INTRODUCTION

Cell surface fucosylated oligosaccharides have received a substantial amount of attention because they play a role in inflammation-mediated cell adhesion and are frequently modified in malignant cells (1-5). The biosynthesis of these glycoconjugates requires the ordered action of several glycosyltransferases, of which fucosylation is the last step (6).

Five human (1,3)-fucosyltransferase genes have been cloned as follows: FUT3 encodes the Lewis (1,3/1,4)-fucosyltransferase or FUC-T3 enzyme (7-10), FUT4 encodes the myeloid (1,3)-fucosyltransferase or FUC-T4 enzyme (11, 12), FUT5 encodes an unspecified type of (1,3)-fucosyltransferase called FUC-T5 (13), FUT6 encodes the plasma (1,3)-fucosyltransferase or FUC-T6 (14, 15), and FUT7 encodes the leukocyte (1,3)-fucosyltransferase or FUC-T7 (16, 17). Three out of these five genes (FUT3, FUT5, and FUT6) constitute a cluster within 1 centimorgan on human chromosome 19p13.3 (18, 19) and share more than 90% sequence identity (14, 20). The individual members of the human (1,2)-fucosyltransferase family, H¹ (21, 22) and Se (23, 24) and the human (1,3)-fucosyltransferase family (FUC-T3, FUC-T4, FUC-T5, FUC-T6, and FUC-T7), are discriminated by differences in substrate specificities, cation requirement, sensitivity to inhibitors, and tissue distribution (25-28). Besides humans and to a lesser extent mice (29), little is known about the molecular mechanisms that determine the tissue-specific (30) and the developmentally regulated expression patterns of fucosyltransferases (31-34).

Previous histochemical data having revealed the absence of (1,4)-fucosylated structures and the prevalence of Gal-type 2 epitope, on bovine red cells and tissues (35), we chose to isolate and characterize the bovine genes homologous to the human FUT3-FUT5-FUT6 cluster to identify the enzymes and glycoconjugate epitopes present in this species. Only one gene, named futb, homologous to the three genes of the human cluster was detected on genomic DNA. We also observed the corresponding mRNA transcript on different bovine tissues such as liver, kidney, brain, and lung. The predicted sequence of the cognate enzyme has a transmembrane type II topology and presents a high degree of identity with the three human enzymes. Nevertheless, the bovine enzyme has a type 2 acceptor substrate specificity, like human FUC-T6, as demonstrated by (i) type 2 acceptor specificity of the activity detected in homogenates of COS-7 cells transfected with futb, (ii) immunofluorescence detection of type 2 (1,3)-fucosylated epitopes (Le^x and sialyl-Le^x) on COS-7 cells transfected with futb, and (iii) the presence of fucosylated type 2 epitopes (Le^x, Le^y, and sialyl-Le^x) on normal bovine tissues.

The position of the bovine gene on the mammalian phylogenetic tree of fucosyltransferase genes suggests that this gene may be the orthologous homologue of an ancestor gene, from which has derived the present human FUT3-FUT5-FUT6 cluster of genes.

EXPERIMENTAL PROCEDURES

The gene described represents the first bovine fucosyltransferase gene. It will be designated futb and the cognate (1,3)-fucosyltransferase enzyme Futb.

PCR was performed as described previously (36) in a mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 9 at 25 °C), 1.5 mM MgCl₂, 150 µM each of dATP, dCTP, dGTP, and dTTP, 50 ng of DNA, 100 pmol of PCR primers (Table I), and 1.25 units of Taq DNA polymerase (Promega) in a total volume of 25 µl. After heating at 95 °C for 4 min, 35 cycles were performed (denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min). The PCR product was analyzed on 1.4% agarose gel electrophoresis. The 459-bp band was eluted from the gel, subcloned into the SmaI site of pBluescript II KS (Stratagene), and sequenced.

