Plant members of the alpha1-->3/4-fucosyltransferase gene family encode an alpha1-->4-fucosyltransferase, potentially involved in Lewis(a) biosynthesis, and two core alpha1-->3-fucosyltransferases.

Three putative alpha1-->3/4-fucosyltransferase (alpha1-->3/4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core alpha1-->3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc. Analysis of the product by 400 MHz (1)H-nuclear magnetic resonance spectroscopy showed that the product is alpha1-->4-fucosylated at the N-acetylglucosamine residue. In vitro, the recombinant B. vulgaris alpha1-->4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis(a) formation on N-linked glycans.

alpha3-galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor.

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose-specific hepatic lectin, the 'asialoglycoprotein receptor'. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galalpha1-->3Galbeta1-->4R is present on glycoprotein glycans. To investigate if alpha3-galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of alpha3-galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galbeta1-->4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N-glycans were alpha3-galactosylated to completion by use of recombinant bovine alpha3-galactosyltransferase. Similarly, desialylated alpha1-acid glycoprotein (orosomucoid) was alpha3-galactosylated in vitro. The alpha3-galactosylation of a branched, Galbeta1-->4-terminated oligosaccharide lowered its affinity for the membrane-bound lectin on whole rat hepatocytes 50-250-fold, and for the detergent-solubilized hepatic lectin 7-50-fold. In contrast, alpha3-galactosylation of asialo-alpha1-acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nM. Fully alpha3-galactosylated alpha1-acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo-alpha1-acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre-injection of an excess asialo-fetuin. Organ distribution analysis showed that the injected alpha1-acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily alpha3-galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte-derived, circulating glycoproteins.

Characterization of a core alpha1-->3-fucosyltransferase from the snail Lymnaea stagnalis that is involved in the synthesis of complex-type N-glycans.

We have identified a core alpha1-->3-fucosyltransferase activity in the albumin and prostate glands of the snail Lymnaea stagnalis. Incubation of albumin gland extracts with GDP-[(14)C]Fuc and asialo/agalacto-glycopeptides from human fibrinogen resulted in a labeled product in 50% yield. Analysis of the product by 400 MHz (1)H-NMR spectroscopy showed the presence of a Fuc residue alpha1-->3-linked to the Asn-linked GlcNAc. Therefore, the enzyme can be identified as a GDP-Fuc:GlcNAc (Asn-linked) alpha1-->3-fucosyltransferase. The enzyme acts efficiently on asialo/agalacto-glycopeptides from both human fibrinogen and core alpha1-->6-fucosylated human IgG, whereas bisected asialo/agalacto-glycopeptide could not serve as an acceptor. We propose that the enzyme functions in the synthesis of core alpha1-->3-fucosylated complex-type glycans in L. stagnalis. Core alpha1-->3-fucosylation of the asparagine-linked GlcNAc of plant- and insect-derived glycoproteins is often associated with the allergenicity of such glycoproteins. Since allergic reactions have been reported after consumption of snails, the demonstration of core alpha1-->3-fucosylation in L. stagnalis may be clinically relevant.

The lactose analog GalNAcbeta1-->4Glc is present in bovine colostrum. Enzymatic basis for its occurrence.

We have isolated from bovine colostrum the lactose analog GalNAcbeta1-->4Glc. The enzymatic basis for its occurrence was studied by assaying the activities of GlcNAcbeta-R beta4-N-acetylgalactosaminyltransferase (beta4-GalNAcT) and GlcNAcbeta-R beta4-galactosyltransferase (beta4-GalT) in primary milk and several lactating bovine mammary gland fractions. As the beta4-GalNAcT, which appears to be tightly membrane bound, is induced by the milk protein alpha-lactalbumin (alpha-LA) to act on Glc, it is concluded that beta4-GalNAcT is responsible for the synthesis of GalNAcbeta1-->4Glc in the gland. The comparatively low level (15-20 mg/l) at which this disaccharide is produced may be due to the relatively poor interaction of beta4-GalNAcT with alpha-LA as well as to the fact that alpha-LA does not inhibit the action of the enzyme on N-acetylglucosaminides.

The acceptor substrate specificity of human beta4-galactosyltransferase V indicates its potential function in O-glycosylation.

