A new extended globoglycolipid carrying the stage specific embryonic antigen-1 (SSEA-1) determinant in mouse kidney

Two neutral glycolipids carrying the stage specific embryonic antigen-1 (SSEA-1) and SSEA-3 determinants, respectively, were purified from mouse kidney by a combination of column chromatographies and droplet counter-current chromatography. The structures of the glycolipids (GL-X and GL-Y) were determined by means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombardment mass spectrometry, a methylation study, and sequential degradation. GL-X was demonstrated to be galactosyl beta 1-3globotetraosylceramide, the structure of which had already been characterized to be that of SSEA-3 by Kannagi et al. [1983) J. Biol. Chem. 258, 8934-8942). GL-Y was a new glycolipid containing fucose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine in a molar ratio of 1:4:1:1:1. The methylation study results indicated that it contained 3 mol of terminal sugars composed of 2 mol of galactose and 1 mol of fucose with two branching points at N-acetylgalactosamine and N-acetylglucosamine. From the data obtained by 1H-NMR spectroscopy, mass spectrometry, and a binding assay using an anti-SSEA-1 monoclonal antibody (PM81) cloned by Ball et al. [1983) J. Immunol. 130, 2937-2941), we propose the structure of GL-Y to be Gal beta 1-4GlcNAc beta 1-6GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-ceramide. (sequence; see text) Fuc alpha 1 Gal beta 1 This is the first report on the isolation and characterization of a glycolipid carrying the SSEA-1 determinant on its globo-core structure.     

Characterization of a major neutral glycolipid in PC12 cells as III3Gal alpha-globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase

Neutral glycolipids in PC12 cells were examined. A major neutral glycosphingolipid, isolated from a chloroform/methanol extract of the cells, was found to contain only galactose and glucose at a ratio of 3:1 and identified as ceramide tetrahexoside by fast atom bombardment (FAB) mass spectrometry. Its saccharide sequence was determined by a new method developed here using endoglycoceramidase (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). The glycosphingolipid was digested with endoglycoceramidase to produce oligosaccharide which was subsequently pyridylaminated. The fluorescence-labeled oligosaccharide was digested with a series of specific exoglycosidases and fractionated by high performance liquid chromatography. The 2-aminopyridyl oligosaccharide was hydrolyzed by alpha-galactosidase to give a 2-aminopyridyl oligosaccharide which was identified as 2-aminopyridyl lactose by high performance liquid chromatography, indicating the glycolipid structure to be Gal alpha Gal alpha Gal beta GlcCer. Ceramide trihexoside obtained by limited digestion of the intact glycolipid was clearly identical with ceramide trihexoside obtained from human erythrocytes, according to NMR spectroscopy and methylation analysis. From these and other data on the intact glycolipid, obtained by methylation analysis and NMR spectroscopy, its structure was confirmed as Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, III3-Gal alpha-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Biosynthesis of the cancer-associated sialyl-Lea antigen

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.     

Detection of bisected biantennary form in the asparagine-linked oligosaccharides of fibronectin isolated from human term amniotic fluid

Fibronectin purified from human term amniotic fluid contains 10 asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction and fractionated by anion-exchange column chromatography and serial lectin affinity chromatography. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bisected and non-bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc beta 1----, GlcNAc beta 1----, Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.     

Identification of UDP-galactose: lactose (lactosylceramide) alpha-4 and beta-3 galactosyltransferases in human kidney

Two galactosyltransferases were identified in human kidney microsomes which both transfer galactose from UDP Gal to lactose as well as to lactosylceramide. Using a solubilized and a partially purified enzyme preparation sufficient product could be obtained for detailed structural analysis. The trisaccharide products were isolated by gel permeation chromatography and separated by preparative high performance thin layer chromatography. The anomeric configuration of the transferred galactose was determined by specific glycosidase digestion and the linkage was identified by methylation and gas-liquid-chromatography. The glycolipid products were not separated but analyzed directly, before and after alpha or beta galactosidase digestion, by methylation, hydrolysis and thin layer chromatography. Into both acceptor substrates galactose was incorporated in alpha 1-4 (30%) and beta 1-3 (70%) linkages. The alpha 1-4 galactosyltransferase is responsible for the synthesis of the Pk antigen Gal alpha 1-4 Gal beta 1-4 Glc-ceramide in human kidney. The beta 1-3 galactosyltransferase has not previously been identified.     