Table I. Primers used to synthesize the 459-bp probe and to check futb transcripts Primers Positiona Fragment size Template used bp U, 5-CTGCTGGTGGCTGTGTGTTTCTTC-3 69-92 Genomic DNA 459 L, 5-CAGCCAGCCGTAGGGCGTGAAGAT-3 504-528 U1, 5-ATGTATCCACCTGGTTGCGCC-3 1-21 464 cDNA L1, 5-CCATCTAGGTCCTTCAGTTTCA-3 443-464 U2, 5-ATGTCTCAAGAGAAGCCCAAGCCC-3 106-129 333 cDNA L2, 5-AGTTGCTGGGTGATTCCATGCTGAACC-3 413-439 a Position of primers is given according to the nucleotide sequence numbering of Fig. 1.

Approximately 2 × 10⁵ bacterial colonies representing more than four bovine genomes (37), with recombinant cosmids bearing bovine DNA fragments of 35-kilobase pair average size (bovine genomic library prepared from semen DNA, Stratagene), were screened. Filters were prehybridized at 42 °C for 1 h in 50% deionized formamide, 2 × Pipes buffer (10 × Pipes buffer is 4 M NaCl, 0.1 M Pipes, pH 6.5), 0.5% SDS, and denaturated sonicated salmon sperm DNA (100 µg/ml). Then they were hybridized at 42 °C for 16 h in prehybridization solution containing the bovine futb -³²P-labeled probe of 459 bp, generated by PCR with primers derived from regions highly conserved in human FUT3, FUT5, and FUT6 genes (see above, Table I and Fig. 1). After hybridization, filters were rinsed three times for 15 min each at 65 °C with 0.1 × SSC, 0.1% SDS and subjected to autoradiography.

Nucleotide sequence of the bovine (1,3)-fucosyltransferase gene (futb) and comparison to human FUT6, FUT5, and FUT3 sequences.

The cosmidic DNA, resulting from a positive colony isolated after a tertiary screening, was digested by BamHI, EcoRI, PstI, HindIII, and XhoI restriction enzymes, fractionated through 0.8% agarose gel electrophoresis, and subjected to Southern blot and hybridization. A single 5.8-kilobase pair BamHI restriction fragment encompassing a potential futb structural gene was found. This insert was cloned into the plasmid pFL44 (38) and was designated pFL44-futb (Fig. 2A).

Both strands of the 5.8-kilobase pair BamHI insert were sequenced by the dideoxy chain termination method using T7 DNA polymerase (Sequenase, Amersham Corp.).

Bovine as well as human genomic DNA (as a control) were digested with restriction endonucleases, fractionated through 0.8% agarose gels, and subjected to Southern transfer. Southern blots were probed with an -³²P-labeled FUT3 probe of 303 bp, generated by PCR (14), and a futb probe of 301 bp obtained by BglI digestion of the 1213-bp AvrII-SacI fragment of pFL44-futb. Both probes are located on the 3 ends of the coding regions of FUT3 and futb and cover part of their putative catalytic domains (Fig. 1).

High stringent hybridizations were performed for at least 12 h at 65 °C in a buffer without formamide, containing 0.26 M Na₂HPO₄, 7% (w/v) SDS, 5% (w/v) dextran sulfate, 1% (w/v) bovine serum albumin, and 0.2 mg/ml salmon sperm DNA. Blots were rinsed three times for 20 min each at 65 °C, in 0.2 × SSC and then were subjected to autoradiography.

One set of low stringent hybridizations was performed for 12 h at 65 °C in the above described buffer. Three washings were made for 20 min at 42 °C in 2 × SSC before autoradiography.

Another set of low stringent hybridizations was performed for 12 h at 42 °C in the same buffer, but they were washed only once in 0.2 × SSC.

