Increased alpha3-fucosylation of alpha1-acid glycoprotein in Type I diabetic patients is related to vascular function.

Diabetic mellitus is attended by the development of endothelial dysfunction which is suggested to be accompanied with a chronic low-degree of inflammation. During a chronic hepatic inflammatory response, specific changes in glycosylation of the acute phase protein alpha1-acid glycoprotein (AGP) can be detected. In this report we studied the changes in glycosylation of AGP in more detail and evaluated the relation between a change in glycosylation of AGP and urinary albumin secretion in Type I diabetic patients. The glycosylation of AGP, studied by crossed affinity immunoelectrophoresis (CAIE) and high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD), showed an increase in alpha3-fucosylation. Staining with an antibody against sialyl Lewis(x) (sLe(x)) implied that part of the alpha3-fucosylation was present in a sLe(x)-conformation. In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP (p<0.0005) could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as an additional marker for the development of vascular complications in Type I diabetic patients.

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.

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains.

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.

Identification of an alpha3-fucosyltransferase and a novel alpha2-fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-->3[Gal(NAc)beta1-->4]GlcNAc sequence.

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-->4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-->2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1-->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3-FucT resembles human FucT V and VI rather than other known FucTs. N-ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-->3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2-FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.

The substrate specificity of the snail Lymnaea stagnalis UDP-GlcNAc:GlcNAc beta-R beta 4-N-acetylglucosaminyltransferase reveals a novel variant pathway of complex-type oligosaccharide synthesis.

UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyltransferase (beta 4-GlcNAcT) of the snail Lymnaea stagnalis is an enzyme with structural resemblance to mammalian beta 4-galactosyltransferases (beta 4-GalT). The enzyme, which is present in the prostate gland of the snail, was expressed in a recombinant form in insect cells using the baculovirus technology. This form was used to determine the enzyme's in vitro substrate specificity in order to assess its possible role in vivo. The enzyme appeared to be a genuine GlcNAcT as no UDP-sugar other than UDP-GlcNAc could serve as an efficient donor substrate. Acceptor specificity studies showed that the enzyme is far more restricted in acceptor usage than beta 4-GalT. Oligomers of beta 4-GlcNAc were relatively poor acceptors, indicating that this enzyme is not involved in the synthesis of chitin-like molecules. Both its polypeptide structure and acceptor specificity suggest that it neither is implicated in the synthesis of the chitobiose core of N-linked glycans. Preferred substrates are those that contain a beta-GlcNAc residue attached to the carbon-6 of Gal or GalNAc residues, as found in vertebrate blood-group I-active and O-linked core 2- and 4-based oligosaccharides, respectively. By contrast, compounds in which GlcNAc is beta 6-linked to Man (as in N-linked glycans) are poor acceptors. Analysis of the products formed in vitro by 1H-NMR spectroscopy and acetolysis showed that the enzyme establishes a GlcNAc beta 1-->4GlcNAc linkage and shows branch specificity with a blood-group I-active trisaccharide substrate. Furthermore, the enzyme differs from beta 4-GalT in that it is not responsive to alpha-lactalbumin. It is proposed that the enzyme functions in a novel, variant pathway of complex-type oligosaccharide synthesis in the snail.

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.

Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins.

BACKGROUND: Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. OBJECTIVE: We sought to characterize the allergen biochemically and immunologically. METHODS: The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. RESULTS: The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. CONCLUSIONS: Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

A Lymnaea stagnalis gene, with sequence similarity to that of mammalian beta 1-->4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyltransferase.

A cDNA encoding a novel glycosyltransferase, that may be involved in a variant pathway for the synthesis of complex type oligosaccharide chains, was cloned from the pond snail Lymnaea stagnalis. By heterologous hybridization, using bovine beta 1-->4-galactosyltransferase cDNA as probe, a genomic clone from a snail library was isolated. This genomic clone was subsequently used to clone the corresponding cDNA from a prostate gland library. The isolated cDNA encodes a polypeptide of 490 amino acids with a type II membrane protein topology typical for glycosyltransferases. The carboxyl-terminal part, encoding the putative catalytic domain, reveals considerable sequence similarity with the corresponding region of mammalian beta 1-->4-galactosyltransferases, suggesting an evolutionary relationship. Expression of this cDNA in COS cells and insect cells revealed that the encoded enzyme transfers GlcNAc, rather than Gal or GalNAc, from the corresponding nucleotide sugars to several beta-N-acetylglucosaminides. Structural characterization by 1H NMR spectroscopy of products formed in vitro demonstrated that the enzyme can be identified as a UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyl-transferase. A new family of glycosyltransferases has hereby been discovered, consisting of enzymes that act on acceptor substrates with a terminal beta-linked GlcNAc residue and establish a beta 1-->4-linkage, but have a different nucleotide sugar requirement.

Heterogeneity of human biliary mucin: functional implications.

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67-74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained alpha-D-N-acetylglucosamine and were fucosylated, sialylated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

Structural characterization of sialylated tetrasaccharides and pentasaccharides with blood group H and Le(x) activity isolated from bovine submaxillary mucin.

In this study we have investigated the structures of a sialylated tetrasaccharide and two sialylated pentasaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. The tetrasaccharide contained NeuGc, while one of the pentasaccharides contained NeuAc and the other contained NeuGc. All three oligosaccharides contained the core type-3 structure (GlcNAc beta 1----3GalNAcol). The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: [formula: see text]. The oligosaccharides occurred in the approximate molar ratios, 1.0:0.6:0.3. This is the first report of these oligosaccharides in bovine submaxillary mucin. 1H-NMR data for structures A1/2c and A1/2e, which are novel structures, are presented for the first time. Oligosaccharide A1/2e contains the blood-group-H type-2 antigenic determinant while oligosaccharide A1/2d contains the Lewis(x) determinant.

Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin.

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.

Determination of the structure of a novel acidic oligosaccharide with blood-group activity isolated from bovine submaxillary-gland mucin.

In this study we investigated the structure of an acidic fucose-containing pentasaccharide released from bovine submaxillary-gland mucin by alkaline-borohydride treatment. The structure, determined by a combination of one-dimensional and two-dimensional 1H-n.m.r. spectroscopy at 270 MHz and methylation analysis involving g.l.c.-m.s., was as follows: Fuc alpha(1----2)Gal beta(1----4)GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol This pentasaccharide is a novel structure and is the first report of a blood-group-H type 2 determinant on a submaxillary-gland mucin.

N-acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct. Application of a sensitive and specific assay by use of high-performance liquid chromatography.

A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three transferase products formed from the common acceptor oligosaccharide substrate GlcNAc beta 1----2Man alpha 1----3(GlcNAc beta 1----2Man alpha 1---- 6)Man beta 1----4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breakdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.