Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

Structural analysis of oligosaccharide-alditols released by reductive {beta}-elimination from oviducal mucins of Bufo bufo: characterization of the carbohydrate sequence Gal({alpha}1-3)GalNAc({alpha}1-3)[Fuc({alpha}1-2)]Gal

Several tri- to hexasaccharide-alditols of the jelly coat surroundingthe eggs of Bufo bufo were studied by methylation analysis,MALDI-TOF mass spectrometry, and ¹H-NMR spectroscopy. As observedfor six other amphibian species, these carbohydrate chains arehighly species-specific. The main characteristics of the speciesBufo bufo consists in the presence of the new carbohydrate sequenceGal(     

Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc and the isomeric Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc.

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).     

Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc.

Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.     

Three-dimensional structure of a glycosphingolipid having a novel carbohydrate linkage, Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta, determined by theoretical calculations

The novel glycosphingolipid, SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta Cer), which was identified by us (Kawakami Y, et al. (1993) J Biochem 114: 677-83), shows a characteristic spectrum on 1H-NMR analysis, in which the anomeric proton resonances of a reducing end galactose and a glucose are split. To elucidate the structural characteristics of SEGLx, we determined its three-dimensional (3D) structure by means of computer simulation, involving such techniques as molecular mechanics (MM2), the semiempirical molecular orbital method (AM1), molecular dynamics (Amber), and computer 3D modelling. With the hypothesis that all OH group(s) of a ceramide participate in intramolecular hydrogen bonds, two kinds of stable conformers, horizontal and right-angled ones, were formed, depending on the ceramide species. The present findings suggest that the chemical species of both the long chain base and fatty acid moieties, mainly the occurrence of OH group(s), affect the chemical shifts of the anomeric proton resonances not only of the reducing terminal galactose but also the penultimate glucose through the formation of intramolecular hydrogen bonds. Computer simulation through theoretical calculation and 3D modelling was shown to be the best means of confirming the results obtained by experimental analysis.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

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.     

Structure of the major neutral oligosaccharide-alditols released from the egg jelly coats ofAxolotl maculatum. Characterization of the carbohydrate sequence GalNAc({eta}l-4)[Fuc({alpha}l-3)] GlcNAc({eta}l-3 6)

Several O-linked oligosaccharides of the jelly coat surroundingthe eggs of Axolotl maculatum were analysed by ¹H-NMR spectroscopy.The four major oligosaccharide-alditols released by reductive-elimination display either the Lewis^x(Le^x) determinant or thesequence GalNAc(l-4)[Fuc(l-3)]GlcNAc This last structure haspreviously been characterized in allergenically active oligosaccharidesisolated from the sea squirt H-antigen, and hi the N-linkedglycans of Schistosoma mansoni and human urokinase. It representsthe major carbohydrate chain found in A.macu-latum,the oviductof which constitutes an excellent source of l-4-acetylgalactosaminyltransferase activity. Moreover, the carbohydrate chains isolatedfrom A.maculatum are quite different from those found hi sevenother amphibian species, in which the presence of species-specificmaterial has been characterized. The role of carbohydrates appearsmore and more apparent during the fertilization process, andthe diversity of the O-linked oligosaccharides supports sucha biological role. amphibian egg jelly coats Axolotl maculatum/Hnmr/oligosccharide structure     

A B72.3 second-generation-monoclonal antibody (CC49) defines the mucin-carried carbohydrate epitope Gal beta(1-3) [NeuAc alpha(2-6)]GalNAc

Mucus glycoproteins from bovine seminal plasma were demonstrated to express the MAbs CC49- and B72.3-defined epitopes on TAG 72 antigen. Inhibition studies with reductively cleaved mucin O-glycans from bovine seminal plasma and submaxillary glands revealed that CC49 binds specifically to a core-type sialyl-oligosaccharide alditol (fraction 2c), which could be defined with regard to its primary structure by FAB- and EI-mass spectrometry combined with methylation analysis: (formula; see text) Structurally related alditols NeuAc alpha(2-6)-GalNAc-ol, Gal beta(1-3)GalNAc-ol, NeuAc alpha(2-3)-Gal beta(1-3) [NeuAc alpha(2-6)] GalNAc-ol, GlcNAc beta(1-3) [NeuAc alpha(2-6)] GalNAc-ol or NeuAc alpha(2-3) Gal beta(1-3)GalNAc-ol were not inhibitory.     

Escherichia coli beta-galactosidase unexpectedly cleaves the hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc without branch specificity

The branch specificity of Escherichia coli beta-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (3) and GlcNAc beta 1-3(Gal-beta 1-4GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha.

