Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma.

Acidic and neutral glycosphingolipids were isolated from a human gastric adenocarcinoma, and binding of Helicobacter pylori to the isolated glycosphingolipids was assessed using the chromatogram binding assay. The isolated glycosphingolipids were characterized using fast atom bombardment mass spectrometry and by binding of antibodies and lectins. The predominating neutral glycosphingolipids were found to migrate in the di- to tetraglycosylceramide regions as revealed by anisaldehyde staining and detection with lectins. No binding of H. pylori to these compounds was obtained. The most abundant acidic glycosphingolipids, migrating as the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori selectively interacted with slow-migrating, low abundant gangliosides not detected by anisaldehyde staining. Binding-active gangliosides were isolated and characterized by mass spectrometry, proton nuclear magnetic resonance, and lectin binding as sialyl-neolactohexaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and sialyl-neolactooctaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer).     

Structure of the tryptic glycopeptide isolated from rabbit transferrin.

The structure of the tryptic glycopeptide isolated from rabbit transferrin was elucidated by use of sequential Edman degradations, specific exoglycosidases, endo-beta-N-acetylglucosaminidases, methylation analyses, and periodate oxidation studies. The glycopeptide consists of a heteropolysaccharide, AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3[AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6]-Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc, attached to a peptide, Asn-Ser-Ser-Leu-Cys, via a linkage involving N-acetyl-glucosamine and asparagine. The stoichiometry of this glycopeptide is 2 mol/mol of protein, indicating that rabbit transferrin contains two structurally identical glycopeptide segments.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

Default biosynthesis pathway for blood group-related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group O Le(a-b-) nonsecretor

Glycoconjugates of the GI tract are important for microbial interactions. The expression of histo-blood group glycosyltransferases governs both the expression of blood group determinants and in part the structure and size of the glycoconjugates. Using neutral glycolipids isolated from the small intestine of a rare blood group O Le(a-b-) ABH secretor-negative (nonsecretor) individual we were able to map the "default" pathway of the individual lacking ABO, Lewis, and secretor glycosyltransferases. Structures were deduced with combined analysis of mass spectrometry (MALDI-TOF and ESI-MS/MS), and 1H NMR (500 and 600 MHz). All structures present at a level >5% were structurally resolved and included two extended structures: Galbeta4(Fucalpha3)GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer and Galbeta3GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer. The first, a novel component, is based on a type 2 chain and bears the Lex glycotopes on both its branches. The second, a major component, is based on a type 1 chain, which bears a 3-linked type 1 precursor (Lec) glycotope and a 6-linked Lex glycotope on its branches. This latter structure is identical to that previously isolated from plasma and characterized by MS and GC-MS but not by NMR. Structural resolution of these structures was supported by reanalysis of the blood group H-active decaosylceramides previously isolated from rat small intestine. Other minor linear monofucosylated penta-, hepta-, and difucosylated octaosylceramides, some bearing blood group determinants, were also identified. The cumulative data were used to define a default biosynthesis pathway where it can be seen that carbohydrate chain extension, in the absence of blood group glycosyltransferases, is controlled and regulated by non-blood group fucosylation and branching with type 2 Galbeta4GlcNAc branches.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.     

Structure of two glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU)

The structures of two glycoasparagines composed of one mole each of N-acetylneuraminic acid, galactose, N-acetylglucosamine, and asparagine were determined by periodate oxidation, enzymatic degradation, and methylation analysis. The structures were NANAalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta leads to Asn and NANAalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta leads to Asn, respectively.     

Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core.

We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso-lacto-N-octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES-MS) and NMR spectroscopy. The branching pattern and blood group-related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high-sensitivity ES-MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H-NMR. Three new structures were identified. The nonfucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc and Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6[Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3]Galbeta1-4Glc, both containing an internal Lex epitope, are also novel structures.     

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines

We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain beta1,6-GlcNAc transferase (Lepp?nen et al., Biochemistry, 36, 13729-13735, 1997).     

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.     

Studies on the structure and I-blood-group activity of poly(glycosyl)ceramides.

Employing a modified technique of acetolysis, which allows almost a complete recovery of constituent sugars from poly(glycosyl)ceramides, the glycolipids were found to contain an excess of N-acetylglucosamine over galactose. On the basis of Smith degradation, methylation study, chromium trioxide degradation and the structures of oligosaccharides released from the glycolipids by partial acid hydrolysis, the presence of two types of sugar sequences has been established in poly(glycosyl)ceramides: a) Galbeta1 leads to 4GlcNAcbeta1 leads to 6Gal3 comes from R1 b) Galbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc1 leads to R2. The repeating unit of poly(glycosyl)ceramides seems to be the GlcNAcbeta1 leads to 3Gal sequence. The specificity of one anti-I serum (Woj) is directed against the non-reducing ending of the first kind of chain. Three other anti-I sera reacted with inner portions of the oligosaccharide chains of the glycolipids.     

