α1,4-Fucosyltransferase Activity: A Significant Function in the Primate Lineage has Appeared Twice Independently

Fabrice Dupuy 1 3 6 Agne`s Germot 1 3 6 Mickael Marenda 1 3 6 Rafael Oriol 0 1 3 Antoine Blancher 1 2 3 Raymond Julien 1 3 7 Abderrahman Maftah 1 3 6 0 Glycobiologie, INSERM U504, Universite Paris XI , Villejuif Cedex , France 1 Laboratoire de Glycobiologie et Biotechnologie, EA 3176, Faculte des Sciences et Techniques, Universite de Limoges , 123 Avenue Albert Thomas, 87060 Limoges Cedex , France 2 Laboratoire d'Immunoge ne tique Mole culaire, Hopital Purpan , Toulouse , France 3 Abbreviations: Fuc , fucose; FUT, fucosyltransferase; FUT3, FUT3-encoded Lewis a3/4-fucosyltransferase; FUT5, FUT5-encoded a3/4-fucosyltransferase; FUT6, FUT6-encoded plasma a3-fucosyl- transferase; FUTb, bovine futb-encoded a3-fucosyltransferase; Gal, ga- lactose; GlcNAc, N-acetylglucosamine; Tricine, N-[2-hydroxy-1,1- bis(hydroxymethyl)ethyl] glycine; The Lewis blood group antigens are Le 4 , Fuca1,2Galb1,3(Fuca1,4) GlcNAc; Le 5 , Galb1,3(Fuca1,4)GlcNAc; Le 6 Laboratoire de Glycobiologie et Biotechnologie , EA 3176 , Institut des Sciences de la Vie et de la Sante , Faculte des Sciences et Techniques , Limoges Cedex , France 7 Unite de Ge ne tique Mole culaire Animale, UMR-INRA 1061, Institut des Sciences de la Vie et de la Sante , Faculte des Sciences et Techniques , Limoges Cedex , France In the animal kingdom the enzymes that catalyze the formation of a1,4 fucosylated-glycoconjugates are known only in apes (chimpanzee) and humans. They are encoded by FUT3 and FUT5 genes, two members of the Lewis FUT5-FUT3-FUT6 gene cluster, which had originated by duplications of an a3 ancestor gene. In order to explore more precisely the emergence of the a1,4 fucosylation, new Lewis-like fucosyltransferase genes were studied in species belonging to the three main primate groups. Two Lewis-like genes were found in brown and ruffed lemurs (prosimians) as well as in squirrel monkey (New World monkey). In the latter, one gene encodes an enzyme which transfers fucose only in a1,3 linkage, whereas the other is a pseudogene. Three genes homologous to chimpanzee and human Lewis genes were identified in rhesus macaque (Old World monkey), and only one encodes an a3/4fucosyltransferase. The ability of new primate enzymes to transfer fucose in a1,3 or a1,3/4 linkage confirms that the amino acid R or W in the acceptor-binding motif ''HH(R/W)(D/E)'' is required for the type 1/type 2 acceptor specificity. Expression of rhesus macaque genes proved that fucose transfer in a1,4 linkage is not restricted to the hominoid family and may be extended to other Old World monkeys. Moreover, the presence of only one enzyme supporting the a1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution. Introduction In humans, six genes encoding a3- and a3/4-fucosyltransferases, FUT3 to FUT7 and FUT9, have been characterized (Goelz et al. 1990; Kukowska-Latallo et al. 1990; Weston et al. 1992a, 1992b; Sasaki et al. 1994; Kaneko et al. 1999). The corresponding enzymes are Golgi resident proteins and have a common structure, including a short cytoplasmic tail, a transmembrane domain, a stem region, and a putative globular domain in the COOH end. With different efficiencies, all these enzymes allow fucose to be substituted in a1,3 on N-acetylglucosamine of the glycoconjugate terminal lactosamine to form Lex antigen (fig. 1). Among them, only a3/4-fucosyltransferases encoded by FUT3 and FUT5 are also able to transfer fucose in a1,4 linkage on the same N-acetylglucosamine to form Lea antigen (fig. 1). The FUT3, or Lewis gene, allows the expression of Lea and Leb antigens in exocrine secretions and on red cells (Oriol 1995). The FUT5 gene directs in vitro synthesis of both fucosylated type 1 (Lea and Leb) and type 2 (Lex and Ley) antigens (Costache et al. 1997b). The FUT6 controls the expression of a plasmatic a3-fucosyltransferase enzyme involved in Lex and Ley antigen synthesis (Mollicone et al. 1990). FUT3, FUT5, and FUT6 genes are organized in a cluster located on the short arm of human chromosome 19 in the band 13.3 (Reguigne-Arnould et al. 1995). They constitute the Lewis fucosyltransferase gene family. Although the corresponding enzymes share about 85% sequence identity, they present different acceptor substrate specificities. Biochemical studies revealed that the main amino acids involved in their type 1/type 2 acceptor substrate specificities are confined to the amino terminal segment of the catalytic domain (Legault et al. 1995; Nguyen et al. 1998). Recently, we defined in this segment the conserved motif HH(W/R)(D/E) (Dupuy et al. 1999), named in the present study as the acceptor-binding motif. On the basis of site-directed mutagenesis, our previous experiments proved that in this motif Trp or Arg is involved in the type 1 or type 2 acceptor substrate specificity, respectively (Dupuy et al. 1999). Oulmouden et al. (1997) and Wierinckx et al. (1999) have shown that the ancestor of futb, a bovine a3-fucosyltransferase gene, is orthologous to the ancestor of the three Lewis genes. The bovine enzyme has the HHRE acceptor-binding motif and transfers fucose only onto type 2 acceptor substrates in vitro (fig. 1), which is in agreement with the absence of Lea and Leb antigens in all the tested bovine tissues (Oulmouden et al. 1997). Other futb orthologous genes were identified in Chinese hamster (Zhang et al. 1999) and mouse (Gersten et al. 1995). Assuming that the human Lewis cluster FUT5-FUT3-FUT6 had originated from an ancestral Lewis gene by two successive duplications after FIG. 1.Synthesis of Lewis antigens by a1,3- and a1,3/4-fucosyltransferases. A, In the case of a1,3 fucosylation, Lewis antigens are synthesized by transfer of fucose in a1,3 linkage on the GlcNAc of different acceptor substrates. From the disaccharide type 2 acceptor substrate, Lex antigen is produced. When the disaccharide is already a1,2 fucosylated on Gal (H-type 2), the antigen produced is Ley. B, The a1,4 fucosylation of type 1 and H-type 1 acceptor substrates produces Lea and Leb antigens, respectively. The enzymes which catalyzed these reactions are indicated. They all use GDP-Fuc as the donor substrate. R refers to the remaining part of the glycoconjugate. the great mammalian radiation, 80 MYA, it was hypothesized that the emergence of a1,4 activity was a late event in the primate lineage. Indeed, FUT3 and FUT5 enzymes, which transfer fucose in a1,4 linkage, were only known in two hominoids, human and chimpanzee (Costache et al. 1997a). Nevertheless, Lea antigens had been immunodetected in the saliva of other Old World monkeys (orangutan for hominoids and baboon, vervet monkey and rhesus macaque for Cercopitecoides) but not in New World monkeys (spider and squirrel monkeys and marmoset) (Moor-Jankowski and Wiener 1968). Taken together, these data mean that functional a3/4-fucosyltransferases could be present in Catarrhines (Old World monkeys) and absent in (...truncated)