Three bovine {alpha}2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Galß1–3GalNAc acceptor substrates

Jean-Pierre Barreaud2, Katiana Saunier2,3, Jacques Souchaire2, Didier Delourme2, Ahmad Oulmouden2, Rafael Oriol4, Hubert Levéziel2,3, Raymond Julien2 and Jean-Michel Petit1,2

2Unité de Génétique Moléculaire Animale-UMR 1061 (INRA/Université de Limoges) Institut Sciences de la Vie et de la Santé, Faculté des Sciences, 87060 Limoges, France, 3Laboratoire de Génétique Biochimique et Cytogénétique, INRA-CRJ, 78350 Jouy-en-Josas, France, and 4Glycobiologie INSERM U 504 / Université Paris Sud XI, 94807 Villejuif, France

Received on October 18, 1999; revised on December 20, 1999; accepted on December 27, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
To investigate the synthesis of {alpha}2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct {alpha}2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in {alpha}1,2 linkage to ganglioside GM1 and galacto-N-biose, but not to the phenyl-ß-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian {alpha}2-fucosyltransferases. Southern blot analyses detected four related {alpha}2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the {alpha}2-fucosyltransferase activity which can also use type 1 and phenyl-ß-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four {alpha}2-fucosyltransferases are present, three adding a fucose on {alpha}1,2 linkage on type 3/4 acceptor (Galß1–3GalNAc) and another able to transfer also fucose on phenyl-ß-D-galactoside and type 1 (Galß1–3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.

Key words: bovine species/{alpha}2-fucosyltransferase/evolutionary rate/GM1 and galacto-N-biose acceptors/phylogeny/tissue expression/cloning


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fucosylated glycoconjugates play an important role in several cell–cell interactions, including differentiation and development, inflammation, cell trafficking and malignant transformation (Varki, 1993Go). However, the significance of glycoconjugates bearing a fucose linked in {alpha}1->2 on the terminal galactosyl residue remains to be clarified.

In humans, at least two {alpha}2-fucosyltransferases (GDP-l-fucose:ß-D-galactosyl-R-{alpha}-L-fucosyltransferase, E.C. 2.4.1.69) are present in several tissues (Oriol et al., 1981Go; Le Pendu et al., 1985Go). One is the FUT1 encoded {alpha}2-fucosyltransferase (H) which regulates the expression of H antigen on red cells and vascular epithelia. The other, the secretor (Se) FUT2 encoded {alpha}2-fucosyltransferase regulates the expression of the H antigen in the endoderm-derived epithelia producing exocrine secretions as stomach, intestine and submaxillary glands (Oriol, 1995Go). FUT1 and FUT2 genes have been cloned (Larsen et al., 1990Go; Kelly et al., 1995Go) and more recently, a third FUT2-like sequence (Sec1), thought to be a pseudogene, has also been cloned (Kelly et al., 1995Go; Rouquier et al., 1995Go). These two genes and the pseudogene share a high degree of DNA sequence identity and are located within a 100 kb region on chromosome 19q13.3 (Reguigne-Arnould et al., 1995Go; Rouquier et al., 1995Go). Conservation of the three {alpha}2-fucosyltransferase genes has been shown in primates (Apoil et al., 2000Go) and lower mammals. In these last species, also three types of {alpha}2-fucosyltransferase genes have been reported that are structurally very similar to the human gene counterparts. Also, three loci (fut1, fut2, sec1) which seem to be equivalent to the human FUT1, FUT2, and Sec1 genes, respectively, have been identified in swine (Cohney et al., 1996Go; Meijerinck et al., 1997Go). Three {alpha}2-fucosyltransferase genes (fut1, fut2, sec1) have also been characterized in rabbit (Hitoshi et al., 1995Go, 1996), in mouse (Domino et al., 1997Go) and rat (Liehr et al., 1997Go; Sherwood and Holmes, 1998Go; Hallouin et al., 1999Go).

Until now, all the {alpha}2-fucosyltransferases identified transfer fucose in the {alpha}1->2 linkage to the terminal galactosyl residues on both type 1 (Galß1–3GlcNAc) and type 2 (Galß1–4GlcNAc) precursors and other structures such as type 3 (Galß1–3GalNAc{alpha}1-R), type 4 (Galß1–3GalNAcß1-R), and phenyl-ß-Gal (Liu et al., 1999Go).

In Caenorhabditis elegans, {alpha}2-fucosylation seems to be rather complex. Among the 19099 genes constituting the entire genome of this nematode, 22 had the 3 conserved {alpha}2-fucosyltransferase peptide motifs and are therefore considered as putative {alpha}2-fucosyltransferases (Wilson et al., 1994Go; Oriol et al., 1999Go, and unpublished observations). Recently DeBose-Boyd et al. (1998)Go has established that C.elegans extracts contain an {alpha}2-fucosyltransferase specific for type 1 glycans but not for the type 2 acceptors.

In our laboratory, we have recently isolated the first bovine {alpha}3-fucosyltransferase gene, futb (Oulmouden et al., 1997Go), and shown that it is the orthologue of the human ancestor of the FUT3-FUT5-FUT6 gene cluster. The complete bovine sequence reveals a possible mechanism for the gene duplications leading to the FUT3, FUT5, and FUT6 primate gene cluster (Wierinckx et al., 1999Go).