Tissue factor pathway inhibitor (TFPI) produced by endothelial cells contains sulfated Asn-linked oligosaccharides. We have determined that greater than 70% of the oligosaccharides on recombinant TFPI expressed in 293 cells terminate with the sequence SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha. Oligosaccharides terminating with this sequence have previously been described on lutropin, thyrotropin, and pro-opiomelanocortin: glycoproteins synthesized in the anterior pituitary. A GalNAc-transferase that recognizes the tripeptide motif Pro-Xaa-Arg/Lys 6-9 residues N-terminal to Asn glycosylation sites accounts for the specific addition of GalNAc to the oligosaccharide acceptor on these glycoproteins, whereas a GalNAc beta 1,4GlcNAc beta 1, 2Man alpha-4-sulfotransferase accounts for the addition of sulfate. The sulfated oligosaccharides present on these hormones are responsible for their rapid clearance from plasma by a receptor in hepatic reticuloendothelial cells. GalNAc- and sulfotransferase activities with the same properties as those expressed in the pituitary are detected at high levels in 293 cells and at lower levels in endothelial cells. Chinese hamster ovary (CHO) cells do not contain detectable levels of either transferase and rTFPI expressed in CHO cells does not contain sulfated Asn-linked oligosaccharides. TFPI contains the sequence Pro-Phe-Lys, 9 residues N-terminal to the glycosylation site at position 228; this tripeptide may act as the recognition sequence for the GalNAc-transferase. rTFPI produced by 293 cells, but not that produced by CHO cells, is bound by the receptor on hepatic reticuloendothelial cells suggesting the sulfated structures play a role in the biologic behavior of TFPI.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

Pro-opiomelanocortin synthesized by corticotrophs bears asparagine-linked oligosaccharides terminating with SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha.

We have determined that greater than or equal to 80% of the Asn-linked oligosaccharides on the glycosylated form of mouse adrenocorticotropin (15-kDa adrenocorticotropin (ACTH)) bear one or more branches terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha (S4GGnM). Proopiomelanocortin (POMC), the precursor of ACTH, is the first example of a glycoprotein that is not a member of the glycoprotein hormone family to bear such sulfated structures. Like lutropin and thyrotropin, 15-kDa ACTH bears dibranched oligosaccharides terminating with SO4-4-GalNAc; however, at least half of the oligosaccharides on 15-kDa ACTH terminating with SO4-4-GalNAc consist of more highly branched structures that have not previously been described. Both the GalNAc beta 1,4GlcNAc beta 1,2Man-4-sulfotransferase and the glycoprotein hormone-specific GalNAc-transferase are expressed in the corticotroph-derived AtT-20 cell line. A tripeptide recognition sequence, Pro-Val-Lys, similar to the Pro-Leu-Arg sequence required for recognition of glycoprotein hormone alpha- and beta-subunits by the glycoprotein hormone-specific GalNAc-transferase, is present 8 residues amino-terminal to the glycosylated Asn of 15-kDa ACTH. Thus, POMC has the features expected for specific addition of the S4GGnM sequence to its oligosaccharides. The recent discovery of a receptor in hepatic endothelial cells that recognizes oligosaccharides terminating with S4GGnM suggests these sulfated oligosaccharides will regulate the circulatory half-life of glycosylated POMC cleavage products.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

Wheat germ agglutinin chromatography of GlcNac beta 1-3(GlcNAc beta 1-6)Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.     

Mucin synthesis. UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from pig and rat colon mucosa

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to GalNAc-ovine submaxillary mucin to form GlcNAc beta 1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAc beta 1-3GalNAc-mucin to form GlcNAc beta 1-3(GlcNAc beta 1-6) GalNAc-mucin. This is the first demonstration of in vitro synthesis of the GlcNAc beta 1-3GalNAc disaccharide and of the GlcNAc beta 1-3-(GlcNAc beta 1-6)GalNAc trisaccharide, two of the four major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-alpha-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide has been named UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase (mucin core 3 beta 3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide has been named UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (mucin core 4 beta 6-GlcNAc-transferase). The beta 3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-alpha-phenyl, GalNAc-alpha-benzyl, and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Vmax values of 59, 62, and 37 nmol h-1 (mg of protein)-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of neutral and acidic oligosaccharide-alditols derived from the jelly coat of the Mexican axolotl. Occurrence of oligosaccharides with fucosyl(alpha 1-3)fucosyl(alpha 1-4)-3-deoxy-D-glycero-D-galacto-nonulosonic acid and galactosyl(alpha 1-4)[fucosyl(alpha 1-2)]galactosyl(beta 1-4)-N-acetylglucosamine sequences.

Six major neutral and acidic oligosaccharide-alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3-deoxy-D-glycero-D-galacto-nonulosonic acid (dNloA) and L-fucose (Fuc). The structures of the six major oligosaccharides were established as follows: [sequence: see text]