An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens.

The erythrocyte PK and P blood group antigens have been identified as ceramide trihexoside (CTH), Gal-(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, and globoside, GalN-Ac(beta, 1 leads to 3)Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, respectively, and the following structure has been proposed for the P1 antigen: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)GlcNAc(beta, 1 leads to 3)Gal(beta, 1 leads to 4)Glc-Cer. Although the P1 and PK determinants have identical terminal disaccharides, CTH did not inhibit anti-P1. The P1 glycolipid and hydatid cyst glycoprotein inhibited the agglutination of P1K erythrocytes by anti-P1 and unabsorbed anti-P1PPK sera, but neither antigen inhibited a specific anti-PK serum. The P1 and PK glycolipids were equally effective in inhibiting the hemagglutinating activity of a lectin with alpha-galactosyl specificity obtained from ova of Salmo trutta. Anti-P sera were inhibited most effectively by human erythrocyte globoside, and to a lesser extent by Forssman glycolipid and rat kidney globoside. In the latter glycolipid the linkage between the internal galactosyl residues is alpha, 1 leads to 3, rather than alpha, 1 leads to 4, as in erythrocyte globoside. No cross-reactions between P and P1 or PK antigens were detected. New hypotheses are offered to explain the genetic regulation and biosynthesis of the P1, P, and PK antigens.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk beta-N-acetylglucosaminide beta 1 leads to 4-galactosyltransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by beta-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is Gal beta 1 leads to 3([14C]Gal beta 1 leads to 4GlcNAc beta 1 leads to 6)GalNAcol. Thus it appears that Gal beta 1 leads to 3(GlcNAc beta 1 leads to 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of alpha-N-acetylgalactosaminylprotein beta 1 leads to 3-galactosyltransferase activity using ovine submaxillary asialomucin as an acceptor can be counteracted by the addition of N-acetylglucosamine.     

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.     

Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase

The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.     

Isolation and characterization of a novel 2-aminoethylphosphonyl-glycosphingolipid from the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I' containing 1 mol of 2-aminoethylphosphonate residue was isolated from the skin of Aplysia kurodai using two silicic acid chromatography systems. Data obtained on methanolysis, permethylation, mild acid hydrolysis, and hydrogen fluoride treatment combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry showed that this glycolipid was 3-O-MeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 1----1Ceramide. Palmitic acid, octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine are its major aliphatic components. The new glycolipid has essentially the same structure as another major phosphonoglycosphingolipid in the skin of Aplysia, SGL-II, that contains 2 mol of 2-aminoethylphosphonate residue, suggesting a metabolic relationship between the two.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

An antigen present in rat adenocarcinoma and normal colon non-epithelial stroma is a novel Forssman-like glycolipid based on isoglobotetraosylceramide

A glycolipid with blood group A activity detected in the non-epithelial stroma of normal rat colon but not in epithelial cells (Hansson, G.C., Karlsson, K.-A., and Thurin, J. (1984) Biochim. Biophys. Acta 792, 281-292), was purified to homogeneity from normal rat colon and rat colon adenocarcinoma. Mass spectrometry and 1H-NMR spectroscopy of the intact permethylated derivative and gas chromatography after degradation revealed the structure GalNAc alpha 1----3GAINAc beta 1----3Gal alpha 1----3Gal beta 1----4Glc beta 1----1Cer, with the predominant ceramide containing sphingosine and non-hydroxylated 24:0 fatty acid. This identifies this glycolipid as a novel Forssman-like glycolipid, which is a tumor-associated antigen by definition, since it is not present in the normal rat large intestinal epithelium cells but in rat adenocarcinoma derived from these cells.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.     

Glycosphingolipids of porcine pancreas

Neutral and acidic glycosphingolipids were purified from porcine pancreas by chromatography on columns of DEAE-Sephadex and Iatrobeads. The chemical structures of the purified glycolipids were determined by carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, NMR and IR. The major glycolipid of porcine pancreas was Gal(alpha,1-4)Gal(beta,1-)ceramide. Gangliosides GM3 and GD3 were major acidic components and galactosylceramide 3-sulfate was also found.