Bacterial homologue of human blood group A transferase.

A bacterial version of human blood group A transferase was identified and found to be able to accept five naturally existing H-antigen core structures as good substrates, demonstrating its versatility for synthesis of blood group A antigens. Furthermore, this enzyme was applied in the engineering of bacterial cell surface polysaccharides by remodeling blood group B mimicry into blood group A mimicry.

Altered expression of blood group A and H antigens on red cells from an acute leukemic patient.

Alternate expressions of the blood group A and H antigens on red cells are described in a patient with acute myelocytic leukemia. The patient's red cells showed mixed field agglutination with anti-A and anti-H sera and lectins, and no agglutination with anti-B serum. The agglutinability of the A red cells with Dolichos biflorus lectin was between A1 and A2 (A intermediate). Inagglutinable red cells were separated with anti-A agglutinin, and the proportion was about 80% of total cells. The agglutinating activity with Ulex europaeus anti-H of red cells, which were inagglutinable with anti-A, was 16 times weaker than that of group O cells. The weaker reaction with Ricinus communis lectin and the stronger reaction with Psathyrella velutina lectin on the inagglutinable cells with anti-A than those on the group O cells suggest that fucosyl alpha (1-2) and galactosyl beta (1-4) residues at the nonreducing end of carbohydrate chains of H antigens on the red cells were diminished, and N-acetylglucosaminyl beta (1-3) residues were sequentially exposed. His saliva contained A and H substances in normal amounts of a secretor. Serum alpha-N-acetylgalactosaminyltransferase activity which converts O red cells to A red cells was the same as those in sera from A1 individuals. These results suggest that the synthesis of H precursors is partially blocked in this patient's red cells.

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue.

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.

Detection of UDP-N-acetylgalactosamine-oligosaccharide N-acetylgalactosaminyltransferase activity in rat stomach and human serum by high-performance liquid chromatography.

The enzymatic transfer of the sugar portion from UDP-N-acetylgalactosamine to pyridylamino (PA) lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-4Glc-PA) was detected by high-performance liquid chromatography. Separation of the fluorescent product from the fluorescence-labeled acceptor was achieved within 10 min by reversed-phase high-performance liquid chromatography. Rat stomach enzyme activity was detected in the microsomal fraction from antrum but not corpus. Ohara et al. (1986, Comp. Biochem. Physiol. 83B, 273-275) reported that the N-acetylgalactosamine content in antrum mucin was greater than that in corpus mucin and antrum mucin had strong blood group A activity. The prominent asymmetrical distribution of the enzyme detected here well supports these findings. The elution position of the fluorescent product was the same as that of the product formed by the action of type A human serum toward the acceptor. Its hydrolysis by alpha-N-acetylgalactosaminidase yielded the acceptor. It is thus evident that the detected enzyme is the same as that producing the blood group A structure.

Blood-group-ABH antigens of human erythrocytes. Quantitative studies on the distribution of H antigenic sites among different classes of membrane components.

The contribution of blood-group-active glycolipids and glycoproteins to the blood-group-ABH character of human erythrocytes was investigated. For that purpose the blood-group-H sites of human O cells were converted in vitro into group-A sites by transfer of alpha-N-acetyl-D-[14C]galactosamine residues with the aid of the blood-group-A gene-dependent alpha-N-acetylgalactosaminyl transferase prepared from human A1 plasma. Upon partition of the red cell membranes between water and organic solvent, about 5% of the label was found in the organic phase and about 20% in the water phase, thus reflecting the distribution of blood-group antigenic sites between glycosphingolipids with short carbohydrate chains and polyglycosylceramides, respectively. The fact that about 70% of the radioactivity remained tightly bound to the membranes and could only be released by treatment with pronase provided good evidence that the bulk of blood-group-H determinants is bound to glycoprotein material. Following these results it can thus be assumed that blood-group-ABH activity of human erythrocytes is determined preferentially by group-specific glycoproteins rather than glycolipids.

Leukemia-induced alterations of serum glycosyltransferase enzymes.

