{alpha}1,4-Fucosyltransferase Activity: A Significant Function in the Primate Lineage has Appeared Twice Independently

Fabrice Dupuy, Agnès Germot, Mickaël Marenda, Rafaël Oriol, Antoine Blancher, Raymond Julien and Abderrahman Maftah3

*Laboratoire de Glycobiologie et Biotechnologie, EA 3176, Institut des Sciences de la Vie et de la Santé, Faculté des Sciences et Techniques, Limoges Cedex, France;
{dagger}Glycobiologie, INSERM U504, Université Paris XI, Villejuif Cedex, France;
{ddagger}Laboratoire d'Immunogénétique Moléculaire, Hôpital Purpan, Toulouse, France;
§Unité de Génétique Moléculaire Animale, UMR-INRA 1061, Institut des Sciences de la Vie et de la Santé, Faculté des Sciences et Techniques, Limoges Cedex, France


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In the animal kingdom the enzymes that catalyze the formation of {alpha}1,4 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 {alpha}3 ancestor gene. In order to explore more precisely the emergence of the {alpha}1,4 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 {alpha}1,3 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 {alpha}3/4-fucosyltransferase. The ability of new primate enzymes to transfer fucose in {alpha}1,3 or {alpha}1,3/4 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 {alpha}1,4 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 {alpha}1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In humans, six genes encoding {alpha}3- and {alpha}3/4-fucosyltransferases, FUT3 to FUT7 and FUT9, have been characterized (Goelz et al. 1990Citation ; Kukowska-Latallo et al. 1990Citation ; Weston et al. 1992a, 1992b;Citation Sasaki et al. 1994Citation ; Kaneko et al. 1999Citation ). 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 {alpha}1,3 on N-acetylglucosamine of the glycoconjugate terminal lactosamine to form Lex antigen (fig. 1 ). Among them, only {alpha}3/4-fucosyltransferases encoded by FUT3 and FUT5 are also able to transfer fucose in {alpha}1,4 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 1995Citation ). 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. 1997bCitation ). The FUT6 controls the expression of a plasmatic {alpha}3-fucosyltransferase enzyme involved in Lex and Ley antigen synthesis (Mollicone et al. 1990Citation ). 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. 1995Citation ). 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. 1995Citation ; Nguyen et al. 1998Citation ). Recently, we defined in this segment the conserved motif "HH(W/R)(D/E)" (Dupuy et al. 1999Citation ), 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. 1999Citation ).



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Fig. 1.—Synthesis of Lewis antigens by {alpha}1,3- and {alpha}1,3/4-fucosyltransferases. A, In the case of {alpha}1,3 fucosylation, Lewis antigens are synthesized by transfer of fucose in {alpha}1,3 linkage on the GlcNAc of different acceptor substrates. From the disaccharide type 2 acceptor substrate, Lex antigen is produced. When the disaccharide is already {alpha}1,2 fucosylated on Gal (H-type 2), the antigen produced is Ley. B, The {alpha}1,4 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

 
Oulmouden et al. (1997)Citation and Wierinckx et al. (1999)Citation have shown that the ancestor of futb, a bovine {alpha}3-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. 1997Citation ). Other futb orthologous genes were identified in Chinese hamster (Zhang et al. 1999Citation ) and mouse (Gersten et al. 1995Citation ). Assuming that the human Lewis cluster FUT5-FUT3-FUT6 had originated from an ancestral Lewis gene by two successive duplications after the great mammalian radiation, 80 MYA, it was hypothesized that the emergence of {alpha}1,4 activity was a late event in the primate lineage. Indeed, FUT3 and FUT5 enzymes, which transfer fucose in {alpha}1,4 linkage, were only known in two hominoids, human and chimpanzee (Costache et al. 1997aCitation ). 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 1968Citation ). Taken together, these data mean that functional {alpha}3/4-fucosyltransferases could be present in Catarrhines (Old World monkeys) and absent in Platyrrhines (New World monkeys).

