GalNAc Alpha Research Articles

Site-directed mutagenesis studies on the lima bean lectin. Altered carbohydrate-binding specificities result from single amino acid substitutions.

The wild-type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant trisaccharide GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-R. We have generated four site-specific mutants of LBL, two of which show altered specificity for extended carbohydrate structures. Four mutants, [C127Y]LBL, [H128P]LBL, [H128R]LBL and [W132F]LBL were expressed in E. coli. Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild-type ligand which is alpha-L-fucose in the A trisaccharide. The mutant [C127Y]LBL showed specificity for the A disaccharide (GalNAc alpha 1-3Gal) and GalNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Gal. The mutant [H128P]LBL bound the Forssman disaccharide structure GalNAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group. The third and fourth mutants, [H128R]LBL and [W132F]LBL, exhibited wild-type specificities, both recognizing the A trisaccharide. All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide-binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high-affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low-affinity binding site for 8-anilinonaphthalene 1-sulfonate (1/monomer). Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the penultimate Gal of the A disaccharide. We have determined, by site-directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide.

The Glycan Moiety of Human Pancreatic Lithostathine. Structure Characterization and Possible Pathophysiological Implications

Lithostathine, also known as pancreatic stone protein, pancreatic thread protein or regenerating protein, is a glycoprotein which is normally found in the exocrine pancreas, whereas in other tissues it appears either only under pathological conditions, such as Alzheimer's disease (brain), cancer (colon) or during regeneration (endocrine pancreas). In the latter case, it has been shown recently that it acts as a growth factor which stimulates islet regeneration. Little is known about its glycan moiety, which conceivably might be involved in this tissue specificity and pathophysiological characteristics. Therefore we isolated the major oligosaccharide chains of human pancreatic lithostathine and determined their sequences by means of NMR analysis. We obtained eleven different glycoforms and we were able to determine the sequence of seven of them. They all were from the same site of glycosylation (Thr5) and displayed the same core 2 structure: GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc alpha-. They ranged in size from 4 to 9 sugar residues. Elongation was found to proceed from a common tetrasaccharidic core: Gal(beta 1-4)GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc-ol through N-acetyllactosamine units. The non-reducing ends of some oligosaccharides carry the antigenic determinant H, with presence of external Fuc linked only in (alpha 1-2) to Gal. All the glycans, except one, carry a sialic acid in (alpha 2-3) linkage to Gal, with one disialylated form which displays a supplementary (alpha 2-6) linkage. These findings are consistent with the polymorphism of the protein, shown by means of SDS gel electrophoresis and isoelectric focusing, either in its native form or after enzymic processing. Moreover, sialylation seems to protect to some extent the Arg11-Ile12 bond from in situ hydrolysis, thus preventing the harmful precipitation of the C-terminal polypeptide in the pancreatic ducts.

Identification of a Novel Glycosaminoglycan Core-like Molecule I: 500 MHz 1H NMR ANALYSIS USING A NANO-NMR PROBE INDICATES THE PRESENCE OF A TERMINAL α-GalNAc RESIDUE CAPPING 4-METHYLUMBELLIFERYL-β- D-XYLOSIDES

beta-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-3H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) beta-xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc alpha(1,4)GlcA beta(1,3)Gal beta(1,3)Gal beta(1,4)Xyl beta MU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional 1H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal alpha-Gal-NAc residue at a position that is normally occupied by beta-GalNAc in chondroitin/dermatan sulfate or by alpha-Glc-NAc in heparin or heparan sulfate chains. An alpha-GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.

