucosamine, and sialic acid as the major constituents as aspartic acid and mannose as minor ones. This suggests that most of the carbohydrate moieties are linked to serine or threonine (O-glycosidic), and that some are linked to asparagine (N-glycosidic). No nearly purely O-glycosidic glycopeptides were found in this fraction from AH 130, through they were the major glycopeptides from the AH 66 plasma membranes. In the fraction included in the gel, glycopeptides containing fucose, galactose, mannose, glucosamine, glaactosamine, and sialic acid were found. The presence of galactosamine suggests that some of the glycopeptides are O-glycosidic though most are N-glycosidic. In the corresponding fraction from AH 66, nearly purely N-glycosidic glycopeptides were found.