In order to assess the function of the different human UDP-Gal:GlcNAc beta4-galactosyltransferases, the cDNAs of two of them, beta4-GalT I and beta4-GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant beta4-GalT V was more than 15 times lower than that of beta4-GalT I, using GlcNAc beta-S-pNP as an acceptor. Whereas beta4-GalT I efficiently acts on all substrates having a terminal beta-linked GlcNAc, beta4-GalT V appeared to be far more restricted in acceptor usage. Beta4-GalT V acts with high preference on acceptors that contain the GlcNAc beta1-->6GalNAc structural element, as found in O-linked core 2-, 4- and 6-based glycans, but not on substrates related to V-linked or blood group I-active oligosaccharides. These results suggest that beta4-GalT V may function in the synthesis of lacNAc units on O-linked chains, particularly in tissues which do not express beta4-GalT I, such as brain.

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin.

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N -acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4-galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc-based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.

Phase variation in Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity in rat brain and in tumour cells that express polysialic acid on neural cell adhesion molecules.

A method for the assay of CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity was developed. Using a 1-day-old rat brain membrane fraction as an enzyme preparation optimal activity was obtained at pH 6.5, 0.3% Triton X-100, and 5 mM MnCl2. However, no absolute cation requirement was found as EDTA only partially inhibited the activity. Within a concentration range of 0.3-3 mg colominic acid (which consists of a mixture of oligomers of alpha 2-->8-linked sialic acid) per 50 microliters a V of 0.61 nmol per mg protein h-1 was estimated while a half-maximal reaction velocity was obtained at a concentration of 1.75 mg per 50 microliters. High performance anion-exchange chromatography of the radioactive products formed in the reaction showed that sialic acid oligomers ranging in size from a degree of polymerization (DP) of 2 up to at least DP 9 could serve as acceptor substrates. Comparison of the acceptor properties of DP 3 and DP 6 showed that the larger oligomer was acted upon with a 10-fold higher efficiency. Periodate oxidation of the products followed by reduction and hydrolysis yielded the C7 analogue of NeuAc as the only radioactive product, indicating that under the conditions of the assay only a single sialic acid residue was introduced into the acceptor molecules. Using the assay it appeared that in rat brain the activity of this sialyltransferase decreased six-fold during postnatal development to the adult stage. The assay method was also applied to lysates of several neuroblastoma and small cell lung tumour cell lines, which differ in the expression of polysialic acid as well as of the neural cell adhesion molecule NCAM, a major carrier of this polymer. Activity of the sialyltransferase appeared to be correlated with the expression of polysialic acid present on NCAM. These results indicate that this sialyltransferase might function in the process of poly-sialylation.

Branching and elongation with lactosaminoglycan chains of N-linked oligosaccharides result in a shift toward termination with alpha 2-->3-linked rather than with alpha 2-->6-linked sialic acid residues.

The activity of bovine colostrum CMP-NeuAc:Gal beta 1-->4GlcNAc beta-R alpha 2-->6-sialyltransferase (alpha 6-NeuAcT) toward oligosaccharides that form part of complex-type, N-linked glycans appears significantly reduced when a bisecting GlcNAc residue or additional branches are present, or when core GlcNAc residues are absent. By contrast human placenta CMP-NeuAc:Gal beta 1-->4GlcNAc beta-R alpha 2-->3-sialyltransferase (alpha 3-NeuAcT) is much less sensitive to structural variations in these acceptors. Furthermore the alpha 3-NeuAcT shows a much higher activity than the alpha 6-NeuAcT with oligosaccharides that form part of linear and branched lactosaminoglycan extensions. These results indicate that, in tissues that express both enzymes, branching and lactosaminoglycan formation of N-linked glycans will cause a shift from termination with alpha 2-->6-linked sialic acid to termination with alpha 2-->3-linked sialic acid residues. These findings provide an enzymatic basis for the sialic acid linkage-type patterns found on the oligosaccharide chains of N-glycoproteins.

Conversion of GalNAc beta(1-4)GlcNAc beta-OMe into GalNAc beta (1-4)-[Fuc alpha(1-3)]GlcNAc beta-OMe using human milk alpha 3/4-fucosyltransferase. Synthesis of a novel terminal element in glycoprotein glycans.