Structural Elucidation of the Biological Repeating Unit of O‐specific Polysaccharide from Citrobacter Serotype O41

Sugar analysis of the O-specific polysaccharide produced by Citrobacter serotype O41 revealed the presence of a hexasaccharide repeating unit, which includes the unusual 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D-galactosyl residue (Fuc3NAcyl). The structure of the repeating unit was determined by extensive use of homo- and heteronuclear two-dimensional NMR spectroscopy, including the application of long-range 1H-13C correlation experiments and NOE studies to establish the sequence of sugar units. Indentification of the Glc beta 1-->2Fuc3NAcyl beta 1-->6GlcNAc alpha 1-->sequence at the non-reducing terminus establishes the biological repeating unit of this O-specific polysaccharide as: -->2Glc beta 1-->Fuc3NAcyl-beta 1-->6GlcNAc alpha 1-->(Glc alpha 1-->2) 4Gal beta 1-->3GalNAc beta 1-->. This structure is similar to that found for the O-specific polysaccharide isolated from Hafnia alvei strain 1211 [Katzenellenbogen, E., Romanowska, E., Dabrowski, U. & Dabrowski, J. (1991) Eur. J. Biochem. 200, 401-407], which differs in having an acetyl substituent at O4 of the Fuc3NAcyl residue and a branch point of GalNAc alpha substituted by Glc beta at O3; these differences are responsible for the only weak serological cross-reactivity of the two strains.

Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T-hapten.

The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)

High Affinity Binding of the Entamoeba histolytica Lectin to Polyvalent N-Acetylgalactosaminides

Entamoeba histolytica trophozoites initiate pathogenic colonization by adherence to host glycoconjugates via an amebic surface lectin which binds to galactose (Gal) and N-acetylgalactosamine (GalNAc) residues. Monovalent and multivalent carbohydrate ligands were screened for inhibition of E. histolytica lectin-mediated human red cell hemagglutination, revealing that: (i) the synthetic multivalent neoglycoprotein GalNAc39BSA (having an average of 39 GalNAc residues linked to bovine serum albumin) was 140,000-fold more potent an inhibitor than monovalent GalNAc and 500,000-fold more potent than monovalent Gal; and (ii) small synthetic multivalent ligands which bind with high affinity to the mammalian hepatic Gal/GalNAc lectin do not bind with high affinity to the E. histolytica lectin. Radioligand binding studies revealed saturable binding of 125I-GalNAc39BSA to E. histolytica membranes (KD = 10 +/- 3 nM, Bmax = 0.9 +/- 0.08 pmol/mg membrane protein). Maximal binding required the presence of calcium chloride (300 microM) or sodium chloride (50 mM), and had a broad pH maximum (pH 6-9). GalNAc39BSA was 200,000-fold more potent than monovalent GalNAc in blocking radio-ligand binding. Among synthetic saccharide-derivatized linear polymers, the GalNAc beta and GalNAc alpha 3Gal beta derivatives were the most potent, with GalNAc alpha and GalNAc alpha 3(Fuc alpha 2)Gal beta derivatives much weaker. The data support a model in which a unique pattern of spaced multiple GalNAc residues are the highest affinity targets for the E. histolytica lectin.

Structural Studies on the Hexasaccharide Alditols Isolated from the Carbohydrate-Protein Linkage Region of Dermatan Sulfate Proteoglycans of Bovine Aorta: DEMONSTRATION OF IDURONIC ACID-CONTAINING COMPONENTS

Five major hexasaccharide alditols were isolated from the carbohydrate-protein linkage region of bovine aorta dermatan sulfate peptidoglycans after reductive beta-elimination and subsequent chondroitinase ABC digestion. These molecules account for at least 55.3% of the total linkage region. Their structures were analyzed by enzymatic digestion in conjunction with high performance liquid chromatography, electrospray ionization mass spectrometry, and 500-MHz one- and two-dimensional 1H NMR spectroscopy. Three of these compounds have the conventional hexasaccharide core; delta HexA alpha 1-3Gal-NAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol. One is nonsulfated, and the other two are monosulfated on C6 or C4 of the GalNAc residue. They represent at least 6.3, 5.2, and 28.8% of the total linkage region, respectively. The other two compounds have the following hitherto unreported hexasaccharide core with an internal iduronic acid residue in common; delta HexA alpha 1-3GalNAc beta 1-4IdoA alpha 1-3Gal beta 1-3Gal beta 1-4Xyl-ol. One is monosulfated on C4 of the GalNAc, and the other is disulfated on C4 of the GalNAc and of the galactose residue substituted by the iduronic acid residue. These two compounds account for 35% of the five isolated hexasaccharide alditols and at least 4.3 and 10.7% of the total linkage region, respectively. The latter two structures form a striking contrast to the currently accepted conception that heparin, heparan sulfate, and chondroitin/dermatan sulfate share the common linkage tetrasaccharide core GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl. The biological significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of dermatan sulfate.