All cDNAs were synthesized using 1 µg of poly(A)⁺ RNAs (CLONTECH) and the Marathon cDNA Amplification Kit (CLONTECH). First strand synthesis was made with the Moloney murine leukemia virus reverse transcriptase and a modified docking oligo(dT) primer that contains two degenerate nucleotide positions at the 3 end: 5-TTCTAGAATTCAGCGGCCGC(T)₃₀N₁N-3 (N₁ = G, A, or C; N = G, A, C, or T). These nucleotides position the primer at the start of the poly(A) tail and thus eliminate the 3 heterogeneity inherent to conventional oligo(dT) priming (39, 40). Second strand synthesis was performed (41) with a mixture of Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase (CLONTECH). One percent of each cDNA preparation was then amplified by a first PCR in the presence of the U1 and L1 primers (Table I). Aliquots of each reaction were then amplified by a second PCR in the presence of U2 and L2 primers (Table I). The 25-µl PCR reaction mixture contained 2.5 nmol each of dATP, dCTP, dGTP, and dTTP; 10 pmol of primers; 25 nmol of MgCl₂, and 1.25 units of Taq DNA polymerase (Promega). The conditions for the first PCR reaction were as follows: one cycle of denaturation at 94 °C for 2 min, annealing at 60 °C for 15 s and extension at 72 °C for 5 min, followed by 35 cycles; denaturation at 94 °C for 10 s, annealing at 60 °C for 15 s and extension at 72 °C for 40 s, supplemented by 1 s at each cycle, plus a last extension at 72 °C for 7 min. The second PCR reaction was started by denaturation at 94 °C for 2 min, annealing at 68 °C for 15 s, and extension at 72 °C for 5 min, followed by 35 cycles; denaturation at 94 °C for 10 s, annealing at 68 °C for 15 s, and extension at 72 °C for 40 s, supplemented with 1 s at each cycle and a last extension at 72 °C for 7 min. Final products were analyzed in 1.5% agarose gel electrophoresis, and PCR fragments were sequenced after gel extraction (PCR pure-bind Kit, CLONTECH).

The 1213-bp AvrII-SacI fragment from the pFL44-futb plasmid containing futb, was cloned between SacI and XbaI sites, into the pFL44 plasmid (Fig. 2A). After amplification in bacteria, the 1247-bp insert was isolated by EcoRI-HindIII digestion and cloned between EcoRI-HindIII sites into the mammalian expression plasmid pcDNAI/Amp (Invitrogen) (Fig. 2B). A plasmid containing a well oriented insert was selected and designated pcDNAI/Amp-futb. COS-7 cells were transiently transfected using DEAE-dextran (42) and expression incubation time of 48 h.

Transfected cells were homogenized at 4 °C in 1% Triton X-100. Each assay contained in a total volume of 60 µl: 50 µg of the protein cell homogenate or 25 µl of plasma, 25 mM cacodylate buffer, pH 6.5, 4 mM ATP, 20 mM MnCl₂, 10 mM L-fucose, 3.5 µM GDP-[¹⁴C]fucose (Amersham Corp., 300 mCi/mmol), and 5 µl of 1 mg/ml solution of the different synthetic 8-methoxycarbonyloctyl trisaccharide acceptors. The mixture was incubated for 2 h at 37 °C, and the reaction was stopped by addition of 3 ml of water, centrifuged, and the supernatant applied to a conditioned Sep-Pak C₁₈ reverse chromatography cartridge (Waters, Milford), attached to a 10-ml syringe. The unreacted GDP-[¹⁴C]fucose and its hydrolysis products were washed out with 25 ml of H₂O, and the radiolabeled reaction products were eluted with two 5-ml portions of methanol collected directly into scintillation vials and counted with 1 volume of Instagel (Packard, IL) in a liquid scintillation beta counter (43).

Trisaccharide acceptor substrates with the 8-methoxycarbonyloctyl aglycone, R = (CH₂)₈COOCH₃, were obtained from Chembiomed (Alberta Research Council, Edmonton, Alberta, Canada).

The Gal-type 2, Gal1-3Gal1-4GlcNAc-R was synthesized by incubating 10 mg of Gal1-4GlcNAc-R with 35 milliunits of (1,3)-galactosyltransferase (44) and 4 mg of UDP-Gal in 30 mM sodium cacodylate buffer, pH 6.5, containing 0.1% Triton X-100 and 20 mM MnCl₂ at 37 °C for 48 h. After 3, 6, 24, and 28 h of incubation, an additional 4.3 mg of UDP-Gal donor was added to the reaction mixture. The product was isolated on three tandem Sep-Pak C₁₈ cartridges, washed with 150 ml of water, and then eluted with 45 ml of methanol, which was evaporated to dryness and the residue chromatographed on an IATROBEAD column (21 × 180 mm) and washed with 60 ml of 4:1 dichloromethane/methanol to remove Triton X-100. The product was eluted with 65:35:2 dichloromethane/methanol/H₂O and evaporated to dryness (final yield: 12 mg of trisaccharide). The NMR spectrum of this product showed signals for the new anomeric H-1 proton of the Gal residue at  5.146 ppm (J = 4.0 Hz) (45).