Until now several {alpha}1,2-fucosylated oligosaccharides have been characterized in bovine submaxillary gland (Menghi et al., 1993Go). Mårtensson et al. (1998)Go have shown that in bovine mucin Galß1–3GalNAc termini structures are preferentially {alpha}1,2 fucosylated. However nothing is known about the genes encoding the proteins which transfer fucose in {alpha}1,2 to such structures.

Here, we report the characterization of three distinct {alpha}2-fucosyltransferase coding sequences, analyze the substrate specificity of the corresponding enzymes, determine their kinetic parameters and compare their evolution rate. Three bovine enzymes transfer fucose in {alpha}1->2 linkage only on Galß1–3GalNAc. Also, a fourth bovine {alpha}2-fucosyltransferase activity was detected in intestine.

Nucleotide sequences reported in this paper have been submitted to the GenBank/EBI Data Bank with accession number AF186465 for bovine fut1, X99620 for bovine fut2, and AF187851 for bovine sec1.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Southern blot analyses of bovine genomic DNA
Nucleotide sequence alignment of human and rabbit {alpha}2-fucosyltransferase genes allowed us to identify areas with very similar sequences. Two of these regions located in the catalytic domain were selected to define the PCR primers F5 and R8 (Table I).


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Table I. Primers used in PCR in this study
 
Using these primers a single PCR product of about 0.7 kb was obtained from bovine genomic DNA. Sequencing of both strands revealed a run of 762 unambiguous nucleotides with 89% and 85% identity to human FUT2 and rabbit fut2. This probe devoid of EcoRI and HindIII restriction sites was used to sample bovine, human, and porcine genomes for cross-hybridizing DNA sequences (Figure 1). Whatever the restriction enzymes used, it revealed three distinct bands corresponding to FUT1, FUT2 and Sec1 genes in human and to fut1, fut2, and sec1 genes in pig. Surprisingly four and five bands in bovine genomic DNA digested by EcoRI and HindIII, respectively, were detected (Figure 1).



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Fig. 1. Southern-blot analysis of human, porcine, and bovine genomic DNA with a bovine FUT2-like probe. Human (H), porcine (P), and bovine (B) genomic DNA were digested to completion with the restriction enzymes EcoRI and HindIII (10 µg of digested DNA/lane) and electrophoresed on agarose gels. Lanes 1, 3, and 5 correspond to EcoRI digestion and lanes 2, 4, and 6 to HindIII digestion (see Materials and methods section). The blot was hybridized with probe I and then washed at high stringency. The five bovine bands obtained with the HindIII digest are indicated by full arrows and the four bands obtained with the EcoRI digest by dotted arrows (see also Figure 5).

 
Rapid amplification of 5' and 3' ends of FUT2-like bovine cDNAs
5' RACE products, resulting from a first round of amplification with AP1 and R6 primers, were used as templates for a second round of PCR with AP2 and R7 (Table I). A similar nested-PCR approach, using AP1 and F7 primers, then AP2 and F6 primers (Table I) was carried out for the 3' ends. For brain, intestine and lung, two 5' and 3' RACE products were obtained differing in size. Each 5' fragment contained the same coding sequence but differed in their 5' untranslated regions (UTR). For 3' RACE products we obtained the same 3' end coding sequence with two UTR. Both 5' and 3' coding sequences of this bovine gene were very similar to the other mammalian FUT2 genes and we have thus named it bovine fut2. Four 5' ends and three 3' ends RACE products were identified in kidney. After subcloning, two distinct coding sequences were found. One corresponds to bovine fut2; the other, named bovine sec1, presented 80% and 78% identity with rabbit and human Sec1, respectively.

Analysis of bovine fut2 and sec1 coding sequences and predicted polypeptides
Appropriate primers (F4/R9 and F8/R13 for bovine fut2 and sec1, respectively; Table I) were chosen to amplify coding sequences from genomic DNA and kidney cDNAs. Whatever the template used, we observed only one fragment of 1035 bp and one of 1107 bp for bovine fut2 and sec1 respectively (Figure 2). Like their human, pig, and rabbit counterparts, the two FUT2-like bovine genes possessed a single coding exon.



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Fig. 2. Amplification of entire coding sequence of bovine fut2 and sec1 genes. PCR reactions for fut2 and sec1 were performed with primer pairs F4/R9 and F8/R13, respectively. (A) The coding sequence of fut2 (1035 bp) was observed with different templates: bovine genomic DNA (G); lung (L); kidney (K) cDNAs. (B) The whole coding sequence of sec1 (1107 bp) was observed with: G, genomic DNA; and K, kidney cDNAs as template. Lane (-) is a negative control without DNA matrix.

 
The bovine Se (344 amino acids) encoded by fut2 shared 88%, 82%, and 80% identity with porcine, human, and rabbit Se {alpha}2-fucosyltransferases, respectively. This protein has a transmembrane domain (residues 7–27) and four potential consensus sites for asparagine-linked glycosylation. Three (189, 283, and 309) are shared by bovine, human, pig, and rabbit Se enzymes, whereas the one at position 255 is only found in the pig Se.