Direct demonstration of increased expression of Thomsen-Friedenreich (TF) antigen in colonic adenocarcinoma and ulcerative colitis mucin and its concealment in normal mucin.

Increased binding of the lectin peanut agglutinin is a common feature in epithelial malignancy and hyperplasia. This may have considerable functional importance in the intestine by allowing interaction between the epithelium and mitogenic lectins of dietary or microbial origin. Peanut agglutinin binds the disaccharide Thomsen-Friedenreich (TF, T or core 1) blood group antigen, Gal beta (1-3) GalNAc alpha-, but is not totally specific for this site. Consequently, there has been controversy about the presence of this structure in colon cancer; studies with anti-TF monoclonal antibodies have failed to detect it. We have examined the presence of TF antigen in colonic mucus glycoprotein (mucin) using endo-alpha-N-acetylgalactosaminidase (O-Glycanase), which specifically catalyzes the hydrolysis of TF antigen from glycoconjugates. Samples of adenocarcinoma, inflammatory bowel disease (ulcerative colitis), and normal mucin were treated with O-glycanase, the liberated disaccharide was separated from the glycoprotein and analyzed using dual CarboPac PA-100 column high performance anion-exchange chromatography coupled with pulsed amperometric detection. O-Glycanase treatment released increased amounts of TF antigen from both colonic adenocarcinoma (8.0 +/- 3.9 ng/micrograms protein, n = 11; P < 0.0001 ANOVA) and ulcerative colitis mucin (3.3 +/- 0.3 ng/micrograms protein, n = 5; P = 0.04) compared with mucin samples from histologically normal mucosa distant from carcinoma (1.5 +/- 1.1 ng/micrograms protein, n = 9). However, after mild acid treatment to remove sialic acids and fucose, releasable TF antigen was increased in all nine of these histologically normal mucin samples (5.5 +/- 2.6 ng/micrograms protein, P < 0.0002). We conclude that TF antigen is an oncofetal antigen which is expressed in colon cancer, but is concealed by further glycosylation (sialylation and/or fucosylation) in the normal colonic mucosa.

A 23 kDa membrane glycoprotein bearing NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells.

Streptococcus sanguis colonizes several human oral surfaces, including both hard and soft tissues. Large salivary mucin-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane‐induced rat colon tumors and human colon cancer cell lines

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R) and B (Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4-21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A-binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer.

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells.

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc alpha 1,3(Fuc alpha 1,2)-Gal beta 1,4GlcNAc beta 1-, GalNAc alpha 1,6Gal beta 1-, Gal beta 1-, Gal beta 1,4GlcNAc- and Gal beta 1,3GalNAc alpha 1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with alpha 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and alpha 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.

Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular modelling of theDolichos biflorus seed lectin and its specific interactions with carbohydrates: ?-D-N-acetyl-galactosamine, Forssman disaccharide and blood group A trisaccharide

The three-dimensional structure of Dolichos biflorus seed lectin has been constructed using five legume lectins for which high resolution crystal structures were available. The validity of the resulting model has been thoroughly investigated. Final structure optimization was conducted for the lectin complexed with alpha GalNAc, providing thereby the first three-dimensional structure of lectin/GalNAc complex. The role of the N-acetyl group was clearly evidenced by the occurrence of a strong hydrogen bond between the protein and the carbonyl oxygen of the carbohydrate and by hydrophobic interaction between the methyl group and aromatic amino acids. Since the lectin specificity is maximum for the Forssman disaccharide alpha GalNAc(1-3) beta GalNAc-O-Me and the blood group A trisaccharide alpha GalNAc(1-3)[alpha Fuc(1-2)] beta Gal-O-Me, the complexes with these oligosaccharides have been also modelled.

