Identification of two functionally deficient plasma alpha 3-fucosyltransferase (FUT6) alleles.

One Indonesian individual without detectable plasma alpha3-fucosyltransferase activity was identified with three point mutations, 730C>G (L244V), 907C>G (R303G), and 370C>T (P124S), in the coding region of one FUT6 allele. Another individual, expressing weak plasma alpha3-fucosyltransferase activity, had the 907C>G together with the 370C>T mutation, but did not have the 730C>G mutation. PCR-RFLP analyses of complete families confirmed the segregation of these alleles and illustrated the existence and inheritance of the [370C>T; 907C>G] mutated allele in three additional families. Altogether, this allele was found heterozygously in nine Indonesian and two Swedish individuals, all with detectable plasma alpha3-fucosyltransferase activities. The FUT6 allele with the three mutations (370C>T; 730C>G; 907C>G) was identified heterozygously in only two Indonesian individuals, both having the inactivating 739G>A mutation in the other allele and both lacking plasma alpha3-fucosyltransferase activity. Enzyme studies made on transiently transfected COS-7 cells demonstrated that the combination of the 370C>T, 730C>G and 907C>G mutations decreased the V(max) by more than 80%, but caused no obvious change of the apparent K(m) values for GDP-fucose and Gal-N-acetyllactosamine. In comparison, chimeric constructs with the isolated 730C>G or 907C>G mutations decreased the V(max) values by about two thirds and one third, respectively.

Cellular expression of H and B antigens in the rat olfactory system during development.

Developmental expression of H and B antigens in the rat olfactory system was studied from the embryonic day 14 up to the postnatal day 30. The H antigen was detected in the olfactory and vomeronasal epithelia as early as fetal day 14, whereas the B antigen first appeared 2 days later. The anti-H reagent reacted strongly with sensory receptors and weakly with supporting cells in both epithelia, whereas the anti-B reagent was specific for olfactory receptors. In the main olfactory epithelium, the H antigen was expressed from fetal day 19 by most of the receptor cells, whereas the B determinant was expressed from fetal day 16 to postnatal day 3 by only a few neuroreceptors mostly located near the epithelial surface. After the postnatal day 3, B positive neurons increased in number from the periphery toward the deeper mucosal layers and they were distributed over 3/4 of the epithelial thickness in 15- and 30-day-old rats. In the main olfactory bulb, a widespread glomerular B staining with variable binding intensity between adjacent glomeruli was already observed at birth. The vomeronasal receptor cells and their axon terminals in the accessory olfactory bulb exhibited a comparable developmental expression of the B antigen. Results suggest that the B antigen could be regarded as a marker of neuronal maturation of both the olfactory and vomeronasal receptor cells; moreover, its first appearance in the receptor cells might be temporally related to the formation of synapses between receptor axons and deutoneurons in the bulb.

Expression of ABH and X (Lex) antigens in various cells.

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.

A simple computer programme for standard curve tracing and calculations in RIA following sigmoid patterns.

A computer programme is described for the analysis of radioimmunoassays assuming a general sigmoid shape. The mathematical treatment uses the results obtained from a sigmoid equation to perform a limited operational search in order to optimize the fitness of the curve. The programme can be installed in most desk top microcomputers and has been tested for the calculation of Ig concentrations in biological fluids.

Molecular cloning of a putative alpha3-fucosyltransferase from Schistosoma mansoni.

Alpha 3-fucosylation of protein or lipid substrates is an important component of the host/parasite interactions during schistosomiasis. In this process, alpha3-fucosyltransferases (alpha3-FucTs) are considered as key enzymes ensuring both parasite survival and adaptation in their (in)vertebrate hosts. In this paper, we report the molecular cloning of a putative alpha3-FucT from Schistosoma mansoni that we termed SmFucTA. The full-length SmFucTA encodes a typical transmembrane type II protein with a short cytoplasmic domain, a transmembrane segment and a long C-terminal catalytic domain. In this region, the GDP-fucose binding site is well conserved whereas the putative acceptor site displays sequence divergence compared to the corresponding region from vertebrate and invertebrate alpha3-FucTs. Southern blot analysis suggested that SmFucTA is present as several copies or has highly related counterparts in the S. mansoni genome. Northern blot revealed a single SmFucTA transcript at 2 kb in adult worms. Affinity purified antibodies directed against recombinant SmFucTA identified a 50 kDa native protein that localizes to the subtegumental and parenchymal regions of adult worms.