The predicted 368 amino acid protein, bovine Sec1, contained also a putative transmembrane region in the NH2-terminal (residues 36 to 54) and three potential N-linked glycosylation sites (amino acids 277, 305, and 331). Comparison of the primary structure of the bovine Sec1 revealed 80% amino acid identity with rabbit Sec1.

Isolation of a bovine FUT1-like gene fragment
Screening of the high density membranes which carry the bovine BAC library constructed by Zhu et al. (1999)Go, using the 762 bp probe allowed the isolation of 10 positive clones. With F2/R3 primer pair designed from human and pig FUT1 comparison, only one clone gave a 173 bp PCR fragment. Its sequence revealed a high identity (>80%) with human FUT1 gene. F3 (sense) and R1 (antisense) primers were synthesized to directly sequence bovine fut1 from the purified BAC. The sequences revealed an ORF of 1083 nucleotides, which had 89%, 84%, and 83% identity with pig, human, and rabbit FUT1, respectively. Bovine fut1 encoded a protein of 360 amino acids with a putative 19 amino acids transmembrane domain (residues 9–27). Three potential consensus sites for asparagine-linked glycosylation (bovine amino acids 65, 301, and 327) were common to the H enzymes of other species. The bovine H was 5 and 13 amino acids shorter than human or pig H (365 amino acids), and rabbit H (373 amino acids), respectively.

Tissue-expression of bovine fut1, fut2, and sec1 genes
The expression patterns were determined in adult tissues by Northern blot and/or RT-PCR analyses. Northern-blot analyses of fut1 and sec1 were negative. Northern blot hybridization studies of bovine fut2 using a 194 bp probe generated by F4/R5 primers revealed a transcript of 1.6 kb in lung, spleen, brain, heart, and kidney (Figure 3). In brain, heart, and lung, another transcript of 1.8 kb was also found. The relative levels of expression, calculated using the ß-actin hybridization signal as reference, indicated that brain, lung, and kidney contain the highest level of transcripts (Figure 3). The two transcripts in heart, lung, and brain have a similar expression level. Moreover RACE analyses confirmed the presence of the 1.6 kb transcript in brain and revealed the two transcript forms in intestine.



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Fig. 3. Northern-blot analysis of bovine fut2 transcript expression in adult bovine tissues. Each lane contains 2 µg of poly(A)+ mRNA isolated from the bovine tissues indicated above each lane. The Northern-blot was prepared and hybridized as described in the Materials and methods section using the 5' coding end of the bovine fut2 as probe (fut2 probe, 194 bp). Transcript size may be estimated from the markers (kb) on the left. The two bovine fut2 transcripts are indicated by full arrows. The Northern blot was reprobed for bovine ß-actin to confirm that the poly(A)+ mRNA was intact and that it had been loaded and transferred equally amongst all lanes in the blot. A relative quantification of bovine fut2 mRNA was carried out owing to ß-actin signal normalization based on a densitometric determination (ImageQuant software from Molecular Dynamics Inc). B, Brain; H, heart; K, kidney; L, lung; S, spleen.

 
Bovine fut1 and sec1 transcripts were only detected by RT-PCR using appropriate primer pairs (F1/R2 and F8/R10, respectively, Table I) located in the 5' part of the ORF. Amplified bands of 243 and 247 bp for bovine fut1 and sec1 respectively were eluted and sequenced. A complete identity among amplified cDNA sequences and their respective gene was found. In brief, bovine fut1 was expressed in brain, intestine and kidney and sec1 only in kidney (Figure 4).



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Fig. 4. Bovine fut1, fut2, and sec1 transcripts in some bovine adult tissues. cDNAs were prepared as described under Materials and methods. (A) RT-PCR reaction for bovine fut1 was performed with primers F1 and R2. The specific amplification product (243 bp) was observed in: brain (B), intestine (I), and kidney (K). (B) RT-PCR reaction for bovine fut2 was performed with primers F4 and R5. The specific amplification product (194 bp) was observed in: brain (B), heart (H), intestine (I), kidney (K), lung (L), spleen (S). (C) RT-PCR reaction for bovine sec1 was performed with primers F8 and R10. The specific amplification product (247 bp) was only observed in kidney (K). Lanes (-) were controls without DNA matrix.

 
Reinvestigation of genomic DNA by bovine fut1, fut2 and sec1 specific probes
Probes for fut1 (243 bp), fut2 (194 bp), and sec1 (247 bp) derived from coding sequences were used to screen bovine genomic DNA by Southern analyses. Each of them revealed only one gene entity (Figure 5) except the sec1 probe which identified two different bands. Since no EcoRI and HindIII restriction sites were found in the sec1 cDNA used as probe, this result suggests that the bovine genome possess two sec1-like genes.