Characterization of the specificity of binding of Moluccella laevis lectin to glycosphingolipids.

The specificity of Moluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal alpha-linked N-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc alpha 3GalNAc beta 3Gal alpha 4Glc beta 1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc alpha 3(Fuc alpha 2)Gal beta. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal alpha-linked galactose. When considering the use of the M. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance.

Use of lectin probes on tissues and sympathetic saliva to study the glycoproteins secreted by rat submandibular glands.

We used a panel of nine lectins to detect the glycosylation patterns of rat submandibular glycoproteins. Binding of lectins was assessed on tissue sections and on Western blots of electrophoretically separated glycoproteins from glandular extracts or sympathetic saliva. Histochemical staining of tissue sections showed that two lectins (DBA and SBA) with affinity for terminal GalNAc residues were localized specifically to acinar cells. In contrast, LTA and UEA-I (alpha Fuc-directed) and LFA (NeuAc-directed) bound exclusively to granular tubule cells. PNA and MPA (beta Gal-directed), LCA (alpha Man- and alpha Glc-directed), WGA (beta GlcNAc- and NeuAc-directed), and sWGA (beta GlcNAc-directed) bound to both acinar and granular tubule cells. On electroblot preparations, LFA, PNA, WGA, DBA, and SBA reacted with high molecular weight acinar mucin components both in glandular extracts and in saliva. LTA, UEA-I, LFA, PNA, MPA, LCA, WGA, sWGA, and DBA bound to lower molecular weight bands on blots known to contain granular tubular proteinases. Lectin binding to acini and granular tubules was reduced in sections and in most bands from glandular extracts after sympathetic nerve stimulation. Our results show that (a) secretory glycoproteins from rat submandibular acinar cells are non-fucosylated and contain abundant alpha GalNAc and NeuAc and a small proportion of beta Gal in their oligosaccharide side chains, and (b) alpha Fuc, NeuAc, beta Gal, and alpha GalNAc form the major carbohydrate moieties of the secretory glycoproteins from granular tubules. The results confirm the considerable potential of lectin probes for studying glycoproteins in secretions and in their cells of origin.

Cloning and expression of Gal beta 1,3GalNAc-specific GalNAc alpha 2,6-sialyltransferase.

A cDNA clone encoding a new type of GalNAc alpha 2,6-sialyltransferase (ST6GalNAc II) with a structure similar to that of a previously cloned GalNAc alpha 2,6-sialyltransferase (ST6GalNAc I; Kurosawa, N., Hamamoto, T., Lee, Y.-C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409) was obtained from chicken testes. The predicted amino acid sequence of ST6GalNAc II encodes a protein with type II transmembrane topology, as found for other glycosyltransferases, and showed 32% identity with that of ST6GalNAc I. Transfection of the full length ST6GalNAc II gene into COS cells led to GalNAc alpha 2,6-sialyltransferase activity with a different substrate specificity from that of ST6GalNAc I. Moreover, asialofetuin after treatment with beta-galactosidase did not serve as an acceptor for this enzyme. 14C-Sialylated oligosaccharides obtained from resialylated asialobovine submaxillary mucin with this enzyme were identical to Gal beta 1,3([14C]NeuAc alpha 2,6)GalNAc-ol but not [14C]NeuAc alpha 2,6GalNAc-ol. These results clearly show that the expressed enzyme is a novel type of sialyltransferase that requires beta-galactoside residues linked to GalNAc residues, whereas sialic acid residues linked to galactose residues are not essential for the activity.

Differential expression of five sialyltransferase genes in human tissues.