Isolectins from Solanum tuberosum with different detailed carbohydrate binding specificities: unexpected recognition of lactosylceramide by N-acetyllactosamine-binding lectins

Glycosphingolipid recognition by two isolectins from Solanum tuberosum was compared by the chromatogram binding assay. One lectin (PL-I) was isolated from potato tubers by affinity chromatography, and identified by MALDI-TOF mass spectrometry as a homodimer with a subunit molecular mass of 63,000. The other (PL-II) was a commercial lectin, characterized as two homodimeric isolectins with subunit molecular masses of 52,000 and 55,000, respectively. Both lectins recognized N-acetyllactosamine-containing glycosphingolipids, but the fine details of their carbohydrate binding specificities differed. PL-II preferentially bound to glycosphingolipids with N-acetyllactosamine branches, as Galbeta4GlcNAcbeta6(Galbeta4GlcNAcbeta3)Galbeta4Glcbeta1C er. PL-I also recognized this glycosphingolipid, but bound equally well to the linear glycosphingolipid Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. Neolactotetraosylceramide and the B5 pentaglycosylceramide were also bound by PL-I, while other glycosphingolipids with only one N-acetyllactosamine unit were non-binding. Surprisingly, both lectins also bound to lactosylceramide, with an absolute requirement for sphingosine and non-hydroxy fatty acids. The inhibition of binding to both lactosylceramide and N-acetyllactosamine-containing glycosphingolipids by N-acetylchitotetraose suggests that lactosylceramide is also accomodated within the N-acetylchitotetraose/N-acetyllactosamine-binding sites of the lectins. Through docking of glycosphingolipids onto a three-dimensional model of the PL-I hevein binding domain, a Galbeta4GlcNAcbeta3Galbeta4 binding epitope was defined. Furthermore, direct involvement of the ceramide in the binding of lactosylceramide was suggested.     

Synthesis of N-acetyllactosamine-containing oligosaccharides, galectin ligands

The following spacered oligosaccharides were synthesized: GlcNAcbetal-3Galbetal-4GlcNAcbeta-sp, GlcNAcbetal-6Galbeta1-4GlcNAcbeta -sp, GlcNAcbeta -3(GlcNAcbeta1-6)Galbeta-4GllcNAcbeta-sp, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbetal-6Galbetal-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbeta -3(Galbeta1-4GlcNAcbeta 1-6)Galbeta1-4GlcNAcbeta-sp, GlcNAcbeta1-3(Galbeta1-4GlcNAcbetal-6)Galbeta 1-4GlcNAcbeta-sp, and Galbeta1-4GlcNAcbetal-3(GlcNAcbetal-6)Galbetal-4GlcNAcbeta-sp (sp = O(CH2)2NH2). They represent N-acetyllactosamines substituted with N-acetylgly-cosamine or N-acetyllalctosamine residue at 03, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.     

Kinetic studies on endo-beta-galactosidase by a novel colorimetric assay and synthesis of N-acetyllactosamine-repeating oligosaccharide beta-glycosides using its transglycosylation activity.

Novel chromogenic substrates for endo-beta-galactosidase were designed on the basis of the structural features of keratan sulfate. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (2), which consists of two repeating units of N-acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl beta-N-acetyllactosaminide. In a similar manner, GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (1), GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (3), Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (4), Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (5), and Galbeta1-6GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (6) were synthesized as analogues of 2. Endo-beta-galactosidases released GlcNAcbeta-pNP or Glcbeta-pNP in an endo-manner from each substrate. A colorimetric assay for endo-beta-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcbeta-pNP or Glcbeta-pNP formed by the enzyme through a coupled reaction involving beta-N-acetylhexosaminidase (beta-NAHase) or beta-d-glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo-beta-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-beta-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (9) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (10) as the major products, which have N-acetyllactosamine repeating units.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Characterization of dog small intestinal fucolipids with human blood group H activity.

Fucolipids with human blood group H activity were isolated from several dog small intestines. On the basis of mass spectrometry, periodate oxidation, enzyme degradation, methylation, and immunologic studies the following structure is proposed: Fucalpha(1 yields2)Galbeta(1 yields 4)Glc-NAcbeta(1 yields 3)Galbeta(1 yields 4)Glc-ceramide. The ceramide was shown by mass spectrometry to contain hydroxyhexadecanoic acid and phytosphingosine as major consitutuents.