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human.

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.

Cellular expression and genetic control of ABH antigens in primary sensory neurons of marmoset, baboon and man.

ABH antigens have been demonstrated in the posterior root ganglia (PRG) of 3 primate species (marmoset, baboon and man). Their expression corresponded to the ABO phenotype of the individual and was independent of the secretor gene. In marmosets more cells were positive for H (33 +/- 9%) than for A (19 +/- 6%). In baboons A or B antigens were more easily detected (66 +/- 9%) than the H antigens (48 +/- 5%). In humans more than two-thirds of PRG cells were positive for H but only a small proportion of these were positive for A or B. The ABH antigens were found mainly in the small and intermediate-size neurons whose central processes project to lamina II of the spinal cord posterior horn. Unipolar neurons of the Gasserian ganglion, neurons of the mesencephalic nucleus of the trigeminal nerve and of some visceral ganglia have also been shown to express these antigens which are also present in the fibre layer and glomeruli of the olfactory bulbs.

Distribution of GICA in normal gastrointestinal and endocervical mucosae and in mucinous ovarian cysts using antibody NS 19-9.

Through the immunoperoxidase method using antibody 19-9, we demonstrated the presence of gastrointestinal cancer-associated antigen (GICA) in normal gastrointestinal and endocervical mucosae and in benign mucinous ovarian cysts of pure endocervical type in Le(a+b-) and Le(a-b+) patients exclusively. However, GICA was more quantitatively expressed in Le(a+b-) than in Le(a-b+) patients. GICA was always encountered in the ducts of esophageal glands, Wirsung's ducts, goblet cells of villosities near the ileo-colonic junction, and the mucus columnar cells of the endocervical mucosae or ovarian mucinous tumor epithelium. In other areas of the gastrointestinal tract, GICA was sometimes found in the mucus cells of esophageal glands, near the neck cells of gastric mucosae, in goblet cells of the upper part of the small intestinal villosities, and in colonic Lieberkühn glands, especially in the cell maturation compartment. GICA therefore appears to be a normal component of gastrointestinal and endocervical mucosae.

Appearance of H and B antigens in primary sensory cells of the rat olfactory apparatus and inner ear.

In the rat olfactory apparatus and inner ear the H antigen was first detected at the 15th day of gestational age. The B antigen appeared at the 16th day. In the olfactory apparatus H and B antigens were first detected in cells of the olfactory mucosa and of the olfactory bulb primordium. Towards the 16th and 17th days many small positive cells were seen between these two areas. In newborn and adult rats, olfactory bulb H and B antigens were restricted to the processes of the peripheral primary sensory cells. Both H and B antigens were detected in the epithelium of the inner ear and were later concentrated in the hair cells of the organ of Corti, the utricle and the saccule.

Histochemical localization of a beta-galactoside-binding lectin and its binding-sites in developing and adult rat cochlea.

The presence of an endogenous rat beta-galactoside-specific lectin (S-Lac) and its specific binding ligands was analyzed, using a rabbit anti-human brain lectin of 14 kDa antibody (anti-HBL-14) and a probe consisting of a biotinylated derivative of the human lectin (HBL-biot) in adult and in developing rat cochleas. At PD1, some epithelial cells of the outer spiral sulcus (OSS) were specifically recognized by the anti-HBL-14 antibody. Then, anti-HBL-14 immunoreactivity progressively appeared in all OSS epithelial cells, and, from PD9 on, it also appeared in inner spiral sulcus (ISS) epithelial cells. Finally, young adult rats exhibited a well defined anti-HBL-14 immunoreactivity in epithelial cells of the inner and outer spiral sulcus. Using the biotinylated probe, from PD3 on, tectorial membrane (TM) and the sensory cells apical surfaces were labelled. The glycoconjugate expression within the developing organ of Corti seems to be highly complex. Lectin was detected in developing and adult epithelial cells surrounding the organ of Corti. In contrast, other glycoconjugates related to B and H human blood group antigens, were transiently present on sensory cells of the organ of Corti. Present findings can be correlated with significant events in development of the cochlea, mainly with cell-cell recognition or cellular adhesion.