Considering that the change of type 2 toward type 1 substrate specificity during mammalian evolution might have been of high significance for primate lineage, we analyzed the molecular status of Lewis-like genes belonging to the three main primate groups that have not been studied to date, prosimians (Eulemur fulvus and Varecia variegata), New World monkeys (Saimiri sciureus), and Cercopitecoides (Macaca mulatta). We show that the {alpha}1,4 fucose transfer is not restricted to hominoids and that this activity might have appeared early in the primate lineage. Also, characterization of these new genes provide new insights into the chronology of the duplication events at the time of origin of the human Lewis gene cluster.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials
Blood samples of brown lemur (E. fulvus), squirrel monkey (S. sciureus), and rhesus macaque (M. mulatta) were obtained from the Centre de Primatologie, Université Louis Pasteur (Strasbourg, France) and the ruffed lemur (V. variegata rubra) from the "Parc du Reynou" (Limoges, France). Type 1 (Galß1,3GlcNAc-(CH2)7-CH3), type 2 (Galß1,4GlcNAc-(CH2)7-CH3), Gal{alpha}1,3 type 1 (Gal{alpha}1,3Galß1,3GlcNAc-(CH2)7-CH3), and Gal{alpha}1,3 type 2 (Gal{alpha}1,3Galß1,4GlcNAc-(CH2)7-CH3) di- and trisaccharide acceptor substrates were kindly provided by Dr. C. Augé (University of Paris XI). Blood group H-type 1 (Fuc{alpha}1,2Galß1,3GlcNAc-sp-biotin) and H-type 2 (Fuc{alpha}1,2 Galß1,4GlcNAc-sp-biotin) trisaccharide substrates were purchased from Syntesome (Munich, Germany).

Southern Blot Analysis
Human and primate genomic DNA were prepared from blood samples (QIAmp Blood Kit, QIAGEN Inc., Hilden, Germany). Ten micrograms was subjected to digestion with EcoRI and BamHI endonucleases, and the fragments were separated in 1% (w/v) agarose gel. DNA was depurinated for 20 min with 0.25 N HCl, denaturated for 30 min with 0.4 N NaOH, and transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Europe GmbH, Orsay, France). A 402-bp human FUT3 catalytic domain probe was generated by PCR using Ps5 and Pas4 primers (table 1 ). Twenty-five nanograms was labeled with [{alpha}-32P]dCTP by random priming (GIBCO BRL, Pontoise, France). Hybridization was carried out for 14 h at 65°C in a buffer containing 10% (w/v) dextran sulfate, 1% (w/v) SDS, 0.5 M NaCl, and 100 µg of sheared salmon sperm DNA. Blot was washed three times at 42°C for 10 min each with 2 x SSC, 2 x SSC–0.1% SDS, and 1 x SSC–0.1% SDS and then analyzed by phosphorimaging.


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Table 1 Primers Used to Amplify and Sequence Primate Lewis-like Sequences

 
Amplification and Cloning of Primate Lewis-like Coding Sequences
The one-step PCR strategy consisted first of the alignment of all {alpha}3- and {alpha}3/4-fucosyltransferase DNA sequences, and second of the designing of specific primers in the highest-conserved regions of Lewis sequences. The rhesus macaque and squirrel monkey coding sequences were amplified by PCR at 61°C using Ps1 and Pas1 primers (table 1 ). Partial lemur coding sequences were obtained with Ps2 and Pas2 primers. PCR was performed by 35 rounds with AGSGoldTM Taq DNA polymerase (Hybaid, Germany) and 50 ng of genomic DNA. PCR products were cloned in the pGEM-T Easy vector (Promega, Madison, Wis.). The recombinant plasmids containing simian Lewis-like sequences were partially sequenced using T7 and SP6+ vector primers (table 1 ) and the dideoxy chain termination method (Sanger, Nicklen, and Coulson 1977Citation ). A dye-labeling chemistry kit (PRISMTMReady Reaction Ampli Taq® FS, USA) and the ABI PrismTM 310 Genetic Analyzer (Perkin-Elmer, Norwalk, Colo.) were used. Primate sequences of interest were inserted into EcoRI restriction site of pcDNA1/Amp mammalian expression vector (Invitrogen, Carlsbad, Calif.). Their orientations were checked, and their complete sequence was determined using the primers given in table 1 .

Molecular and Phylogenetic Analyses
Nucleotide and amino acid sequences of characterized {alpha}3- and {alpha}3/4-fucosyltransferases were retrieved from GenBankTM database by BLAST (Altschul et al. 1990Citation ), using the sequences determined in this study. Their accession numbers are Homo sapiens FUT3 (X53578), FUT5 (M81485), and FUT6 (L01698); Pan troglodytes FUT3 (Y14033), FUT5 (Y14034), and FUT6 (Y14035); Bos taurus FUTb (X87810). Alignments were performed with ClustalW version 1.7 (Thompson, Higgins, and Gibson 1994Citation ) and refined with further manual adjustments using the ED program (Philippe 1993Citation ) of the MUST 2000 package (http://sorex.snv.jussieu.fr/must2000.html). Positions that could not be confidently aligned, i.e., the stem indels, and missing data for lemur sequences were removed from the phylogenetic analyses. Phylogenetic trees were constructed with maximum-likelihood (ML), maximum-parsimony (MP), and distance-based (DB) methods with the programs NUCML from MOLPHY version 2.2 (Adachi and Hasegawa 1996Citation ), and DNAPARS and FITCH from PHYLIP version 3.5c (Felsenstein 1989Citation ), respectively. The distances were computed with the two-parameter substitution model of Kimura (1981Citation ). ML trees were obtained by the quick-add operational taxonomic units (OTUs) search with the Hasegawa-Kishino-Yano model of nucleic acid substitution (Hasegawa, Kishino, and Yano 1985Citation ) retaining the 1,000 top ranking trees. A single most parsimonious tree (length = 415) was obtained, and the length (L) of the ML tree was ln L = -2650.94. Bootstrap proportions (Felsenstein 1985Citation ) were calculated by the analysis of 1,000 resampled data sets for MP and DB methods. For ML, bootstrap proportions were computed by using the RELL method (Kishino, Miyata, and Hasegawa 1990Citation ).