Studies on blood group A and H glycosyltransferase enzymes in 54 patients with acute myeloid leukemia were carried out on serum derived from blood samples taken prior to treatment, and in 16 cases, further tests were performed during clinical remission and at the time of relapse. The enzyme assay procedures, using low-molecular-weight compounds as sugar acceptors and radioactive nucleotide sugars as the donor substrates, have been described by Chester et al. (Eur. J. Biochem., 69:583, 1976). Abnormally low values of H enzyme (expressed as percentage of radioactive sugar incorporated into product; (that is, 1 to 3%) were observed in practically all presentation sera, but the values reverted to normal levels (3 to 15%) at the time of clinical remission and then became low once more with the development of drug resistance and clinical relapse. A enzyme levels measured in presentation sera which had demonstrated abnormal H enzyme were mostly within the normal range. In 2 of 5 A1 patients; sera and in all of three A2 patients increases in enzyme levels were observed in remission as compared with presentation serum samples. The depression of biosynthetic enzymes in acute leukemic sera could not be accounted for on the basis of competitive inhibitors or catabolic enzymes. It is proposed that changes of serum glycosyltransferase enzymes reflect alterations in a leukemic cell population and that knowledge of these changes may be of value in prognosis in acute leukemia.

Probable biosynthetic pathway for the synthesis of the B antigen for Bh variants.

Red cells and serum from two Bh variants (B+H-cells) have been investigated for B and H blood group glycosyltransferases. The H enzyme could not be detected using either type 1 or type 2 chain acceptors. The B enzyme was present in normal amount when 2'-fucosyllactose was used as substrate, neither 6'-fucosyllactose nor 6'-fucosyllactosamine could act as acceptors for the B enzyme. Upon treatment of the Bh red cells by the B-degrading enzyme from Trichomonas foetus the B antigen was destroyed while H determinants were uncovered (B-H + cells). The cells thus treated could be further converted into A&H-red cells by the action of the A transferase from human blood group A serum. Previous treatment of the B-H + cells by the H-degrading enzyme from T. foetus, however, led to B-/-erythrocytes and prevented their conversion into A red blood cells by the A enzyme. The results clearly demonstrate that, as found in normal B individuals, the B antigen from Bh cells is built up from the H precursor and provide additional evidence that H is not a completely silent gene in Bh individuals.

Human blood-group A- and H-specified glycosyltransferase levels in the sera of newborn infants and their mothers.

The level of blood-group A1-specified alpha,3'-N-acetyl-D-galactosaminyl-transferase in the serum of recently-delivered women was found to be appreciably lower than the level of this enzyme in the serum of non-pregnant adults and of newborn infants; a similar but less striking decrease was observed in the levels of the A2-specified alpha,3'-N-acetyl-D-galactosaminyltransferase and the H-specified alpha,2'-L-fucosyltransferase. Although the red cells of newborn infants are known to have relatively few A and H antigen sites, the serum of neonates was found to have a level of A1- and A2-dependent N-acetylgalactosaminyltransferases and H-dependent fucosyltransferase as high as, if not higher than, the serum of non-pregnant adults. This finding is compatible with the fact that the haemopoietic tissue contributes only about 20% of the serum transferase level.

[Demonstration of N-acetylgalactosaminyltransferase activity in the murine lymphoma YC8]. / Mise en évidence d'une activité N-acétyl-galactosaminyltransférase au cours du lymphome murin YC8.

The N-acetyl-galactosaminyltransferase activity has been studied in the biological fluids of YC8-lymphoma bearing Balb/c mice. It is enhanced during tumor development from 1 to 30 times in peritoneal fluids and from 1 to 10 times in sera. This activity is nil in urines. Optimal requirements for activity have been determined. Results suggest the existence, during tumor process not only of an enhancement of enzymatic activity, but also of a new molecule synthesis, molecules which are endogenous acceptors for the enzyme.