Transfected cells were trypsinized and distributed in 96-well conic-bottom microtiter plates (3 × 10⁵ cells/well). Oligosaccharide epitopes were stained by 30 min incubation with first monoclonal antibodies (50 µl per well), washed twice in phosphate-buffered saline, pH 7.5, and incubated for 30 min with fluorescein isothiocyanate-labeled sheep anti-mouse Ig's second antibodies (Pasteur Diagnostics, Marnes la Coquette, France). Each reaction was stopped by sucking off the reagent, after 10 min centrifugation of the plates at 2000 rpm, and then cells were resuspended and washed (3 ×) in phosphate-buffered saline. Stained and washed cells were resuspended in 10 µl of phosphate-buffered saline/paraformaldehyde 4%. Then 5 µl of Mowiol 4:80 (Hoechst, Frankfurt, Germany) were added, and they were mounted under coverslides for observation, on a Leitz SM-Lux epifluorescence microscope. Positive and negative cells were counted with a 25 × oil-immersion NPL-fluotar objective.

Routine formalin-fixed/paraffin-embedded sections were deparaffinated and stained by indirect (monoclonal antibodies) or direct (lectin) immunofluorescence. They were incubated for 30 min in a wet chamber with the first monoclonal antibody, washed, and stained for 30 min with fluorescein isothiocyanate-labeled sheep anti-mouse Ig's second antibody (Pasteur Diagnostics, Marnes la Coquette, France). Direct staining was performed for 30 min with fluorescein isothiocyanate-labeled Ulex europaeus lectin 1 (UEAI) and fluorescein isothiocyanate-labeled Griffonia simplicifolia isolectin I-B4 (GSI-B4) (Vector Laboratories, Burlingame, CA). Stained slides were washed again and mounted under coverslides with 1 drop of Mowiol 4:80 (34) and observed under the epifluorescence microscope.

Anti-Le^x were obtained from Sigma (CD15), Valbiotech (SSEA1), and Chembiomed (82H5); anti-Le^a (069, 070, 071), anti-A (013), and anti-B (026) were obtained from the Second International Workshop on Monoclonal Antibodies Against Human Red Blood Cells (46); anti-sialyl-Le^x was from Valbiotech (KM93) and P. I. Terasaki (UCLA) (TT19A6); anti-Le^y (75.12) was from P. Avner (Pasteur Institute, Paris, France); anti-Le^a (7LE), anti-Le^b (2.25LE), and anti-sialyl-Le^a (19.9) were from J. Bara (INSERM U55, St. Antoine Hospital, Paris, France).

Twelve selected complete coding sequences available from the GenBank/EMBL data base (Table II), were aligned with the Clustalw 1.5 program, and the genetic distances in the matrix were analyzed with the Phylip phylogeny package, using the Fitch-Margoliash (47) least square method with evolutionary clock.² The phylogenetic tree was drawn from the Phylip dendrogram with the NJ plot program in a Power Macintosh 6100/66 computer.

Table II. Accession numbers on the GenBank/EMBL data base of the DNA sequences containing the coding sequences (cds) of the published functional fucosyltransferase genes of different species, used to build the phylogenetic tree of Fig. 6 Accession number Species Locus DNA cds Protein amino acids Ref. bp M355[GenBank]31 Human FUT1 1098 365 64 U178[GenBank]94 Human FUT2 999 332 24 X535[GenBank]78 Human FUT3 1086 361 7 M585[GenBank]96 Human FUT4 1218 405 11 M814[GenBank]85 Human FUT5 1125 374 13 L016[GenBank]98 Human FUT6 1080 359 14 X780[GenBank]31 Human FUT7 1029 342 16 X802[GenBank]26 Rabbit rfut1 1122 373 65 X802[GenBank]25 Rabbit rfut2 1065 354 65 U334[GenBank]57 Mouse mfut4 1302 433 29 U459[GenBank]80 Mouse mfut7 1170 389 66 X878[GenBank]10 Bovine futb 1098 365 This article

RESULTS

Using the bovine futb probe (459 bp), obtained as described under "Experimental Procedures" and corresponding to the stem domain of FUT3, FUT5, and FUT6 human genes, the hybridization screening of a bovine genomic library for an (1,3)-fucosyltransferase gene yielded one positive cosmid.