Site-directed Mutagenesis
The mammalian expression vector pcDNA1/Amp (InVitrogen) containing the coding sequence of the rhesus macaque FUT5 was directly used for PCR-based mutagenesis. The Arg124->Trp mutation was performed with the QuickChange Kit (Stratagene, La Jolla, Calif.). The reaction was carried out with 50 ng of vector, 125 ng of sense primer 5'-GACGCGGTCATCGTGCACCACTGGGATATCATGTAC-3', 125 ng of antisense primer 5'-GTACATGATATCCCAGTGGTGCACGATGACCGCGTC-3', and 2.5 U of Pfu DNA polymerase (Stratagene). After 30 s of denaturation at 95°C, the reaction was cycled for 18 rounds (30 s at 95°C, 1 min at 55°C, and 15 min at 68°C). Parental DNA was removed by DpnI endonuclease treatment. Escherichia coli DH5{alpha} strain was directly transformed with the amplified vector. The mutation was verified by sequencing as described previously.

Expression of Mutated Enzymes
Highly pure recombinant plasmids were obtained by anion exchange chromatography (plasmid midi Kit, QIAGEN Inc.). They were used to transiently transfect COS-7 cells using SuperFect Transfection Reagent (QIAGEN Inc.). After 48 h of transfection, proteins were extracted in a lysis buffer (1% [v/v] Triton X100, 10 mM sodium cacodylate (pH 6), 20% [v/v] glycerol, and 1 mM DTT) for 2 h at 4°C. Cellular fragments were eliminated by centrifugation at 4°C (12,000 rpm for 10 min). Protein content was determined with bovine serum albumin as a standard (Bio-Rad, Hercules Calif.) (Bradford 1976Citation ).

Western Blot Analysis
Anti-human FUT3 antibodies were obtained by immunization of rabbits with a truncated form of FUT3 (Pro45-Thr361). This shortened enzyme was produced in E. coli BL21(DE3) cytoplasm associated with a NH2-(His)6-Tag and was purified by affinity chromatography on Ni-NTA-agarose (QIAGEN Inc.). IgG was purified from serum using protein G (HiTrap Protein G, Amersham Pharmacia Biotech. Europe GmbH, Orsay, France). Fifty micrograms of soluble proteins extracted from COS-7 cells was boiled for 3 min after addition of ß-mercaptoethanol (5% v/v) and bromophenol blue (0.02% w/v). Electrophoresis was carried out in Tris/Tricine–10.5% SDS–polyacrylamide gel. Separated proteins were then electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The immunoblot was processed by chemiluminescence detection (Chemiluminescence Blotting Substrate [POD], Roche Molecular Biochemicals, Mannheim, Germany). The blot was first incubated with rabbit anti-human FUT3 antibodies (4 µg/ml) and then with the secondary antibody, a pig anti-rabbit IgG conjugated to horseradish peroxidase (dilution 1:1,000) (Dako, Denmark).

Fucosyltransferase Assays
Fucosyltransferase assays were performed in a mixture (60 µl) containing 25 mM sodium cacodylate (pH 6.5), 5 mM ATP, 20 mM MnCl2, 10 mM {alpha}-l-fucose, 3 µM GDP-[14C]-fucose (310 mCi/mmol; Amersham Pharmacia Biotech., U.K.), and 20 µg of crude protein extracts from transfected COS-7 cells. The mixture was incubated for 1 or 3 h at 37°C depending on the activity levels. Acceptor substrate concentrations were 0.1 mM. The reaction was stopped by the addition of 3 ml of cold water. The reaction mixture was then applied to a conditioned Sep-Pack C18 reverse chromatography cartridge (Waters Millipore, Bedford, Mass.) attached to a 10-ml syringe. Unreacted GDP-[14C]-fucose was washed off with 15 ml of water. The radiolabeled reaction product was eluted with two times 5 ml of ethanol, collected directly into scintillation vials and counted with two volumes of Biodegradable Counting Scintillant (Amersham Pharmacia Biotech.) in a liquid scintillation beta counter (liquid scintillation analyzer, Tri-Carb-2100TR, Packard, USA).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Southern Blot Analysis
Genomic DNA extracted from blood cells of species (rhesus macaque, squirrel monkey, and ruffed lemur) belonging to different primate subfamilies was fragmented by EcoRI/BamHI endonucleases. After transfer, DNA was hybridized with a human FUT3 probe corresponding to the sequence encoding the highly conserved catalytic domain shared by FUT3, FUT5, and FUT6 enzymes.