[Serum glycosyltransferase activity associated with antigen biosynthesis in blood groups A and B. Study of normal B group and cis AB group subjects]. / Activités glycosyltransférasiques sériques associées à la biosynthése des antigénes de groupes sanguins A et B. Application à l'étude de sujets B normaux et cis AB

Two groups of alpha-D-galactosyltransferase activity were described in the sera of 132 caucasian B donors of the National Centre of Blood Transfusion, Paris. The group having the lowest activity represented 84% of the caucasian population, the second one: 16%. They were related neither to the secretory status nor to the genotype of the individuals studied. In some of the 20 african sera studied a third group of activity could be defined. In the caucasian population, these groups were clearly correlated to the agglutinability of the red cells by anti B serum. But no clearcut agglutinability groups could be defined. In the african heterogeneous population, there was no relationship between the two parameters. We did not find the third group of Gibbs. The agglutinability of the B red cells by anti-H (Ulex europaeus) varied inversely as transferase activity and two relationships were distinguished according to the ethnical origin. Only in the caucasian population, this phenomenon was in close relationship with the B agglutinability. Glycosyltransferases activities associated to the biosynthesis of A and B blood group antigens were looked for in 12 cis AB samples from 5 families. The alpha-N-acetyl-D-galactosamyltransferase activity was always weaker than that of normal A and AB sera except for A/cis AB genotype individuals. The presence of alpha-D-galactosyltransferase activity was demonstrated using 2' fucosyllactose as acceptor and by transformation of O red cells into weak B active ones.

[Properties of alpha-N-acetylgalactosaminyltransferases in sera of group A and weak A subjects]. / Etude des propriétés alpha-N-acétylgalactosaminyltransférasiques des sérums de sujets A et "A faible"

Activities of A gene-specified alpha-N-acetylgalactosaminyltransferase in sera of subjects with "normal A" (A1, A2, A1B, A2B) and "weak A" phenotypes (A3, Ax, Aend, Am, Ay and Ael) have been investigated using both 2'fucosyllactose and O red blood cells as exogeneous acceptors with H serological specificity. Among the normal A samples, the enzymatic studies provided two main conclusions: 1) A1 and A2 gene-products respectively found in A1 or A1B and A2 or A2B sera, could be distinguished from each other according to their kinetic properties, namely: (i) optimum pH activity (6.0 or 7.0 respectively); (ii) metal requirement (effect of Mg++); (iii) in vitro conversion of O into A RBC. As a matter of fact, A1 and A1B sera gave high RBC converted titer, but A2 sera lead to very weak or negative values. However an unexpected difference of behavior noticed between A2 and A2B sera,--the latter being a good source of A enzyme for RBC conversion,--suggested a possible interaction between A2 and O gene products on the one hand, A2 and B gene products on the other. 2) A simple and reproducible assay, established on pH ratio dependent values, allows the direct recognition of A1A2 genotypes among A1 subjects, which means that both A1 and A2 enzymes are present in heterozygote sera. Further studies on "weak A" samples sera can be summarized as follows: a) Two kinds of Am bloods--tentatively named AmA1 and AmA2--were identified according to the kinetic properties of their enzymes which were respectively similar to those described in A1 and A2 sera. However, the activity of "Am" enzymes was only 1/2 to 1/3 of that of controls A1 or A2. The genetical background of Am phenotypes was then discussed to the light of results leading to the experimental identification of A2AmA1 genotypes. b) In prolonged incubation times, a very weak A enzyme activity (1/50 to 1/200 of controls) was noticed in Ay sera. Therefore the recessive "yA" gene appears to be not completely silent, but blocks the A enzyme synthesis in most of the cell lines of the organism. c) A3 samples are highly heterogeneous. Two groups of individuals were first identified in which the A enzyme activities was weak (group I, activity 1/8 to 1/20 of controls) or absent (group II). Moreover the occurrence of an A3serum exhibiting strong A enzyme activity (1/2 to 1/3 of controls) and kinetic properties similar to those described in A1 sera suggest the possible existence of a third group. d) In standard or prolonged incubation times, alpha-N-acetylgalactosaminyltransferase seems to be absent in several samples of Ax, Aend or Ael sera. In these cases, as well as in the A3 group II samples sera, no hydrolase or inhibiting substances for transferase activity was demonstrated. Finally, from all these results, the significance of the transferases activities and properties measurements performed directly on whole sera was discussed.