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Fig. 5. Southern blot of bovine genomic DNA. Bovine genomic DNA was digested with the restriction enzymes EcoRI (lanes 1) and HindIII (lanes 2) and electrophoresed on agarose gel. Results are derived from a master gel and blots containing reiterated sets of the two digests (10 µg of digested DNA/lane). Each strip containing two digests was separately hybridized and washed at high stringency (see Materials and methods) with a FUT2-like probe of 762 bp as in Figure 1 a 243 bp bovine fut1 probe, a 194 bp bovine fut2 probe and a 247 bp bovine sec1 probe.

 
Expression of bovine fut1, fut2, and sec1 genes in COS-7 cells
EcoRI fragments containing the entire ORF of each bovine gene were cloned into the mammalian expression vector pcDNAI/Amp and introduced by transfection into COS-7 cells deficient in endogenous {alpha}2-fucosyltransferase activity (Clarke and Watkins, 1999Go). Extracts from the transfected COS-7 cells were assayed for {alpha}2-fucosyltransferase activity using several different acceptor substrates (phenyl-ß-D-galactoside, type 1, type 2, galacto-N-biose and monosialoganglioside: GM1). No significant activity with phenyl-ß-D-galactoside, type 1 or type 2 was detected. The three genes encoded proteins that only can {alpha}1,2-fucosylate type 3/4, GM1 and galacto-N-biose acceptors (the kinetic constants for both acceptors are given in Table II).


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Table II. Kinetic parameters of bovine recombinant {alpha}2-fucosyltransferases
 
Enzymatic activities in different tissues
We looked for {alpha}2-fucosyltransferase activities in fresh bovine tissue extracts from brain, heart, liver, intestine, lymphocytes, lung, spleen, kidney, submaxillary glands, and testis, with GM1, phenyl-ß-D-galactoside, type 1, type 2 and galacto-N-biose. For almost all the tissues tested, we found {alpha}1,2 fucose transfer on GM1 and galacto-N-biose (Table III). However, no activity was found with lymphocyte extract whatever the substrate used. Moreover, with intestine extracts, significant activities were measured with all substrates except for type 2. For the same acceptor concentration of 0.6 µM for phenyl-ß-D-galactoside and type 1, we found 1.2 and 4.8 nmol of fucose transferred per hour and per milligram of protein.


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Table III. Relative bovine {alpha}2-fucosyltransferase tissular activities toward different acceptors
 
Phylogeny
The same long distance, represented by a discontinuous branch in Figure 6, was observed between the Caenorhabditis elegans sequence and all the other {alpha}2-fucosyltransferases. This is in good agreement with the rather old divergence of this worm from the common evolutionary trunk, and suggests that after this divergence a duplication of the ancestor of the mammalian {alpha}2-fucosyltransferase genes has originated the two main families found today. The H enzyme, present in all the species studied, forms a single cluster and no other duplications were detected in this H branch. Unlike this, a new duplication in the other branch originated two subfamilies of enzymes, Se and Sec1, also present in all the species studied and constituting two clusters. Each of these two clusters and the H cluster have a similar overall species branching pattern, but the Se branches exhibit consistently shorter apparent genetic distances than the Sec1 branches.



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Fig. 6. Phylogenetic tree based on the alignment of amino acid sequences of the products of FUT1 (H), FUT2 (Se), and Sec1 gene families. Multiple alignments of predicted protein sequences were carried out using ClustalW. The genetic distances were calculated with the Phylip package using the Fitch-Margoliash last squares method with an evolutionary clock. The GenBank/EBI accession numbers of the sequences used are listed in Table V. The Caenorhabditis elegans putative {alpha}2-fucosyltransferase F (Z92830_5) was used as an outgroup.

 
Each of the three bovine enzymes occupies a similar position in each of the three clusters, which is compatible with the known phylogenetic position of Bovidae in evolution.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Three genes fut1, fut2, and sec1 have been identified in bovine which respectively present a high degree of sequence identity with human (81%), rabbit (79%), and pig (85%) FUT1 genes; human (84%), rabbit (82%), and pig (89%) FUT2 genes; and human (83%), rabbit (85%), and pig (79%) Sec1 genes. Whereas pig and human Sec1 are pseudogenes, bovine sec1 encodes a functional {alpha}2-fucosyltransferase. The three bovine genes are assigned within approximately 85 kb on chromosome 18q24, both fut2 and sec1 genes being located in the 3' region of fut1 (unpublished observations). By comparative mapping, this region is homologous to the human chromosome 19q13.3. We have recently shown that the futb gene is the orthologous homologue of the ancestor gene leading to the present human FUT3-FUT5-FUT6 gene cluster. It is located on bovine chromosome 7 (Oulmouden et al., 1995Go), which is homologous to a portion of the short arm of the human chromosome 19. Thereby, a chromosome rearrangement interesting a primate ancestor of fucosyltransferase genes might be at the origin of the two clusters on long arm ({alpha}2-fucosyltransferase genes) and short arm ({alpha}3/4-fucosyltransferase genes) of human chromosome 19. We could not exclude that such a rearrangement might interfere with the gene regulation expression.

