Novel oligosaccharide has suppressive activity against human leukemia cell proliferation.

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with beta1-4, alpha1-6 and beta1-6 glycosidic bond were synthesized; Galbeta1-4GlcNH(2), Galalpha1-6GlcNH(2), Galalpha1-6GlcNAc, Galbeta1-6GlcNH(2), Galbeta1-4Galbeta1-4GlcNH(2) and Galbeta1-4Galbeta1-4GlcNAc. Galalpha1-6GlcNH(2) (MelNH(2)) and glucosamine (GlcNH(2)) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH(2). Adding Galalpha1-6GlcNH(2) or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galalpha1-6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galalpha1-6GlcNH(2) were stained. The difference in the stainability of the K562 cells by Galalpha1-6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galalpha1-6GlcNH(2) and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galalpha1-6GlcNH(2) binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (gamma) antibodies).

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically.

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B4 lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8 x 10(5) M-1 at 4 degrees C, whereas that for GalNAcalpha-1-phosphate was 1.3 x 10(5) M-1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B4 lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.

Simple purification method of the antiphospholipid antibody from normal human plasma.

We have reported that rabbit serum contains a phospholipid (PL)/ganglioside-binding protein which adsorbs to Sephacryl S-400 gel and agglutinates human red blood cells. A new protein similar to the PL/ganglioside-binding protein was simply purified from normal human plasma using Sephacryl S-400, Sepharose CL-4B and DEAE-Sepharose CL-6B columns. The purified protein was found to agglutinate rabbit red blood cells. The hemagglutination was specifically inhibited by two PL, phosphatidylserine and phosphatidylinositol, but not by any other PL, gangliosides, saccharides or glycoproteins tested. From analyses of the N-terminal amino acid sequence and immunological specificity, the protein was identified to be a human immunoglobulin M.

Identification and purification of a novel phospholipid/ganglioside-binding protein in rabbit serum.

We have isolated a novel phospholipid/ganglioside-binding protein from rabbit sera or platelet-free plasma. Using an affinity chromatography of a commercial gel (Sephacryl S-series gel, Pharmacia) column and a preparative polyacrylamide gel electrophoresis, the protein can be easily purified. The protein agglutinates human red cells irrespective of the ABO blood types, and its hemagglutination reaction is specifically inhibited by some phospholipids (phosphatidylserine and phosphatidylglycerol) and ganglioside (N-acetylneuraminyl-galactosylglucosyl ceramide, GM3). The hemagglutination and its inhibition reactions are independent on any divalent cations (Ca2+, Mg2+, Mn2+, Ni2+). The protein seems to be assembled as multimers of disulfide-bonded molecular of 86 kDa and 59 kDa subunits.

A novel lectin in rabbit serum binds H type 1, H type 2 and N-acetyl lactosamine structures.

A novel lectin (RSL) which recognizes blood group H type 1 and type 2 (Fuc alpha 1-->2Gal beta 1-->3/4GlcNAc beta-R), and N-acetyllactosamine (Gal beta 1-->4GlcNAc beta-R) was purified from rabbit serum using affinity chromatography on Synsorb H type 2 beads, gel filtration and preparative polyacrylamide gel electrophoresis. The lectin agglutinated human O type red cells, and the hemagglutination reaction was inhibited by H type 1 and type 2 haptens, N-acetyllactosamine and human salivas from secretor individuals. The molecular weight of the lectin was estimated to be approximate 650,000 and 65,000 on Sephacryl S-400 gel filtration and SDS-polyacrylamide gel electrophoresis, respectively.

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: kinetic evidence for the responsibility of N-acetylglucosaminyl-transferase.