Three fragments were detected in human (fig. 2 ) corresponding to the three Lewis genes (Weston et al. 1992bCitation ). A similar pattern was also observed in rhesus macaque, which could indicate that genes orthologous to the three human Lewis ones are present. An additional weaker band of high molecular size was detected and might result from a partial genomic DNA digestion. Interestingly, in the case of ruffed lemur and squirrel monkey, only two fragments were labeled (fig. 2 ), irrespective of the endonucleases used (data not shown).



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Fig. 2.—Southern blot analysis of primate genomic DNA. DNA samples from human, ruffed lemur, squirrel monkey, and rhesus macaque were digested by EcoRI/BamHI, fractionated on agarose gel (10 µg of digested DNA/lane) and transferred onto a nylon membrane. The probe corresponds to a highly conserved catalytic domain of human Lewis sequences (nucleotides 655–1,057 of the human FUT3 ORF). Labeled DNA fragments are indicated by arrows

 
Cloning of Primate Lewis-like Genes
Nine prosimian (lemurs) and simian (squirrel monkey and rhesus macaque) Lewis-like fucosyltransferase coding sequences (one allele of each gene is presented) were amplified by a single-step PCR strategy. Only three different monoexonic ORFs were obtained for the rhesus macaque. Two are 1,119 bp long (accession numbers AF345881 and AF345883) and encode putative proteins of 372 amino acids as in chimpanzee FUT3 (Costache et al. 1997aCitation ). The third ORF (1,125 bp, accession number AF345882) is of the same length as that of human and chimpanzee FUT5 and encodes an enzyme of 374 amino acids. As expected, two monoexonic ORFs (FUT sq1, accession number AF345884 and FUT sq2, accession number AF345885) were obtained for squirrel monkey. They are 1,113 and 1,087 bp long, respectively, and share 97% sequence identity. FUT sq1 sequence encodes an enzyme of 370 amino acids. A 17-bp deletion was found in FUT sq2, which generates a frameshift and a subsequent truncated protein of 280 amino acids. Finally, partial sequences of 542 bp, including the start codons of FUT va1 and FUT va2 (accession numbers AF345887 and AF344886) and FUT eu1 and FUT eu2 (accession numbers AY050244 and AY050243), were amplified from ruffed and brown lemur genomic DNA, respectively. The inferred amino acid sequences correspond to the first 180 amino-terminal residues, i.e., to cytoplasmic, transmembrane, and variable domains of putative fucosyltransferases.

Comparison of Lewis-like Peptide Sequences
The new primate peptide sequences were aligned with human, chimpanzee, and bovine Lewis enzymes (fig. 3 ). The motifs I and II, which characterize {alpha}3/4-fucosyltransferases, were found in all sequences except in FUT sq2. In this sequence the 17-bp deletion beside the His116 codon induces the truncation of the acceptor-binding motif and the loss of motifs I and II. By a virtual deletion of the nucleotide at position 349, a putative FUT sq2 sequence is obtained, which encodes a typical {alpha}3/4-fucosyltransferase with only a deletion of six amino acids in the acceptor-binding motif (FUT sq2 frame 2, fig. 3 ). Therefore, FUT sq2 gene might be a recently shaped pseudogene. All the other new {alpha}3/4-fucosyltransferase sequences possess the highly conserved acceptor-binding motif. The two conserved N-glycosylated sites of human Lewis enzymes (Asn154 and Asn185 in human FUT3, Christensen et al. 2000Citation ) are also found in the new primate enzymes (fig. 3 ). Likewise, the four Cys residues (Cys81-Cys338 and Cys91-Cys341) involved in disulfide bonds of human FUT3 (Holmes et al. 2000Citation ) are conserved at the corresponding positions. In the variable domain, upstream of the acceptor-binding motif, a conserved region of nine amino acids named motif III was identified. This motif slightly differs from the region III described by Trottein et al. (2000)Citation .