Five specificity patterns of (1----3)-alpha-L-fucosyltransferase activity defined by use of synthetic oligosaccharide acceptors. Differential expression of the enzymes during human embryonic development and in adult tissues.

The use of synthetic trisaccharides as acceptors led to the definition of five main (1----3)-alpha-L-fucosyltransferase activity patterns in human adult tissues: (I). Myeliod cells, granulocytes, monocytes, and lymphoblasts, transfer an alpha-L-fucopyranosyl group to O-3 of a 2-acetamido-2-deoxy-D-glucosyl residue of H blood-group Type 2 oligosaccharide [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R] with Mn2+ as activator. (II) Brain has the same acceptor specificity pattern as myeloid cells, but can also use Co2+ as activator. (III) Plasma and liver transfer an alpha-L-furopyranosyl group to H blood-group Type 2 and to sialyl-N-acetyllactosamine [alpha-NeuAc-(2----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R]. (IV) Intestine, gall bladder, kidney, and milk have the same activity as (III), but also transfer an alpha-L-fucopyranosyl group to O-4 of a 2-acetamido-2-deoxy-D-glucose residue of H blood-group Type 1 [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R] and sialyl Type 1 [alpha-NeuAc-(1----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R]. (V) Stomach mucosa is not able to use sialyl-N-acetyllactosamine, but can transfer an alpha-L-fucopyranosyl group to the other Type 1 and Type 2 acceptors. Unlike in adult tissue, a single myeloid-like pattern of (1----3)-alpha-L-fucosyltransferase activity was found at early stages of development in all tissues tested. This embryonic enzyme is later progressively replaced by enzymes or mixtures of enzymes having the corresponding adult patterns of enzyme expression. All lymphoblastoid cell lines and half of the tumor epithelial cell lines tested expressed the myeloid-like pattern of enzyme found in normal embryonic tissues. The remaining tumor epithelial cell lines expressed different forms of (1----3/4)-alpha-L-fucosyltransferase acceptor specificity patterns.

Molecular genetics of H, Se, Lewis and other fucosyltransferase genes.

Seven human fucosyltransferase genes have been cloned and registered in the Genome Data Base (GDB) as FUT1 to FUT7. According to their acceptor specificity, two main groups of enzymes can be distinguished. The alpha-2-fucosyltransferases: FUT1 (H) of red cells and vascular endothelium and FUT2 (Se) of exocrine secretions. The alpha-3-fucosyltransferases: FUT3 (Lewis) of exocrine secretions; FUT4 (myeloid) of white cells and brain; FUT5 whose tissue distribution has not been defined as yet; FUT6 (plasma) present in plasma, renal proximal tubules and hepatocytes; FUT7 (leukocyte) found in neutrophils. A high DNA sequence homology has been detected among the genes within each of these two groups, while no homology has been detected between the genes of the two groups. Point mutations responsible of inactivating genetic polymorphisms have been found for FUT1, FUT2, FUT3 and FUT6, while FUT4 and FUT7 seem to be genetically monomorphic. FUT4 has been detected in all tissues of 5 to 10 weeks old human embryos suggesting that it may play a role in development. FUT7 is a candidate for the control of the synthesis of the receptors of selectin mediated cell adhesion.

Advances in molecular genetics of alpha-2- and alpha-3/4-fucosyltransferases.