Previously we reported the occurrence of UDP-GalNAc:Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase activity in human plasma. Here, the donor substrate specificity of the enzyme partially purified from blood group O plasma was investigated by means of competition experiments with analogs of donor. The enzyme activity was found to be inhibited most strongly by UDP-GlcNAc among the nucleotide sugars tested, and UDP was the best inhibitor among test nucleotides. UDP-GlcNAc was much more inhibitory than UDP, whereas UDP-GalNAc was much less inhibitory than UDP. These results show that the donor-binding site of enzyme has a high affinity for UDP-GlcNAc, but not UDP-GalNAc, suggesting that the enzyme essentially functions in the transfer of N-acetylglucosamine. Indeed, UDP-GlcNAc:Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylglucosaminyltransferase activity is known to occur in human plasma. The ratio of N-acetylgalactosamine to N-acetylglucosamine transferred to lactose with O or B plasma was 1:20 when assayed separately at 1 microM of donor, but increased to 1.2:1 at 5 mM. The nearly identical ratio was obtained with the partially purified enzyme preparation.

A novel method for blood group typing with the aid of monoclonal antibody immobilized dyed microspheres.

A novel and simple method for blood group typing has been developed. The procedure involves the use of type-specific monoclonal antibodies covalently linked to dyed microspheres of polyglycidyl methacrylate. The presence of ABH and Lewis blood group antigens in saliva and plasma could be determined easily and specifically with respective monoclonal antibodies bound to dyed microspheres. The present method could also be applicable for the determination of the presence of invisible antigens for the forensic and diagnostic purpose with visible agglutination reaction.

Use of synthetic H disaccharides as acceptors for detecting activities of UDP-GalNAc:Fuc alpha 1-->2Gal beta-R alpha 1-->3-N-acetylgalactosaminyltransferase in plasma samples from blood group A subgroups.

Concentrations of blood group A-specified alpha(1-->3)-N-acetylgalactosaminyltransferase (A enzyme) were measured in human plasma of blood groups A1, A2, and A3 by using chemically synthesized H disaccharides and H type 1 and type 2 trisaccharides attached to hydrophobic aglycones as acceptors. When the trisaccharides were used as acceptors, enzyme activities were reduced in samples from A2 and A3. However, the H disaccharides were shown to be good acceptors even for enzymes from A2 and A3, and no significant difference in enzyme concentration was detected in any of the plasma tested.

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.

Isolation and characterization of anti-H antibody from egg yolk or immunized hens.

An anti-H antibody was demonstrated to be produced in egg yolk as well as in serum of hens which were immunized with human type O red blood cells. The antibody in egg yolk was isolated with polyethylene glycol and ethanol and was purified by affinity chromatography using immunoadsorbant beads immobilized with H type 2 hapten (Fuca1----2Gal beta 1----4GlcNAc beta). Hemagglutination reaction of the antibody from egg yolk was inhibited by human saliva samples from secretor types irrespective of their ABO blood types, and by immunoadsorbant beads which contain Fuca1----2Gal beta structures.

Characterization of anti-Leb antibody purified from serum of immunized rabbits.

An anti-Le(b) antibody was produced in sera of rabbits by immunization with human saliva from blood group O Le(a-b+) secretor and purified by sequential use of silica beads immobilized with H type 1, Le(a) and Le(b). The purified antibody agglutinated only Le(a-b+) red cells irrespective of their ABO blood type. Hemagglutination reaction with the antibody of blood group O Le(a-b+) red cells was inhibited not only by saliva samples from blood group Le(a-b+) secretors and Synsorb beads immobilized with Le(b) hapten, but also weakly by Synsorb immobilized with Y and H type 2 haptens.

Complete purification and characterization of alpha-3-N-acetylgalactosaminyltransferase encoded by the human blood group A gene.