BamHI digestion released a 5.8-kilobase pair fragment (Fig. 2A) that also cross-hybridizes with the futb probe. Nucleotide sequence analysis of the genomic fragment identified a single long open reading frame including a sequence fully homologous to the bovine probe (Fig. 1). This open reading frame was located within a sequence context largely consistent with Kozak's consensus rules for mammalian translation initiation. Like its human counterpart, the bovine gene apparently maintains a single coding exon.

Hydropathy analysis (48) of the 365-amino acid protein sequence, predicted by the open reading frame, identified a single 19-amino acid hydrophobic segment at the NH₂ terminus, corresponding to amino acids 15-34 flanked by histidine and arginine, implying that the polypeptide has the type II transmembrane topology typical of mammalian glycosyltransferases (49). Sequence comparisons made between the predicted bovine protein and human FUC-T3, FUC-T5, and FUC-T6 enzymes revealed that they share 67.3, 69, and 69.3% amino acid sequence identity, respectively (Fig. 3).

Comparison of protein sequences of bovine (1,3)-fucosyltransferase enzyme (Futb) and human FUC-T6, FUC-T5, and FUC-T3 enzymes.

Seven cysteine residues are common to human FUC-T3, FUC-T5, and FUC-T6 (16); the bovine enzyme has these same 7 cysteines (Fig. 3), including the bovine Cys-147, which determines N-ethylmaleimide sensitivity (50). As expected, 90% of the Futb activity determined on type 2 acceptors was inhibited by pretreatment with 8 mM N-ethylmaleimide for 1 h at 37 °C. Consequently, Futb behaves as FUC-T3, FUC-T5, and FUC-T6 and is different from the human myeloid (FUC-T4) and leukocyte (FUC-T7) enzymes, which are resistant to N-ethylmaleimide (26).

Two potential consensus sites for asparagine-linked glycosylation (bovine amino acids 158 and 189) are common to the bovine Futb and the human FUC-T3, FUC-T5, and FUC-T6 enzymes. Another bovine glycosylation site (Asn-100) is close to a similar glycosylation site in human FUC-T5 (Asn-105) and FUC-T6 (Asn-91). Finally, a fourth glycosylation site is only present in human FUC-T5 (Asn-46) and FUC-T6 (Asn-60) (14), and it is absent from human FUC-T3 and bovine enzymes (Fig. 3).

Protein sequence alignment of bovine Futb and human FUC-T3, FUC-T5, FUC-T6 across the subdomains 4 and 5 (51) also revealed an intermediate primary structure of the bovine enzyme. Indeed, between bovine positions 115 and 155 there are 14 amino acids common to the four enzymes (37%), and only 10 are specific for the bovine sequence (24%). One amino acid is identical only to FUC-T3 (Futb position 122), four are identical to FUC-T3 and FUC-T5 (Futb positions 127, 131, 146, 153), five are identical to FUC-T5 and FUC-T6 (Futb positions 140, 141, 143, 145, 155), and five are common only with the human FUC-T6 (Futb positions 115, 116, 117, 124, 151) (Fig. 3).

After position 46, the FUC-T5 enzyme has a unique insertion of 11 amino acids, which is not present in any of the other two human enzymes (14) nor in the bovine enzyme. In this area the bovine peptide segment, between positions 36 and 69, shows the strongest divergency from human enzymes. Indeed, only 2 amino acids (6%) are common to the four sequences (Fig. 3).