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Fig. 3.—Amino acid alignment of Lewis fucosyltransferases. The nine new fucosyltransferases (bold-type characters) are compared with the human, chimpanzee, and bovine Lewis enzymes. Numberings refer to position in the alignment, above the sequences, and to the length of each sequence, at the end of the line. Dashes indicate common residues to the human FUT3, dots show missing positions, question marks represent nonsequenced characters, and crosses point out residues that cannot be aligned. The FUT sq2 sequence was made translatable (frame 2) by deletion of one nucleotide at position 349 in addition to the 17-bp gap. The {alpha}3- and {alpha}3/4-fucosyltransferase conserved motifs I and II (Oriol et al. 1999Citation ), the acceptor-binding motif (abm) (Dupuy et al. 1999Citation ), and the new motif III are shaded in gray for all sequences. The Trp/Arg residue of acceptor-binding motif involved in acceptor substrate specificity is indicated in bold type. The two conserved Asn-linked glycosylation sites are shown by arrows, and stars correspond to conserved Cys involved in human FUT3 folding (Holmes et al. 2000Citation ). The putative transmembrane domain is underlined. Shaded characters indicate selectively conserved residues characteristic of the three Lewis orthology classes defined by Hominoid sequences (FUT3: white characters on gray background; FUT5: black characters on gray background; FUT6: white characters on black background). Nomenclature: bos, Bos taurus; ch, chimpanzee; eu, Eulemur fulvus; hu, human; rh, rhesus macaque; sq, squirrel monkey; va, Varecia variegata

 
Assignment of the New Enzymes to Lewis Orthology Classes
Among the three rhesus macaque sequences, FUT3 rh and FUT5 rh are clearly orthologous to hominoid FUT3 and FUT5 enzymes, respectively. The comparison of the Lewis proteins with the bovine FUTb sequence allowed the identification of amino acids specific to each orthology class. We identified 18, 9, and 17 selectively conserved residues, which define the Lewis FUT3, FUT5, and FUT6 orthology classes. The FUT3 rh has 13/0/1 and FUT5 rh has 1/6/2 of these specific residues (fig. 3 ). Moreover, FUT3 rh possesses the specific deletion of two amino acids at positions 76 and 77. None of the selectively conserved FUT5 residues was found in FUT3' rh, and this enzyme showed an intermediate situation with 7 on 18 and 4 on 17 residues, which support its assignment to FUT3 and FUT6 orthology classes, respectively. They are not randomly distributed; the FUT3-specific residues are found upstream of the acceptor-binding motif and the FUT6-specific residues downstream. The sequence is devoid of the FUT6 characteristic deletions in the stem region and at position 375 but shows the typical FUT3 deletion at positions 76 and 77. Consequently, we chose to name this sequence FUT3' rh because of its closest relationship with the FUT3 class. Different attempts to reveal the possible presence of FUT6 rh have failed. Indeed, primers chosen in known FUT6 coding sequences and overlapping their specific deletions do not permit any amplification. Only FUT3' rh is obtained when primers designed to amplify chimpanzee FUT6 gene (Costache et al. 1997aCitation ) are used.

Both squirrel monkey sequences are distinguished by a common gap of four amino acids from position 45 to 48. In some respects, the FUT sq1 is similar to FUT5 rh. It possesses the conserved "HHRD" acceptor-binding motif and the two characteristic residues of FUT5 enzymes (Pro146 and Thr147). Compared with FUT sq1, FUT sq2 sequence has two additional deletions leading to the absence of three amino acids at the N-terminal end and to a truncated protein (see previously). Thus, its assignment to one orthology class remains tricky. The partial peptide sequences of lemurs show nine additional amino acids (residues 60–68) in the stem domain (fig. 3 ). The "HHWD" acceptor-binding motif is present in FUT va1 and FUT eu1 and "HHRD" in FUT va2 and FUT eu2. These enzymes cannot really be assigned to one of the Lewis orthology classes. Nevertheless, they do not possess any conserved residues characteristic of the FUT6 class.

Phylogenetic Analyses of Lewis-like Sequences
Because of the truncated lemur sequences and the suppression of the stem indels for phylogenetic analyses, only 459 nucleic acid positions have been analyzed; 247 positions correspond to variable sites and 149 are informative for parsimony. Considering that Artiodactyls constitute one of the closest sister group of primates, the bovine sequence was chosen as the outgroup for phylogenetic reconstructions. Furthermore, the ancestor of futb sequence is supposed to be orthologous to the ancestor of primate Lewis genes (Oulmouden et al. 1997Citation ). Phylogenetic analyses were carried out using ML, MP, and DB methods. The three analyses showed the same dichotomy with the prosimian sequences always separated from all the other primate sequences (fig. 4 ). FUT va1 and FUT eu1 are grouped, with bootstrap values ranging from 59% to 88%. The squirrel monkey fucosyltransferase sequences form a monophyletic group strongly supported by whatever method was used. The three classes of orthology, FUT3, FUT5, and FUT6, are always recovered with bootstrap values ranging from 98% to 100%. Within the three sequences identified in the rhesus macaque, FUT3 rh and FUT5 rh consistently cluster with their hominoid counterparts, always emerging as their sister group. The third rhesus sequence, FUT3' rh, is related to the FUT3 orthology class, and their clustering is supported by high bootstrap values, 88%–97%. The relative branching order of the three orthology classes and the New World monkey group is not resolved (bootstrap values between 15% and 48%). However, analyses performed without lemur sequences support the fact that the Saimiri sequences are the sister group of FUT5 (data not shown).