Human alpha-3-N-acetylgalactosaminyltransferase has been purified 27,000,000-fold from A1 plasma by (NH4)2SO4 fractionation and affinity chromatography on Sepharose 4B, anti-human group O plasma antibodies-Sepharose 4B, and Blue Dextran-Sephadex G-25. A modified procedure in the Sepharose 4B step was developed by batch adsorption and desorption experiments. Cibacron Blue F3G-A, the chromophore of Blue Dextran, was found to bind to the enzyme. UDP is an effective inhibitor of this binding. The pure transferase has an apparent molecular weight of 35,000 as judged by SDS-PAGE in the presence of a reducing agent. The specific activity is 16 pmol/min.ng enzyme, which is comparable to that (30 pmol/min.ng enzyme) of alpha-3-N-acetylgalactosaminyltransferase from porcine submaxillary glands [Schwyzer and Hill (1977) J. Biol. Chem. 252, 2338-2355]. The apparent Km values for UDP-GalNAc, 2'-fucosyllactose, and lacto-N-fucopentaose I are 13, 270, and 350 microM, respectively. The reaction velocity was found to fall off again at high concentrations of oligosaccharide acceptor substrates. The apparent Ki values for UDP and UDP-galactose are 8.6 and 6.2 microM, respectively. The pure enzyme also catalyzes the transfer of galactose in alpha-linkage to 2'-fucosyllactose though the transfer rate of galactose is much lower than that of N-acetylgalactosamine.

Identification of a soluble UDP-Gal: Gal (beta 1-4)Glc (or GlcNAc) (alpha 1-3) galactosyltransferase of bovine colostrum.

A soluble UDP-Gal: Gal (alpha 1-3) galactosyltransferase was first detected in bovine colostrum and this enzyme activity was simply assayed by using rho-nitrophenyl-beta-lactoside (Gal(beta 1-4)Glc-C6H5NO2, rho NP-lactoside) as an acceptor. Treating the radioactive product with alpha- or beta-galactosidase, the radioactivity (greater than 95%) was released by only alpha-galactosidase and was identified as [3H]galactose. This shows that galactosyl residue was alpha-linked to rho-nitrophenyl-beta-lactoside. Methylation, hydrolysis, thin layer chromatography and fluorography of the reaction product (Gal(alpha 1-)-[3H]Gal(beta 1-4)Glc-rho NP) yielded 2,4,6-tri-O-methyl[3H]galactose, indicating that galactosyl residue had been transferred to the carbon-3 position of the terminal nonreducing beta-galactosyl residue in rho-nitrophenyl-beta-lactoside. These results confirmed that the structure of the reaction product was Gal(alpha 1-3)Gal(beta 1-4)Glc-rho NP. The enzyme requires Mn2+ for its activity, and shows pH optimum from 6.5 to 7.5. rho-Nitrophenyl-beta-lactoside and asialo alpha 1-acid glycoprotein were more effective as an acceptor than N-acetyllactosamine. The bovine colostrum (alpha 1-3) galactosyltransferase could not convert human O red cells into B active cells, indicating that this enzyme preparation did not contain the activity to synthesize human blood group B erythrocytes.

Separation into two major forms of beta(1-3)N-acetylglucosaminyltransferase from human serum.

The major beta(1-3)N-acetylglucosaminyltransferase [beta(1-3)GlcNAc-transferase] activity in human serum was isolated by DEAE- and CM-Sepharose column chromatography. This enzyme fraction consisted of two forms of the enzyme, which were separated from each other on a DEAE-Sepharose column and designated as GNAc-TI and GNAc-TII, respectively. They have the same molecular weights (about 90,000), optimum pH values (between pH 7.5 and 8.5) and apparent Km values for N-acetyllactosamine and lactose (7.1-8.8 mM and 10.9-11.5 mM, respectively). Glycosidase and methylation analyses of the reaction products demonstrated that both the enzyme catalyze exclusively the transfer of one N-acetylglucosamine to position C-3 of the terminal galactose of lactose in the beta linkage.

The relationship between the (beta 1-3) N-acetylglucosaminyltransferase and the presence of oligosaccharides containing lacto-N-triose II structure in bovine and human milk.