The special position of the pig Sec1 enzyme shifted to the end of the Sec1 cluster (Figure 6) might be due to a particular problem of the published sequence. It lacks the start codon and could not be expressed in mammalian cells (Meijerinck et al., 1997Go). Thereby, it has been considered as the product of a pseudogene. On the other hand, overall similar phylogenetic branching patterns compatible with the known evolutionary appearance of the different species were observed, for all the other sequences, in each of the clusters of H, Se and Sec1 enzymes (Figure 6). Since the separation of each of the different primate and mammalian species in evolution is a fixed and unique event, the apparently different genetic distances observed for each of the three {alpha}2-fucosyltransferases in the same species, has to be interpreted as differences in relative evolutionary rates of the three genes. Comparison of the apparent genetic distances to the main branching points (Table IV) and the overall results suggest that (1) the gene encoding for Sec1 evolves twice as fast as Se, and (2) H has an intermediate evolutionary rate, although it is closer to Se than to Sec1. An ancestral Se gene is thought to have been duplicated in two related genes Se and Sec1. The high evolution rate of Sec1 gene family may be explained by successive missense mutations without effect on the functionality of the encoded enzyme, such as seen in the bovine and rabbit species. Then, a nonsense inactivating mutation might have occurred in the ancestor of chimpanzee, gorilla, and human Sec1 genes, followed by independent frame shifts in gorilla and human leading to the present Sec1 pseudogenes found in these three species (Apoil et al., 2000Go).


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Table IV. Relative evolutionary rates of {alpha}2-fucosyltransferases calculated from the apparent genetic distances separating the present Sec1 (Sec1), H (FUT1), and Se (FUT2) proteins from their ancestors
 
Using hydrophobic cluster analysis (HCA) and fold recognition methods, it has been proposed that vertebrates, invertebrates, and bacteria {alpha}2-fucosyltransferases are related proteins sharing the same type of fold and three conserved peptide motifs (Breton et al., 1998Go; Oriol et al., 1999Go). These three motif signatures are found in the three bovine enzymes. We also found another well preserved motif described by Wang et al. (1999)Go in the stem domain. However, unlike man, pig, and rabbit {alpha}2-fucosyltransferases, the bovine enzymes did not transfer fucose onto type 1 and type 2 acceptors in vitro but only onto type 4 Galß1–3GalNAcß1-R termini structures such as GM1 and galacto-N-biose, like the rat hepatoma H35 cell {alpha}2-fucosyltransferase (Sherwood and Holmes, 1998Go). The type 1 and type 2 substrate acceptors tested can receive fucose linked in {alpha}1,2 to the Gal and in {alpha}1,3/4 to the GlcNAc. However, the lack of activity on both Galß1–3GlcNAc and Galß1–4GlcNAc prove that these bovine enzymes have no {alpha}3/4-fucosyltransferase activity towards GlcNAc residue. Until now, the structure Fuc{alpha}1–3GalNAc has only been found at the nonreducing terminal of a salmon ganglioside (Niimura et al., 1999Go), but fucose linked in {alpha}1,4 onto the internal GalNAc of GM1 or type 3/4 precursor chains has never been found as yet, suggesting that the enzyme activity detected was an {alpha}2-fucosyltransferase corresponding to the {alpha}2-fucosyltransferase peptide motifs found in these bovine enzymes. Moreover, analyses of bovine oligosaccharides isolated from mucines show that the {alpha}2-fucosylation is always observed onto the Gal terminal and never onto the internal GalNAc (Mårtensson et al., 1998Go). Interestingly, bovine H is shortened as compared to the other mammalian H enzymes. Such a modification in the COOH end may modify the catalytic properties of the enzyme and could explain its affinity for Galß1–3GalNAc structures.

A survey of the tissue-specific expression pattern of these three genes reveals an unexpectedly wide distribution. There were some discrepancies regarding the expression of {alpha}2-fucosyltransferase previously reported. In humans, the FUT1 blood-type {alpha}2-fucosyltransferase is expressed in tissues of mesodermal origin (Le Pendu et al., 1982Go; Mollicone et al., 1986Go; Watkins, 1995Go) while rabbit fut1 is expressed in brain (Hitoshi et al., 1995Go) and more particularly in dorsal root ganglia (Hitoshi et al., 1999Go). The bovine counterpart was depicted in brain, but also in kidney and intestine. In swine, fut1 is expressed in intestine and two alleles have been described (Meijerinck et al., 1997Go). One has two mutations G307-A and G857-A. It has been shown that swine which possess the G307-A mutation are resistant to colibacillosis. This mutation replaces a neutral-nonpolar residue (alanine 103) by a neutral polar residue (threonine) and might induce a functional modification such as the presence or absence of an {alpha}2-fucosylation on specific acceptor substrates implicated in bacterial adhesion. In bovine, alanine was at the same position, however we could not exclude that a similar mutation may exist and confer the resistance to colibacillosis. Bovine fut2 was expressed in different exocrine tissues as human FUT2 gene (Koda et al., 1997Go), whereas the products of the rabbit gene are restricted to gastrointestinal tract (Hitoshi et al., 1996Go). The bovine sec1 transcript, was found in kidney, whereas in rabbit, sec1 was expressed in mammary and submaxillary glands. In bovine intestine, a high level of activity using phenyl-ß-D-galactoside and type 1 substrate as acceptors was measured, suggesting the existence of a fourth {alpha}2-fucosyltransferase. While structural genes are apparently well conserved among species, their tissue range of expression is different. A similar observation has been previously registered for mammalian {alpha}(2,6) sialyltransferase genes (Mercier et al., 1999Go). These changes in tissue expression patterns may correspond to the vertebrate evolution. The sequential appearance of {alpha}2-fucosylated epitopes in different vertebrate species shows a progression from endodermal to ectodermal tissues (Oriol et al., 1992Go). The evolution is achieved in humans, in which the erythrocytes (mesodermal origin) are the last cells to acquire the histo-blood group ABH antigens. The fucosylation of human erythrocytes is only dependant on the FUT1 gene expression. In contrast, in other mammals except apes, the tissue antigens possess mainly the epitope Gal{alpha}1–3Gal, which is responsible for the hyperacute vascular rejection in xenotransplantation (Joziasse and Oriol, 1999Go). This may explain the tissue restriction and the low expression level of bovine fut1 whereas fut2 encoding the Se-type enzyme is always expressed whatever the species.