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Fig. 4.—Phylogenetic tree based on partial nucleotide sequences of Lewis fucosyltransferases. The tree was reconstructed using a maximum-likelihood method with bovine sequence as the outgroup. The new sequences obtained in this study are distinguished by bold-type characters. Bootstrap values are shown for all nodes in bold, normal, and italic types for maximum-likelihood, maximum-parsimony, and distance-based methods. When all methods gave 100%, only the bold type value is indicated. The scale bar represents the number of substitutions per position for a unit branch length. The three orthology classes FUT3, FUT5, and FUT6, identified in hominoids, are indicated by vertical brackets

 
Acceptor Substrate Specificity of the New Primate {alpha}3/4-Fucosyltransferases
As none of the known inactivating natural mutations was found in the four complete primate Lewis-like peptide sequences (Mollicone et al. 1994a, 1994b;Citation Orntoft et al. 1996Citation ; Pang et al. 1998a, 1998b, 1999Citation ; Elmgren et al. 2000Citation ), transient expressions of these enzymes in COS-7 cells were performed in order to determine their ability to transfer fucose in {alpha}1,3 or in {alpha}1,4 linkage or in both (fig. 5 ). Activities were determined using H-type 1 and H-type 2 trisaccharides as acceptor substrates in order to only analyze the formation of {alpha}1,4 or {alpha}1,3 fucose linkages on GlcNAc, respectively. The rhesus macaque FUT3, which has the "HHWD" acceptor-binding motif, transfers fucose preferentially onto H-type 1 acceptor substrate, whereas FUT5 rh and FUT3' rh, which share the "HHRD" acceptor-binding motif, add fucose only onto H-type 2 acceptor substrate (table 2 ). Interestingly, the mutated form of FUT5 rh, where the Arg124->Trp substitution generated the "HHWD" acceptor-binding motif, loses its {alpha}1,3 activity but does not gain {alpha}1,4 activity. The presumed {alpha}3-fucosyltransferase encoded by the squirrel monkey FUT sq1 gene, which has the "HHRD" acceptor-binding motif, transfers fucose exclusively onto H-type 2 acceptor substrate. Because the DNA sequences corresponding to the lemur {alpha}3/4-fucosyltransferases do not encode full-length proteins, the proteins could not be characterized for enzyme activity.



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Fig. 5.—Western blot analysis of primate Lewis fucosyltransferases expressed in COS-7 cells. Lane 1, negative control, 50 µg of proteins extracted from COS-7 cells transfected with pcDNAI/Amp; lane 2, human FUT3 (MW = 42.1 kDa); lane 3, human FUT5 (MW = 43.0 kDa); lane 4, human FUT6 (MW = 41.8 kDa); lane 5, rhesus monkey FUT3 (MW = 43.1 kDa); lane 6, rhesus monkey FUT5 (MW = 43.0 kDa); lane 7, rhesus monkey FUT3' (MW = 43.3 kDa); lane 8, squirrel monkey FUT sq1 (MW = 43.0 kDa). The molecular weights are deduced from amino acid sequences. Lewis-like fucosyltransferases were labeled by anti-human FUT3 antibodies. Revelation was performed by a secondary antibody, a pig anti-rabbit IgG conjugated to horseradish peroxidase

 

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Table 2 {alpha}1,3 and {alpha}1,4 Activities of New Lewis-like Fucosyltransferases

 
In order to compare the acceptor substrate specificities between these new {alpha}3/4-fucosyltransferases and their human counterparts, activities were measured with different type 1 and type 2 disaccharide and trisaccharide substrates (table 3 ). The FUT3 rh, which preferentially uses type 1 substrates, has a higher activity with trisaccharide acceptors, like human FUT3. Compared with human FUT5, FUT5 rh transfers fucose only onto type 2 acceptor substrates and can use {alpha}Gal-type 2 substrate with the same efficiency as H-type 2 substrate. Although FUT3 rh and FUT3' rh sequences are very close, the fucose transfer activity on type 2 acceptors of FUT3' rh is similar to that of human FUT6. However, FUT3' rh differs from human FUT6 in its ability to preferentially use the {alpha}Gal-type 2 trisaccharide acceptor. FUT sq1 transfers fucose onto type 2 di- and trisaccharide acceptor substrates with lower efficiency compared with human FUT5 and FUT6 and rhesus macaque FUT5 and FUT3'. This discrepancy could be the result of cloning the allele because some variations in activity may exist between alleles, as already reported for chimpanzee FUT3 and FUT6 (Costache et al. 1997aCitation ).