Incubation of GalNAc beta(1-4)GlcNAc beta-OMe with GDP-Fuc in the presence of human milk alpha 3/4-fucosyltransferase resulted in the formation of GalNAc beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OMe. Under conditions that led to complete alpha 3-fucosylation of Gal beta(1-4)GlcNAc beta-OEt, GalNAc beta(1-4)GlcNAc beta-OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one- and two-dimensional 1H-NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Gal beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OEt and GalNAc beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C-2', i.e. a hydroxyl function versus a N-acetyl group.

Enzymatic synthesis of the alpha 3-galactosyl-Lex tetrasaccharide: a potential ligand for selectin-type adhesion molecules.

Using recombinant UDP-Gal:Gal beta 1-->4GlcNAc alpha 1,3-galactosyltransferase and human milk alpha 1,3-fucosyltransferase the disaccharide Gal beta 1-->4GlcNAc has been converted in vitro into a tetrasaccharide product. The product has been characterized by gel filtration chromatography and HPLC and was analyzed using 1H-NMR. Based on NMR spectral data along with the known linkage specificity of the alpha 1,3-galactosyltransferase and the alpha 1,3-fucosyltransferase used, the chromatographic behaviour of the product, and the 1:1 molar ratios of the galactose and fucose residues calculated from incorporated radioactivity, it is concluded that the structure of the tetrasaccharide product is Gal alpha 1-->3Gal beta 1--4[Fuc alpha 1-->3]-GlcNAc. The tetrasaccharide is a non-charged analogue of the sialyl-Lex determinant that potentially may act as a ligand structure in selectin-mediated cell-cell adhesion.

Human myeloid alpha 3-fucosyltransferase is involved in the expression of the sialyl-Lewis(x) determinant, a ligand for E- and P-selectin.

The sialyl-Lex determinant (NeuAc alpha 2-->3Gal beta 1-->4[Fuc alpha- 1-->3]GlcNAc) has been identified as a major ligand in the selectin-mediated adhesion of neutrophils and monocytes to activated endothelium or platelets. This carbohydrate epitope is formed by the sequential action of alpha 3-sialyltransferase and alpha 3-fucosyltransferase on N-acetyllactosamine (Gal beta 1-->4GlcNAc) disaccharide termini of glycoconjugates. We have addressed the role of the human myeloid alpha 3-fucosyltransferase in the expression of this epitope at the leucocyte surface by determining its activity in human-mouse leukemic cell hybrids (WEGLI), normal human granulocytes and chronic myeloid leukemia (CML) cells using sialylated and desialylated glycoproteins and oligosaccharides as acceptor substrates. In contrast to what has been reported for the myeloid-type enzyme, we found that the alpha 3-fucosyltransferase of the cells studied can use sialylated acceptors be it that the activity is several times lower than with asialo-substrates. Characterization of the product obtained with a sialylated oligosaccharide indicated that the enzyme can catalyze the formation of the sialyl-Le(x) structure. Flow cytometry of the WEGLI cells using a sialyl-Le(x)-specific monoclonal antibody (MoAb) showed that these cells indeed express sialyl-Lex at their surface, provided that they contain human chromosome 11. Earlier the presence of this chromosome had been correlated with the expression of alpha 3-fucosyltransferase activity. In addition to sialyl-Le(x), WEGLI cells containing chromosome 11 showed high-expression levels of related structures recognized by antibodies VIM-2 and VIM-8, suggesting that fucose addition can occur at both distal and proximal GlcNAc residues in poly-N-acetyl-lactosaminoglycan sequences. Based on the human chromosome contents it could be ruled out that the alpha 3-fucosyltransferase of WEGLI cells is a Lewis-type alpha 3/4- or plasma-type alpha 3-fucosyltransferase, the genes of which have been mapped to chromosome 19. It is concluded that the enzyme studied is of the myeloid-type and indeed is involved in the synthesis of sialyl-Le(x) (and also VIM-2 and VIM-8 structures) in leukocytes provided that its expression is at a sufficiently high level.

Human liver and human placenta both contain CMP-NeuAc:Gal beta 1-->4GlcNAc-R alpha 2-->3- as well as alpha 2-->6-sialyltransferase activity.