The 762 bp probe revealed four bands from which two can be easily associated to fut1 and fut2. However, it was more difficult to identify what signal corresponded to the sec1 gene. A probe corresponding to the 5' coding region was unable to discriminate (Figure 5) between sec1 and the unknown putative gene. However, a probe corresponding to the 3' end of the coding sequence and a part of subsequent 3' UTR hybridized only the upper bands of each digest (data not shown). We conclude that they corresponded to the sec1 gene. Thus, we postulate that the gene whose product transferred fucose in the {alpha}1->2 linkage on type 1 and phenyl-ß-D-galactoside acceptors may be related to this unidentified band. Bovine sec1 and this new entity might be homologous in a large part of their coding sequence since the 762 bp probe and the sec1 probe hybridized even at high stringency conditions. A few nucleotides modifying some amino acids might specify the acceptor substrate recognition, as it has been recently described for human Fuc-TIII (Dupuy et al., 1999Go). The expression of this gene could be under the control of regulatory elements specific to intestinal tract cells since its product was found only in this tissue.

We have characterized three of the four enzymes which transfer fucose in the {alpha}1->2 linkage in the bovine species. Although they were similar to their mammalian counterparts, they were only able to transfer fucose on Galß1–3GalNAc termini whereas other mammalian enzymes transfer fucose on both substrates Galß1–3/4GlcNAc and Galß1–3GalNAc. The possibility of a fourth type of {alpha}2-fucosyltransferase is suggested by enzymatic studies and Southern blot analyses. In the present work we show that at least in one mammalian species, a fourth entity distinct from FUT1, FUT2, and Sec1 is expressed.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Nomenclature
The three genes described in this study were designated bovine fut1, fut2, and sec1 and the cognate {alpha}2-fucosyltransferase enzymes bovine H, Se, and Sec1, respectively.

Materials
The oligonucleotides used in this study are listed in Table I. Before sequencing, all PCR products were cloned into the pMOSBlue T-vector (Amersham, Uppsala, Sweden), pEasy T-vector (Promega, Madison, WI, USA) or pTarget T-vector (Promega, Madison, WI). Transfections of COS-7 cells were carried out using pcDNAI/Amp expression vector (Invitrogen). GDP-[14C]fucose (285 mCi/mmol) and [32P]dCTP (3000 Ci/mmol) were purchased from Amersham, United Kingdom. Type 3/4 acceptor or Galacto-N-biose (Galß1–3GalNAc), phenyl-ß-D-galactoside and GM1 (Galß1–3GalNAcß1–4[NeuAc{alpha}2–3]Galß1–4Glcß1-Cer) were purchased from Sigma (St. Louis, MO). Type 1 acceptor (Galß1–3GlcNAc-(CH2)7CH3) was a gift of Dr. Claudine Augé, University of Paris-Sud, Orsay, France. Type 2 acceptor (Galß1–4GlcNAc-(CH2)8COOCH3) was a gift of Dr. Monica Palcic, University of Alberta, Edmonton, Alberta, Canada. Bovine brain, heart, kidney, lung, and spleen mRNAs were obtained from CLONTECH (Palo Alto, CA). Intestine RNAs were prepared using TRIZOL Reagent (Life Technologies, Grand Island, NY) according to the manufacturer protocols. High density membranes and BAC clones containing genomic bovine inserts were provided by RZPD (Berlin, Germany), BAC library no. 750.

Southern blot analysis of genomic DNAs
Bovine, human, and porcine genomic DNA were subjected to restriction digestion with EcoRI and HindIII and fractionated by electrophoresis through 0.7% (w/v) agarose gel. After electrophoresis, depurination (15 min) with 0.25N HCl and denaturation (30 min) with 0.4N NaOH, DNA was subjected to Southern transfer on Hybond-N+ membranes (Amersham, Arlington Heights, IL). Blots were hybridized with probes generated by PCR amplification of genomic DNA using the primers presented in Table I: F5/R8 (762 bp probe), F1/R2 (fut1 probe), F4/R5 (fut2 probe), and F8/R10 (sec1 probe). The 762 bp probe corresponded to the bovine fut2 catalytic domain. Probes fut1 (243 bp), fut2 (194 bp), and sec1 (247 bp) corresponded to the 5' coding end of bovine fut1, fut2, and sec1 genes, respectively.