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Table 3 Acceptor Substrate Specificities of Human, Squirrel Monkey, and Rhesus Macaque Lewis-like Fucosyltransferases for Type 1/Type 2 Di- and Trisaccharide Acceptor Substrates

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In animals, FUT3 and FUT5 Lewis enzymes are the only known fucosyltransferases which attach fucose in {alpha}1,4 linkage on the subterminal N-acetylglucosamine in the last steps of the biosynthesis of glycoconjugates. Its peripheral position confers on fucose important roles in several biological processes, such as development (Candelier et al. 2000Citation ), cell adhesion, and carcinogenesis (Wietrzyk et al. 2000Citation ). Until now, FUT3 and FUT5 have only been characterized in human and chimpanzee, and it was hypothesized that {alpha}1,4 fucosylation would be specific to hominoids. In the present work we have cloned in rhesus macaque the two genes orthologous to FUT3 and FUT5 genes. FUT3 rh encodes an enzyme which uses preferentially type 1 acceptor substrates, whereas FUT5 rh produces an enzyme which is active only with type 2 acceptor substrates. As for all {alpha}3- and {alpha}3/4-fucosyltransferases, activities of the rhesus macaque enzymes are well correlated with the presence of Arg or Trp residue in the acceptor-binding motif. FUT3 rh, which transfers fucose onto type 1 acceptor substrates, has the "HHWD" acceptor-binding motif, whereas enzymes, which present the "HHRD" motif (FUT5 rh, FUT3' rh), transfer fucose only onto type 2 acceptor substrates.

Although the Trp residue of acceptor-binding motif is not sufficient by itself to determine {alpha}1,4 fucose transfer (Dupuy et al. 1999Citation ), the presence, in the two lemur species, of a Lewis-like sequence with the "HHWD" acceptor-binding motif typical of {alpha}3/4-fucosyltransferases suggests the possible existence of {alpha}1,4 fucosylation in pro-simians. Conversely, in New World monkeys the {alpha}3/4-fucosyltransferase gene could have evolved into a pseudogene, as illustrated by the FUT sq2, and this may justify the absence of Lea antigens (Moor-Jankowski and Wiener 1968Citation ). Thus, the ability to transfer fucose in {alpha}1,4 linkage is not restricted to hominoids and may be generalized at least to catarrhines and possibly to all primates.

The {alpha}1,4 fucosylated glycoconjugates are also present in plants. Recently, two {alpha}1,4-fucosyltransferase activities were characterized from Vaccinium myrtillus (Palma et al. 2001Citation ) and Arabidopsis thaliana (Léonard, personal communication). Interestingly, plant {alpha}4-fucosyltransferase genes (Bakker et al. 2001Citation ) encode proteins with the characteristic motif II and a putative acceptor-binding motif similar to the primate one. These shared motifs suggest a common origin of the genes responsible for {alpha}1,4 fucosylation in primates and plants. However, because such activity is not found in other vertebrates, i.e., other mammals belonging to Carnivores, Artiodactyls, Lagomorphs, and Rodents, which are Lea negative (Oriol et al. 1999Citation ), we have to admit that the {alpha}1,4 fucosylation has independently appeared twice, in plants and in primates.

In animals the simplest model of Lewis fucosyltransferase gene evolution (fig. 6A ), which explains the emergence of {alpha}1,4 fucosylation, would involve two successive duplications of a Lewis ancestral gene, after the great mammalian radiation 80 MYA (Oulmouden et al. 1997Citation ; Wierinckx et al. 1999Citation ). In primates after the first duplication of a gene orthologous to the bovine futb ancestral gene, a second duplication would be at the origin of FUT3 and FUT5, the corresponding enzymes carrying the {alpha}1,4 activity (Dupuy et al. 1999Citation ). The presence of two Lewis-like sequences in pro-simians (brown and ruffed lemurs) and in New World monkeys (squirrel monkey) suggests that the first duplication event occurred before the emergence of pro-simians, 70 MYA (fig. 6B ). The three genes identified in rhesus macaque could set the second duplication event before the separation of Cercopitecoides from hominoids 35 MYA and after the emergence of Platyrrhines 47 MYA. However, species-specific independent gene duplications could not be formally ruled out.