A high pH anion exchange chromatographic (HPAEC) system for the separation of isomeric sialo-oligosaccharide products was developed. Employing this system, using Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Man beta 1-->4GlcNAc as a substrate, a Gal beta 1-->4GlcNAc-R alpha 2-->3-sialyltransferase activity was detected for the first time in human liver. This activity is expressed together with the prevalent alpha 2-->6-sialyltransferase. Furthermore, in addition to the major alpha 2-->3-sialyltransferase, a low but distinct activity of alpha 2-->6-sialyltransferase was detected in human placenta. This activity could not be found by methods based on methylation analysis or high resolution NMR spectroscopy. It is concluded that HPAEC, in combination with the use of the pentasaccharide as an acceptor substrate, is suited for the specific detection of minor, Gal beta 1-->4GlcNAc-specific sialyltransferase activities.

Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:N-acetyllactosaminide beta 1----3-N-acetylglucosaminyltransferase from Novikoff tumor cell ascites fluid.

An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type.

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.

Porcine submaxillary mucin contains alpha 2----3- and alpha 2----6-linked N-acetyl- and N-glycolylneuraminic acid.

Four acidic trisaccharides have been obtained by alkaline borohydride reductive cleavage (beta-elimination) of a fraction of porcine submaxillary mucin precipitating at 60-75% ethanol. Their structures have been investigated using the techniques of methylation analysis involving gas-liquid chromatography/mass spectrometry along with high-resolution 1H-NMR analysis. Two of the four oligosaccharides, B1-B4, contain N-acetylneuraminic acid (NeuAc) while two contain N-glycolylneuraminic acid (NeuGc). The following structures are proposed for the acidic trisaccharide fraction: (B1) NeuAc alpha(2----3)Gal beta(1----3)GalNAcol, (B2) NeuGc alpha(2----3)Gal beta(1----3)GalNAcol, (B3) Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol and (B4) Gal beta(1----3)[NeuGc alpha(2----6)]GalNAcol, (GalNAcol = reduced N-acetylgalactosamine. These oligosaccharides were present in a molar ratio of 69:22:4:5. Although oligosaccharide B4 has previously been found in porcine submaxillary mucin B1, B2 and B3 have not. Furthermore, oligosaccharide B2 is a novel structure.

Branch specificity of bovine colostrum CMP-sialic acid: N-acetyllactosaminide alpha 2----6-sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins.

By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Man alpha 1----3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N-acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1----3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme.

Identification and characterization of an UDP-Gal: N-acetyllactosaminide alpha-1,3-D-galactosyltransferase in calf thymus.

Calf thymus was found to contain a high activity of a galactosyltransferase, which transfers galactose from UDP-galactose to asialo-alpha 1-acid glycoprotein and N-acetyllactosamine. By means of competition and acceptor-specificity studies the enzyme could be distinguished from an N-acetylglucosaminide beta-1,4-galactosyltransferase and an N-acetylgalactosamine-protein beta-1,3-galactosyltransferase, which in addition occur in calf thymus, as well as from the blood-group-B-associated alpha-galactosyltransferase. Identification of the products revealed that the enzyme accomplishes an alpha 1 leads to 3 linkage resulting in a terminal Gal(alpha 1 leads to 3)Gal(beta 1 leads to 4)GlcNAc sequence. The enzyme is membrane-bound and is activated by Triton X-100. It shows optimal activity over a broad pH range (5.5-7.0) and has a pronounced requirement for Mn2+ ions (Km = 6.1 mM) for its action. It is suggested that the alpha-1,3-galactosyltransferase functions in the biosynthesis of calf thymocyte cell-surface glycoconjugates including glycoproteins.

Novikoff ascites tumor cells contain N-acetyllactosaminide beta 1 leads to 3 and beta 1 leads to 6 N-acetylglucosaminyltransferase activity.

Novikoff ascites tumor cell homogenate was found to catalyze the transfer of [14C]N-acetylglucosamine from UDP-[14C]GlcNAc to asialo-alpha 1-acid glycoprotein. Mucins appeared to be poor acceptors. Methylation and hydrolysis of the product formed in an incubation with UDP-GlcNAc and asialo-alpha 1-acid [3H]glycoprotein yielded 2,4,6-trimethyl [3H]galactose and 2,3,4-trimethyl [3H]galactose, indicating that N-acetylglucosaminyl residues were introduced to position C-3 and C-6 of the terminal galactoses on the glycoprotein. It is concluded that Novikoff cells contain two N-acetylglucosaminyltransferases which might be involved in the synthesis of linear and branched forms of cell surface polylactosaminoglycans and blood group I/i antigenic structures.

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.