After electrophoresis on a 1.5 % (w/v) and gel-extraction, 25 ng of each probe were labeled with [{alpha}-32P]dCTP (Amersham) by random priming (Random Primers DNA Labeling System, Life Technologies, Grand Island, NY) and purified to avoid unincorporated isotope (QIAquick Nucleotide Removal Kit, Qiagen, Hilden, Germany) at a specific activity of 5 x 108 c.p.m./mg or higher. For each blot, high stringent hybridization was carried out for 16 h at 65°C in a buffer containing 10% (w/v) dextran sulfate, 0.5% (w/v) SDS, 0.5 M sodium chloride and 100 µg/ml of sheared salmon sperm DNA. Blots were washed three times at 65°C with (2x) SSC/0.1% SDS, (1x) SSC/0.1% SDS, (0.2x) SSC/0.1% SDS each for 15 min and then subjected to autoradiography.

Rapid amplification of 5' and 3' cDNA ends (RACE)
The Marathon cDNA Amplification kit (CLONTECH) was used to obtain a library of adapter-ligated double-stranded cDNA from bovine brain, heart, intestine, kidney, lung, and spleen tissues. One microgram of poly(A)+ RNA was used as a template for the first strand synthesis, with the 52-mer CDS primer and 100 U of the MMLV reverse transcriptase in a total volume of 10 µl. Synthesis was carried out at 42°C for 1 h. Then, the second strand was synthesized at 16°C for 90 min in a total volume of 80 µl containing the enzyme mixture (RNase H, Escherichia coli DNA polymerase I, and E.coli DNA ligase), the second strand buffer, the dNTP mixture, and the first strand reaction. cDNA ends were then made blunt by adding to the reaction 10 units of T4 DNA polymerase and incubating at 16°C for 45 min. The double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in 10 µl of water. Half of this volume was used to ligate the CLONTECH adapter to the cDNA ends in a total volume of 10 µl using 1 unit of T4 DNA ligase. The ligation reaction was incubated 16 h at 16°C. The resulting cDNA library was diluted to a final concentration of 0.1 mg/ml.

The 5'-end of bovine fut2 was amplified by PCR using 2.5 µl of the library as a template with the oligonucleotide AP1 and R6 (see Table I, binding antisense to the 5'-end of bovine fut2). The 50 µl reaction mixture contained 5 nmol each of dNTPs, 20 pmol of each primer, 50 nmol of MgCl2, and 2.5 U of cDNA Advantage Taq DNA polymerase (CLONTECH). After a pre-PCR cycle (1 min at 94°C, 15 s at 65°C, 2 min at 68°C) 35 cycles were performed (10 s at 94°C, 15 s at 65°C, and 1 min at 68°C), supplemented with 1 s at each cycle, followed by extension at 68°C for 5 min. The resulting PCR product (0.01 µl) was reamplified under the same conditions using the nested oligonucleotides AP2 and R7 (nested to R6, see Table I). Final PCR products were analyzed on a 1.2% agarose gel, and RACE fragments were gel-extracted (QIAquick, Qiagen), cloned, and sequenced using T7 and pUCM13rev sequencing primers. For the determination of the 3'-end of bovine fut2 the same procedure was achieved except that specific primers were F7 and F6. For bovine sec1 5'-end amplification, the same approach was carried out using R12 and R11 (Table I) as specific primers for the first and second PCR rounds, respectively.

DNA sequence analysis
Sequencing was achieved using T7 and U-19 (pMOSBlue T-vector) or T7 and pUCM13rev (pEasy T-vector) sequencing primers, a dye terminal labeling chemistry (kit PRISMTM Ready Reaction Ampli Taq® FS) and the ABI PRISMTM 310 Genetic Analyser (Perkin Elmer, Norwalk, CO).

Northern blot analysis
Two micrograms of poly(A+) RNA, purchased from CLONTECH, were denaturated and fractionated with 0.8% formaldehyde agarose gel electrophoresis and transferred to Hybond-N+ membranes. The blots were prehybridized for 4 h at 42°C in formamide buffer (50% (v/v) deionized formamide, 1% (w/v) SDS, 1x Denhardt’s, (5.5x) SSC, 21.5 mM Na2HPO4, 11% (w/v) dextran sulfate, 7 µg/ml salmon sperm DNA). fut2 probe (194 bp), labeled as described above, was used as bovine fut2 specific probe. Hybridization to ß-actin messenger was used as control. Bovine ß-actin probe was synthesized by PCR using kidney cDNAs and primers corresponding to the most conserved domain of various bovine tissue actin (sense primer: 5'-TTTACAACGAGCTGCGTGTGGCC-3'; antisense primer: 5'-GATCTTCATGAGGTAGTCTGTCAGG-3'). Hybridization was performed at 42°C for 18 h. Blots were washed at 42°C with (2x) SSC/0.1% SDS, (1x) SSC/0.1% SDS, (0.2x) SSC/0.1% SDS for 15 min each and finally with (0.5x) SSC at 65°C and then analyzed using a PhosphorImager 445 SI (Molecular Dynamics). Autoradiography was performed with intensifying screens at –80°C for 24 h.