Fucosyltransferases are involved in the last steps of the biosynthesis of ABH and Lewis oligosaccharide antigens. Seven human genes (FUT1 to FUT7) and one pseudogene (Sec 1) have been cloned and localized on different chromosomes (9q34.3; 11q21; 19p13.3 and 19q13.3). Their locations and their high degree of primary sequence identity, suggest that they have appeared by successive duplications followed by translocation and divergent evolution. Their expression is tissue specific and they present a switch during human embryo-foetal development similar to that of hemoglobins. Polymorphic genes FUT1-FUT2 and FUT3-FUT5-FUT6 are organized in two clusters and each gene is partially or totally inactivated by different types of point mutations (nonsense, missense and frame shift), complete gene deletion or a fusion gene. The products of the monomorphic genes FUT4 and FUT7 seem implicated in cell-cell interactions during embryo-foetal development and in the leukocyte adhesion phenomena to endothelial cells in the adult. A phylogenetic tree of the 28 available nucleotide coding sequences of fucosyltransferases has allowed us to situate the duplication events with respect to the separation of species from the main evolutionary path (nematods, birds, mammals, primates and humans). Recently, using a computer approach a general structure of fucosyltransferases has been proposed, inspired from the crystalline structure of the beta-glucosyltransferase of bacteriophage T4. This folding contains two domains with an alternate succession alpha and beta chains. In this model the GDP-fucose binding site would be located between the two domains.

Molecular genetics of alpha-L-fucosyltransferase genes (H, Se, Le, FUT4, FUT5 and FUT6).

Six human alpha-L-fucosyltransferase genes have been registered in the GDB as FUT1 to FUT6 according to the chronology of their description. FUT1 and FUT2 encode the alpha(1,2)fucosyltransferases H and Se respectively. The FUT2 gene has not been cloned, but it is expected to be closely linked to FUT1 on the long arm of chromosome 19. FUT3, FUT4, FUT5 and FUT6 encode different alpha(1,3)fucosyltransferases which share between 60 and 90% homology with each other, but none with FUT1. Missense and nonsense point mutations have been found to inactivate the cognate enzymes of FUT1, FUT3 and FUT6. FUT3 and FUT6 are closely linked on the short arm of chromosome 19 and encode the Lewis and plasma enzymes respectively. The FUT5 gene has been cloned and sequenced, but its tissue expression has not been defined as yet. FUT4 has been mapped to 11q21 and encodes a monomorphic myeloid enzyme. All but FUT4 are genetically polymorphic. The deficient alleles of FUT1 and FUT6 have a very low incidence and they have been found mainly around the Indian Ocean. A myeloid enzyme is present in 5 to 10 week old human embryos and is later progressively replaced by different patterns of adult fucosyltransferase enzymes in all tissues, except in leukocytes and brain which continue to express a FUT4 like enzyme in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical approach to the structure of human blood group B and H glycoconjugate antigens in the three days old rat cochlea.

The possible structure of human blood-group antigens, as found in cochlear hair cells of 3-day-old rats, is suggested. Data were obtained from immunocytochemical studies using 77 antibodies against the major human blood group antigens of the ABO, H, I and Lewis genetic systems. Neither the anti-A-related nor the anti-Lewis-related antibodies showed any positive immunoreaction on hair cells. In contrast, anti-B, anti-AB and anti-H antibodies displayed specific positive immunoreactive patterns on the hair cells. The results suggest that, in immature hair cells, two main glycoconjugate structures of the lactoseries are present: H type 2 antigen, which is the precursor of the B type 2 antigen, and the B type 2 antigen itself. Similar H and B carbohydrate structures have been reported in rat olfactory receptors. The type 2 glycoconjugates carrying these H and B antigens of auditive and olfactory receptors are resistant to fixation and paraffin embedding, suggesting that they might be glycoproteins. These auditive and olfactory H and B antigens must be different from the B-related antigens that are expressed by pseudo-unipolar neurons of rat posterior root ganglia, that are built from type 4 core chains, and that are destroyed by routine paraffin embedding procedures.

Molecular genetics of H.

BACKGROUND AND OBJECTIVES: Formal genetics of ABO, H-h and Se-se systems illustrate that these three systems are genetically independent MATERIALS AND METHODS: Population analysis of phenotypes and family segregation of the ABH related genetic markers RESULTS: Inactivating mutations of FUT1 and FUT2 are compatible with a structural gene model assuming that FUT1 and FUT2 genes encode for two distinct enzymes, one encoding for the H antigen expressed in red cells (FUT1) and the other encoding for the H gene expressed in saliva (FUT2) CONCLUSION: Most inactivating mutations of FUT1 and FUT2 genes are located in the coding region of the genes and are nonprevalent sporadic mutations of relative recent appearance.