We measured UDP-GlcNAc:Gal (beta 1-4) Glc (or GlcNAc) (beta 1-3) N-acetylglucosaminyltransferase activities in bovine (Holstein and Jersey cow) and human colostrums, and found in human colostrums sufficient activity to study the enzyme properties while not in bovine colostrums. The properties (requirements, pH optimum, acceptor specificity and Km values for lactose and N-acetyllactosamine) of the enzyme from human colostrum were very similar to those from human serum and urine. The reaction product was hydrolyzed by beta-N-acetylhexosaminidase, indicating that the N-acetylglucosaminyl residue was beta-linked to lactose. Methylation and hydrolysis of the reaction product from lactose [3H] labeled at the terminal galactose yielded 2, 4, 6-tri-O-methyl [3H] galactose. Thus the structure of the product was demonstrated to be GlcNAc (beta 1-3) Gal (beta 1-4) Glc (lacto-N-triose II). On the other hand, bovine sera contained N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to lactose. The enzyme activities were approximately 1/6-1/4 of that contained in human serum. The presence of (beta 1-3) N-acetylglucosaminyltransferase in human colostrum and its absence in bovine colostrums, apparently corresponds with the presence and absence of oligosaccharides containing lacto-N-triose II structure in colostrum.

Identification and characterization of UDP-GalNAc: NeuAc alpha 2-3Gal beta 1-4Glc(NAc) beta 1-4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human blood plasma.

Normal human plasma was found to contain beta 1-4N-acetylgalactosaminyltransferase catalyzing the transfer of N-acetylgalactosamine from UDP-GalNAc to 3'-sialyl-lactose, NeuAc alpha 2-3Gal beta 1-4Glc. The transferred N-acetylgalactosaminyl residue was cleaved from the desialylated reaction product by the beta-N-acetylhexosaminidase from jack beans. Methylation and hydrolysis of the desialylated reaction product yielded only 2,3,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose as neutral sugars, indicating that the N-acetylgalactosaminyl residue was introduced at position C-4 of the galactosyl residue of 3'-sialyllactose. The enzyme required Mn2+ ions for its activity and showed a pH optimum between 6.5 and 8.5. By using a wide variety of oligosaccharides and glycoconjugates, the acceptor specificity of the beta 1-4N-acetylgalactosaminyltransferase was investigated. No detectable amount of N-acetylgalactosamine was transferred to either 6'-sialyllactose or lactose. The enzyme did not act on ganglioside GM3, NeuAc alpha 2-3Gal beta 1-4Glc-ceramide, suggesting that the hydrophobic ceramide portion of GM3 interferes with the enzyme reaction. On the other hand, glycoproteins carrying terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc structures on their N-linked oligosaccharide chains, e.g. Tamm-Horsfall glycoprotein, were efficient acceptors.

Assay of alpha-D-galactosyltransferase of subgroup B sera.

Carriers of weak B antigen were found in three generations of a family. The red cells of the propositus reacted with anti-A human serum and Dolichos biflorus lectin as strongly as normal A1B red cells, but they agglutinated at 8-fold dilution against anti-B human serum (1:128) and did not have a mixed-field agglutination. The red cells of her niece agglutinated at 32-fold dilution against the same anti-B serum and did not have a mixed-field pattern. Her red cells were provisionally designated B2, analogous to A2 of subgroup A. B antigen of the propositus appeared clearly depressed, and she was provisionally designated A1B2. When papain-treated O red cells were used as acceptors instead of untreated O red cells, group A1B2 sera could convert them into B-active cells, which were agglutinated by anti-B human serum. alpha-D-galactosyltransferase activity in A1B2 serum was about one-eighth that in normal B serum.

The presence of N-acetyllactosamine and lactose: beta (1-3)N-acetylglucosaminyltransferase activity in human urine.

Normal human urine was found to contain beta (1-3)N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to N-acetyllactosamine and lactose. Lacto-N-tetraose which carries the terminal Gal beta (1-3)GlcNAc structure was a poor acceptor. The product of the transferase reaction with N-acetyllactosamine as acceptor was identified by methylation analysis as GlcNAc beta (1-3)Gal beta (1-4)GlcNAc. The beta-linkage of the GlcNAc in the synthesized trisaccharide was confirmed by the action of the specific beta-N-acetylhexosaminidase. The enzyme requires Mn2+ ions for its activity, shows a broad pH optimum from 7 to 9, and appears to have a molecular weight of about 200,000 as estimated by Sephadex gel filtration.