Transfection and expression of the bovine {alpha}2-fucosyltransferase genes
Recombinant plasmids were isolated (Qiagen plasmid midi Kit, Qiagen) and used to transiently transfect COS-7 cells with SuperFect Transfection Reagent (Qiagen) using the protocol described by the manufacturer. After 48 h, proteins were extracted in lysis buffer (1% [v/v] Triton X-100, 10 mM sodium cacodylate pH 6, 20% [v/v] glycerol, 1 mM DTT) for 2 h at 4°C. The suspension was then centrifuged (12,000 x g for 10 min) at 4°C. Proteins in the supernatant were estimated using the Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as standard (Bradford, 1976Go).

Fucosyltransferase enzyme assay
The fucosyltransferase assays (Hitoshi et al., 1995Go) were performed in a mixture of 25 mM sodium phosphate (pH 6.1), 5 mM ATP, 30 µM GDP-fucose, 3 µM GDP-[14C]fucose (285 mCi/mmol), 50 µg COS-7 extract proteins, and 2 mM acceptor GM1 or galacto-N-biose in a final volume of 100 µl. Each reaction was incubated at 37°C for 2 h and then applied to a Silica Gel 60 or PEI-cellulose thin layer chromatography plate (Merck) and was developed with chloroform/methanol/0.5% (w/v) CaCl2 (55:45:10) or phosphate buffer 1 mM, pH 8.0, respectively. The radioactivity on the plate was determined with the PhosphorImager. The amounts of added enzyme were adjusted (25 µg of protein extract) to ensure that reactions were linear throughout the incubation period (2 h at 37°C). Under these conditions, less than 10% of the substrate was converted. Apparent Km values for {alpha}2-fucosyltransferase activity were calculated from Lineweaver-Burk plots of initial velocity for various acceptors within concentrations of 0.1–10 mM galacto-N-biose or 0.6–10 mM GM1 with a saturating concentration of GDP-fucose (104 µM). For tissue extracts the same conditions were applied, substrate acceptor concentrations were 2 mM GM1, 7.8 mM phenyl-ß-D-galactoside, 0.78 mM type 1 and galacto-N-biose.

For type 1 disaccharide, H type 1, H type 2, and H type 3 trisaccharides and phenyl-ß-D-galactoside, 3 ml of water were added, at the end of the reaction. After centrifugation, supernatant was applied to conditioned Sep-Pak C18 reverse chromatography cartridge (Waters, Milford, CT). The unreacted GDP-[14C]fucose was washed out with 20 ml of H2O, and the radiolabeled reaction products were eluted with two 5 ml portions of methanol, collected into scintillation vials, and counted with 1 volume of BCS (Amersham) in a liquid scintillation beta counter.

Sequence analysis
In addition to the bovine species (this article), eleven animal species were selected because they had the three {alpha}2-fucosyltransferases: (1) H (FUT1), (2) Se (FUT2), and (3) Sec1 (Sec1) cloned and the corresponding sequences were available in GenBank (Table V). The Caenorhabditis elegans putative {alpha}2-fucosyltransferase F (Z92830_5) containing the three peptide conserved motifs (Oriol et al., 1999Go) was added as an outgroup. The sequences of the gorilla, chimpanzee and human Sec1 pseudogenes were corrected in order to get the products of the complete ORF in phase (Apoil et al., 2000Go). This induces a slight underestimation of genetic distances, but avoids the large overestimation induced by frameshifts and premature stop codons.


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Table V. List of the GenBank/EBI accession numbers of the enzyme peptide sequences used for the construction of the phylogenetic tree and the evaluation of relative evolutionary rates of {alpha}2-fucosyltransferases
 
Multiple enzyme sequence alignments were performed with ClustalW 1.7 (Thompson et al., 1994Go). The genetic distances were calculated with the Phylip package using the Fitch-Margoliash last squares method with an evolutionary clock and the tree was drawn with NJplot. Bootstrap analysis was performed by generation of 100 data sets. All software is available in the server from Infobiogen (http://www.infobiogen.fr/).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Paul François Gallet and Marie-Pierre Laforêt for their technical assistance. This work has been achieved in the frame of the French network "GT-rec" and was supported by grants from MENRT, INRA, Conseil Régional du Limousin, and the DG XII of the European Union.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
H, FUT1 encoded {alpha}2-fucosyltransferase; Se, FUT2 encoded {alpha}2-fucosyltransferase; Sec1, FUT2 related human pseudogene; FUT1 and FUT2 are the genome data base (GDB) names of the two human {alpha}2-fucosyltransferase genes. Lowercase fut1, fut2, and sec1 preceded by the animal name indicate each of the animal homologous genes; ORF, open reading frame; UTR, untranslated region; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.


    Footnotes
 
1 To whom correspondence should be addressed at: Bâtiment de Biotechnologie, Faculté des Sciences, 123 avenue Albert Thomas, F-87060 Limoges, France Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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