Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Purification and characterization of the epitectin from human laryngeal carcinoma cells

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

Structural characterization of a blood group A heptaglycosylceramide with globo-series structure. The major glycolipid based blood group A antigen of human kidney

A blood group A glycosphingolipid with the globo-series structure has been isolated from human kidney and structurally characterized. The structure was shown by mass spectrometry and proton NMR spectroscopy of the intact permethylated and permethylated-reduced derivatives together with degradation studies to be, GalNAc alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 Ceramide. This glycolipid reacts with both polyclonal and monoclonal anti-A blood group typing antisera and it is the major glycolipid based blood group A antigen present in the human kidney.     

Immunochemical studies on blood groups. Purification and characterization of radioactive 3H-reduced di- to hexasaccharides produced by alkaline beta-elimination-borohydride 3H reduction of Smith degraded blood group A active glycoproteins

Treatment of blood group A active glycoprotein from human ovarian cyst fluid by one stage of Smith degradation followed by alkaline beta-elimination in the presence of NaB[ 3H4 ] (Carlson degradation) liberated tritiated oligosaccharide alditols. The carbohydrate mixture was fractionated by gel filtration, elution from charcoal, paper chromatography, and high pressure liquid chromatography. Structures were established based on sugar composition, periodate oxidation, methylation analysis, and analysis of oligosaccharide alditols as permethylated and N-trifluoroacetylated derivatives by gas-liquid chromatography-mass spectrometry. The following structures have been deduced: Gal beta 1----3GalNAc-ol, GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1---- 3GlcNAc beta 1----6(3-deoxy)GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1----3[GlcNAc beta 1----6]GalNAc-ol, Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3[Gal beta 1----4GlcNAc beta 1----6]Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol. The smaller structures represent pieces of the larger structures. Together they provide direct evidence for the core structure of the carbohydrate side chains in the blood group substances as proposed by K. O. Lloyd and E. A. Kabat [1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1470-1477). Oligosaccharides previously isolated after Carlson degradation of intact human ovarian cyst fluid HLeb , Lea, and B substances and from human and horse B substances contained various alpha-linked L- fucopyranose and alpha-linked Gal substitutions on the composite structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Structure of a triphosphonopentaosylceramide containing 4-O-methyl-N-acetylglucosamine from the skin of the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I containing 3 mol of 2-aminoethylphosphonate residues was isolated from the skin of a sea gastropod, Aplysia kurodai. The saccharide moiety of the glycolipid was characterized as 4-O-methyl-GlcNAc alpha 1----4GalNAc alpha-1----3 [6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6)Gal beta 1----4(2-aminoethylphosphonyl----6) Glc beta 1----1-ceramide. The major aliphatic components of the ceramide portion were palmitic acid, stearic acid, octadeca-4-sphingenine, and anteisononadeca-4-sphingenine. This glycolipid is unique in containing 4-O-methyl-N-acetylglucosamine and 3 mol of 2-aminoethylphosphonate residues, one of which is attached to C-6 of glucose.     

Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Gal beta 1----4Glc-Cer (lactosylceramide) alpha-2----3sialyltransferase; Sat-1), and CMP-NeuAc:Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4Glc-Cer (GM1 ganglioside) alpha 2----3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of cis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments.     

Enzymatic basis for the accumulation of glycolipids with X and dimeric X determinants in human lung cancer cells (NCI-H69)

Many human carcinomas accumulate a large quantity of glycolipids having X (Gal beta 1----4[Fuc alpha 1----3] GlcNAc) as well as di- or trimeric X determinant (Gal beta 1----4 [Fuc alpha 1----3] GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal) (e.g. Hakomori, S., Nudelman, E., Levery, S. B., and Kannagi, R. (1984) J. Biol. Chem. 259, 4672-4680). The enzymatic basis of this phenomenon has been investigated with human small cell lung carcinoma NCI-H69 cells, in which a series of these structures has been found to accumulate. An alpha 1----3 fucosyltransferase solubilized from the membrane fraction with Triton X-100 catalyzed not only the transfer of a fucosyl residue from GDP-fucose to the penultimate GlcNAc residue of lactoneotetraosylceramide (nLc4) and lactonorhexaosylceramide (nLc6), but also to the internal GlcNAc residue (III-GlcNAc) of y2 glycolipid (V3FucnLc6) and that of sialosyl2----6lactonorhexaosylceramide (VI6NeuAcnLc6). No transfer of fucose to the internal GlcNAc (III-GlcNAc) of lactonorhexaosylceramide occurred, unless the above substitutions (V3Fuc or VI6NeuAc) were present. Fucosylation at V-GlcNAc and III-GlcNAc of nLc6 could be catalyzed by the same enzyme, based on the following observations: (i) fucosylation at both III- and V-GlcNAc was competitively inhibited by V3FucnLc6 and III3V3Fuc2nLc6; (ii) the same conditions (pH, bivalent cation, detergent) were optimal for fucosylation at both III- and V-GlcNAc; (iii) the Km values of the enzyme for nLc4, nLc6, and V3FucnLc6 were approximately the same; and (iv) the activity of the enzyme catalyzing fucosylation at both III- and V-GlcNAc was adsorbed on GDP-hexanolamine-Sepharose and was not inhibited by N-ethylmaleimide. The enzyme preferentially transferred fucose to the penultimate VGlcNAc, followed by transfer to the internal III-GlcNAc of nLc6. Thus, the pathway for synthesis of dimeric X proceeds as follows: nLc6----V3FucnLc6----III3V3Fuc2nLc6. No mechanism was found to operate for chain elongation of the X hapten structure through addition of GlcNAc residues to the terminal Gal of the X hapten.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.