Immunohistologic pattern of type 1 (Lea, Leb) and type 2 (X, Y, H) blood group-related antigens in the human pyloric and duodenal mucosae.

Type 1 antigens (Lea and Leb) of the pyloric mucosa are restricted to the mucus cells of the surface epithelium and the neck. In the duodenum they are present in absorptive and goblet cells of duodenal villi as well as in the glands of Lieberkühn. Type 2 antigens (X, Y, and H) are also present in these areas of the mucosa, and both type 1 and type 2 antigens, in this superficial location, are under the control of the secretor gene. In addition, type 2 antigens are also present in the mucus cells of pyloric and Brunner's glands, but in this deep location they are not controlled by the secretor gene. These results suggest the existence of two differentiation patterns for the expression of glycosyltransferases in the stem cells. The upward differentiation process would favor the expression of the alpha-2-L-fucosyltransferase coded by the Se gene, and the downward differentiation process would favor the expression of the alpha-2-L-fucosyltransferase coded by the H gene. The knowledge of the distribution and the genetic control of type 1 and type 2 antigenic determinants in normal gastrointestinal tract may help to interpret the modifications in the expression of these antigens in pathologic conditions such as neoplasia.

Genetics of ABO, H, Lewis, X and related antigens.

The present knowledge on chemical, enzymatic, serologic and genetic aspects of ABH antigens is reviewed in an effort to produce a simple and coherent genetic model for the biosynthesis of these antigens and chemically related structures. The genetic control of type 1 (Le(a), Le(b), Le(c) and Le(d)), type 2 (X, Y, I, and H), type 3 and type 4 ABH and related antigens in different animal and human tissues is analyzed, taking into account the properties of the glycosyltransferases which are involved in their synthesis and considering possible competition for common acceptor and donor substrates. The phylogeny of ABH determinants shows that they appeared as tissular antigens much earlier than as red cell antigens. The ontogeny of ABH antigens suggests that they behave as differentiation antigens, and an effort is made to correlate their tissular distribution in the adult with the embryological origin of each tissue.

Evolution of alpha 2-fucosyltransferase genes in primates: relation between an intronic Alu-Y element and red cell expression of ABH antigens.

Coding sequences of the paralogous FUT1 (H), FUT2 (Se), and Sec1 alpha 2-fucosyltransferase genes were obtained from different primate species. Analysis of the primate FUT1-like and FUT2-like sequences revealed the absence of the known human inactivating mutations giving rise to the h null alleles of FUT1 and the se null alleles of FUT2. Therefore, most primate FUT1-like and FUT2-like genes potentially code for functional enzymes. The Sec1-like gene encodes for a potentially functional alpha 2-fucosyltransferase enzyme in nonprimate mammals, New World monkeys, and Old World monkeys, but it has been inactivated by a nonsense mutation at codon 325 in the ancestor of humans and African apes (gorillas, chimpanzees). Human and gorilla Sec1's have, in addition, two deletions and one insertion, respectively, 5' of the nonsense mutation leading to proteins shorter than chimpanzee Sec1. Phylogenetic analysis of the available H, Se, and Sec1 mammalian protein sequences demonstrates the existence of three clusters which correspond to the three genes. This suggests that the differentiation of the three genes is rather old and predates the great mammalian radiation. The phylogenetic analysis also suggests that Sec1 has a higher evolutionary rate than FUT2 and FUT1. Finally, we show that an Alu-Y element was inserted in intron 1 of the FUT1 ancestor of humans and apes (chimpanzees, gorillas, orangutans, and gibbons); this Alu-Y element has not been found in monkeys or nonprimate mammals, which lack ABH antigens on red cells. A potential mechanism leading to the red cell expression of the H enzyme in primates, related to the insertion of this Alu-Y sequence, is proposed.