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Fig. 6.—Hypothetical models of Lewis fucosyltransferase gene evolution. Compared with the simplest model (A), the new model stemming from this work (B) sets more precisely the duplication events at the origin of the three Lewis fucosyltransferase genes. In this representation FUT3 and FUT6 genes share a recent common ancestor contrary to the classical model where FUT3 and FUT5 genes were supposed to derive from a common ancestor. Vertical thick lines correspond to a duplication event. Mya: million years ago

 
On the basis our present results that show (1) the presence of FUT3 rh and FUT3' rh related to FUT3 orthology class, (2) our inability to characterize a clear ortholog of FUT6 in rhesus macaque, (3) the existence of selectively conserved residues between FUT sq1 and FUT5 orthology class, and (4) the absence of conserved residues characteristic of FUT6 in pro-simians, we reconsider the current hypothesis, and we propose an alternative scenario for the Lewis gene evolution (fig. 6B ). The first duplication, at the base of the primate lineage, would have given the FUT3 and FUT5 ancestral genes. The second duplication, in Old World Monkeys, would concern the FUT3 ancestral gene and might be at the origin of the present FUT6 gene. This scenario is reinforced by the high similarity found between i2 and i3 introns of futb and the 5'-untranslated exons of human FUT6 and FUT3, respectively (Wierinckx et al. 1999Citation ). In the same way, homologies were found between the exon c of futb and both FUT3 and FUT6 5'-unstranslated exons but not with FUT5 ones. Therefore, it could be hypothesized that FUT3 and FUT6 have a recent common genetic origin. This close relationship is also highlighted in our phylogenetic analyses.

Interestingly, the {alpha}1,4 fucosylation is supported by one gene in Cercopitecoides (FUT3 rh), and potentially in pro-simians, whereas in Hominoids two genes (FUT3 and FUT5) are involved. In our hypothesis, the ancestor of FUT3 enzyme might already have an {alpha}1,4 activity, whereas FUT5 would have gained this ability after the emergence of Cercopitecoides. Indeed, the rhesus macaque FUT5 enzyme, unlike chimpanzee and human FUT5, possesses the HHRD acceptor-binding motif and uses exclusively type 2 acceptor substrates. The Arg124->Trp substitution in FUT5 rh, which generates the HHWD acceptor-binding motif characteristic of {alpha}3/4-fucosyltransferases, produces an enzyme without type 1 activity and which has lost its type 2 substrate specificity. This result shows that one mutation from Arg to Trp is sufficient to abolish the {alpha}1,3 activity, but it is not enough to acquire the {alpha}1,4 fucosylation, which probably needs the mutation of other amino acids. The substitution Arg->Trp in the acceptor-binding motif of FUT5 might have occurred only recently in hominoids. Therefore, the {alpha}1,4 fucosylation might have appeared twice independently in the primate lineage. Nevertheless, in regard to the rhesus macaque sequences, evolution of Lewis genes seems more complicated than was suggested before and requires new genetic information from other primates.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank L. Forestier and M.-P. Laforêt for their technical assistance, D. Petit for his helpful discussion, and G. Costa and C. Riou-Khamlichi for their critical reading of the manuscript. This work has been performed in the frame of the French network "GT-rec" partially supported by MENRT (ACC SV number 9514111) and CNRS (Program PCV) grants, the Association for Research on Cancer (ARC) grant 5348, and the Conseil Régional du Limousin.


    Footnotes
 
Pierre Capy, Reviewing Editor

Abbreviations: Fuc, fucose; FUT, fucosyltransferase; FUT3, FUT3-encoded Lewis {alpha}3/4-fucosyltransferase; FUT5, FUT5-encoded {alpha}3/4-fucosyltransferase; FUT6, FUT6-encoded plasma {alpha}3-fucosyltransferase; FUTb, bovine futb-encoded {alpha}3-fucosyltransferase; Gal, galactose; GlcNAc, N-acetylglucosamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine; The Lewis blood group antigens are Lea, Galß1,3(Fuc{alpha}1,4)GlcNAc; Leb, Fuc{alpha}1,2Galß1,3(Fuc{alpha}1,4) GlcNAc; Lex, Galß1,4(Fuc{alpha}1,3)GlcNAc; Ley, Fuc{alpha}1,2Galß1,4(Fuc{alpha}1,3)GlcNAc. Back

Keywords: acceptor substrate specificity {alpha}3/4-fucosyltransferase evolution Lewis phylogeny primate Back

Address for correspondence and reprints: Abderrahman Maftah, Laboratoire de Glycobiologie et Biotechnologie, EA 3176, Faculté des Sciences et Techniques, Université de Limoges, 123 Avenue Albert Thomas, 87060 Limoges Cedex, France. maftah{at}unilim.fr Back


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Accepted for publication January 4, 2002.