Cell type-specific expression of specific carbohydrate structures on cell surface glycoproteins and glycolipids is increasingly recognized for providing information relevant to cell-cell interactions in developing and adult organisms. Sialyltransferases contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions on glycolipid and on glycoprotein (N-linked and O-linked) carbohydrate groups. In this report, differential expression of five sialyltransferase genes in human tissues is evaluated as a potential mechanism to account for cell type-specific variation in terminal sialoside structures produced by a cell. For this analysis, the complete cDNA of the human Gal beta 1,3GalNAc alpha 2,3-sialyltransferase and a partial cDNA of the developmentally regulated STX gene were cloned. Northern analysis was performed using these cDNAs and those of three previously cloned human sialyltransferase genes as probes. Each of the five sialyltransferase genes exhibits dramatic differential expression in the 16 adult and 5 fetal human tissues examined, and expression of each gene appears to be independently regulated. Comparison with fragmentary earlier studies of the expression of several of the same enzymes in rat tissues suggests that the overall pattern of expression is largely conserved.

Monoclonal antibody specific for alpha 2-->8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry.

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing alpha 2-->8-linked polysialic acid (-->8Neu5Gc alpha 2-->)n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (-->8KDN alpha 2-->)n-->6(KDN alpha 2-->3Gal beta 1-->3G alpha lNA-c alpha 1-->3) GalNAc alpha 1-->residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of porcine kidney neutral glycosphingolipids: identification of a carbohydrate antigen recognized by human natural antibodies.

We have investigated the glycosphingolipids of pig kidney with a special interest in identifying compounds which may be involved in the rejection of tissue in xenotransplantation. Nine neutral glycosphingolipids have been characterized in porcine kidney and structurally characterized by a combination of techniques including 1H-NMR, permethylation analysis and thin-layer chromatography (TLC) immunostaining with carbohydrate sequence-specific monoclonal antibodies. The major components are members of the globo family and represent the human pk and P antigens. Three other compounds were found to contain a neolacto core structure; the major neolacto compound carries a nonreducing terminal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc) recognized by the naturally-occurring human antibody, anti-Gal, and a second neolacto compound carries the blood group A trisaccharide (GalNAc alpha 1-3(Fuc alpha 1-2)Gal). These results are discussed with respect to tissue transplantation.

Kinetic Properties and Acceptor Substrate Preferences of Two Kinds of Gal.beta.1,3GalNAc .alpha.2,3-Sialyltransferase from Mouse Brain

The cDNAs encoding two kinds of Gal beta 1,3GalNAc alpha 2,3-sialytransferases (ST3GalA.1 and ST3GalA.2) have been cloned from mouse brain, both of which could synthesize the NeuAc alpha 2,3Gal beta 1,-3GalNAc sequence of gangliosides as well as O-glycosidically linked oligosaccharides of glycoproteins [Lee et al. (1993) Eur. J. Biochem. 216, 377-385; Lee et al. (1994) J. Biol. Chem. (in press)]. Kinetic analysis of the two sialyltransferases using Gal beta 1,3GalNAc, asialoGM1, or asialofetuin revealed that ST3GalA.1 exhibits the highest Km value for asialoGM1 (Km = 1.25 mM) and the lowest one for asialofetuin (Km = 0.10 mM), whereas the Km values of ST3GalA.2 for the substrates are very similar (Km approximately 0.5 mM). The synthesis of GM1b from asialoGM1 by ST3GalA.1 was clearly inhibited in the presence of Gal beta 1,3GalNAc or asialofetuin, but that by ST3GalA.2 was not at all. On the other hand, the activity of ST3GalA.2 toward Gal beta 1,3GalNAc or asialofetuin was inhibited by asialoGM1 or GM1. The results of acceptor competition experiments involving asialoGM1, Gal beta 1,3GalNAc, and asialofetuin indicated that ST3GalA.2 exhibits noncompetitive inhibition between asialoGM1 and Gal beta 1,3GalNAc or between asialoGM1 and asialofetuin, whereas ST3GalA.1 exhibits competitive inhibition between all kinds of acceptors. These results strongly indicate that acceptor preference of ST3GalA.1 is different from that of ST3GalA.2, although their acceptor substrate specificities are the same; i.e., gangliosides serve as predominant acceptors for the latter over O-glycosidically linked oligosaccharides of glycoproteins, which are much better acceptors for the former.