FUT3 and futb catalytic domain probes were used to sample both bovine and human genomes for cross-hybridizing DNA sequences. Regardless of the restriction enzyme used, both probes identify the same three bands corresponding to human FUT3, FUT5, and FUT6 genes in human genomic DNA (14). Alternatively, but also regardless of the restriction enzyme used, the same two probes recognize only one band corresponding to the bovine gene identified above in bovine genomic DNA (Fig. 4A). As a control, the low stringency hybridization of bovine DNA with futb probe revealed a similar pattern (Fig. 4, B and C). At these low stringencies two other bands were detected resulting from unspecific hybridization due to the high amount of repeated satellite DNA. The 1.4-kilobase pair band (Fig. 4, B and C) corresponded to an already described bovine satellite (52), and the other around 1.8-kilobase pair (Fig. 4B) is not well characterized. Altogether, these results suggest that in the bovine genome, there is a single gene, futb, related to the human enzyme family of (1,3)-fucosyltransferases.

cDNAs obtained by reverse transcriptase-PCR on mRNA transcripts from bovine liver, kidney, lung, and brain were probed by nested PCR using specific primer pairs corresponding to the futb stem domain (Table I and Fig. 1). In all cases, the clear-cut amplified band of 333 bp (Fig. 5) was eluted and sequenced. A complete identity between sequences of the amplified DNAs and futb was found, certifying that the bovine gene is effectively transcribed in the probed bovine tissues.

To confirm that the bovine sequence encodes a functional (1,3)-fucosyltransferase, the 1247-bp EcoRI-HindIII fragment encompassing the bovine open reading frame was cloned into the mammalian expression vector pcDNAI/Amp (Fig. 2B), and the resulting plasmid (pcDNAI/Amp-futb) was introduced into COS-7 cells. The cells were analyzed to assess in vitro substrate acceptor pattern of enzyme activity (Table III) and the appearance of fucosylated type 1 and type 2 epitopes on transfected cells (Table IV), comparatively to COS-7 cells transfected with the human FUT3, FUT5, and FUT6 genes.

Table III. (1.3/1.4)-Fucosyltransferase activities of extracts of COS7 cells transfected with pcDNAI/Amp vector alone or containing either human FUT3, FUT5, or FUT6, or bovine futb constructs Enzyme activity is expressed as cpm of [14C]fucose incorporated onto synthetic oligosaccharide acceptors, after 2 h incubation at 37 °C. Acceptor Oligosaccharides Transfected constructions pcDNAI/Amp Human FUT3 Human FUT5 Human FUT6 Bovine futb Type-1 Fuc1-2Gal1-3GlcNAc-Ra 0 50,000 11,960 0 0 Type-2 Fuc1-2Gal1-4GlcNAc-R 0 2,690 19,100 24,900 3,000 Gal1-3Gal1-4GlcNAc-R 0 1,350 3,920 19,100 2,390 NcuAc2-3Gal1-4GlcNAc-R 0 650 4,450 22,900 1,880 a R, (CH2)8COOCH3.

Table IV. Percentage of fluorescent positive cells detected with monoclonal antibodies Anti-type 1 (Lea and sialyl-Lea), anti-type 2 (Lex and sialyl-Lex), and anti-human blood group B, on COS-7 cells are transfected with the pcDNA1/Amp vector alone or containing either human FUT3, FUT5, or FUT6 or the bovine futb constructs. Monoclonal antibodies Transfected constructions Chain Epitope Name pcDNAI/Amp Human FUT3 Human FUT5 Human FUT6 Bovine futb Type 1  Lea 7LE 0 15 4 0 0 Lea 069 0 32 4 0 0 Lea 070 0 40 6 0 1 Lea 071 0 40 4 0 1 Sialyl-Lea 19.9 0 53 22 0 0 Type 2  Lex 82H5 0 29 36 35 22 Lex SSEA1 0 42 54 40 28 Lex CD15 0 18 22 25 14 Sialyl-Lex TT19A6 0 12 30 25 20 Sialyl-Lex KM93 0 31 28 23 18 Human blood group B B 026 0 0 0 0 0

Both analyses demonstrate that pcDNAI/Amp-futb can determine transient expression of type 2 (Le^x and sialyl-Le^x) but not type 1 (Le^a or sialyl-Le^a) epitopes. These results are similar to those obtained with the human FUT6 construct (Tables III and IV). However, in quantitative terms the bovine enzyme is about 10-fold less efficient than the human FUC-T6 for the three type 2 acceptors tested. The amount of fucose incorporated by the bovine enzyme on H-type 2 is 7-fold lower than that of FUC-T5 and similar to that obtained with the FUC-T3 human enzyme. The amounts of fucose incorporated onto Gal-type 2 and sialyl-type 2, by the bovine enzyme, are intermediate between those observed with human FUC-T3 and FUC-T5 enzymes (Table III).

We have added to the human paralogous fucosyltransferase tree (20) the sequences of orthologous animal fucosyltransferase genes to make a combined phylogenetic tree. This new tree suggests that separation of mouse, rabbit, and bovine species from the main evolutionary pathway, during the great mammalian radiation, about 80 millions years ago (Fig. 6), occurred after the duplication events which originated the ancestral H and Se (1,2)-fucosyltransferase genes and the divergency of the ancestors of the myeloid, leukocyte, and Lewis (1,3)-fucosyltransferase loci but before the duplication events that originated the present FUT3, FUT5, and FUT6 human genes. Consequently, the bovine fucosyltransferase gene, described in this paper, may be the orthologous homologue of an ancestor gene, which originated the present human FUT3-FUT5-FUT6 cluster.

Human FUC-T6 activity is mainly found in plasma, whereas human FUC-T3 is absent from plasma. Using the same conditions established for detection of human (1,3)-fucosyltransferase activity, no enzyme activity was detected in two different samples of bovine plasma. Alternatively, (1,3)-fucosyltransferase activity has been reported in mesenteric lymph nodes, suggesting the presence of another calf enzyme functionally homologous to the human FUC-T4 (53).

Bovine tissues present an immunofluorescent pattern of carbohydrate epitopes similar to other lower mammals and new world monkey tissues but quite different from human and old world monkey tissues (35). Vascular endothelium, leukocytes, and red cells are strongly positive with the anti-Gal isolectin GSI-B4 and are completely negative with ABH and either of type 1 or type 2 Lewis-related reagents.

Bovine pancreas present strong staining with GSI-B4 of vascular endothelium, canaliculi, and ducts and weak staining of acinar cells and ducts, as illustrated on Fig. 7A. For comparison strong staining of acinar cells and ducts with anti-A and no staining at all of vascular endothelium are illustrated in Fig. 7B. None of the reagents tested stained the endocrine cells of the islets of Langerhans (white star, Fig. 7A).

Kidney cortex shows strong staining, with GSI-B4, of vascular endothelium of glomeruli, intertubular capillaries, and larger vessels and weak staining of the epithelial cells of proximal convoluted tubules (Fig. 7C). The only stain observed in kidney, with other Lewis-related reagents, was seen with anti-sialyl-Le^x, and it was specifically located on cells of the macula densa of the juxtaglomerular apparatus (Fig. 7D), but the glomeruli and the rest of the renal parenchyma were negative.

Liver vascular endothelium and hepatocytes were strongly stained with GSI-B4, whereas biliary ducts were weakly stained with GSI-B4 (Fig. 7E). No other staining was detected with other Lewis-related reagents, with the exception of a weak staining of biliary ducts with anti-sialyl-Le^x (Table V).

Table V. Immunofluorescent detection of oligosaccharide epitopes, on tissue sections of normal bovine tissues, with specific reagents for fucosylated type 1, fucosylated type 2, Gal, and human blood group A epitopes Reagents Vascular endothelium erythrocytes and leukocytes Pancreas Kidney tabules Liver Lung Small intestine Colon Chain Epitope Name Acini Ducts Proximal Distal Hepatocytes Biliary ducts Bronchial epithelium Brush border Goblei cells Brush border Goblei cells Type 1  Lea 9LE                         Leb 2.25LE                         Sialyl-Lea 19.9                         Type 2  Lex 82H5                 +++   ++   Ley 75.12                 ++   ++   Sialyl-Lex KM93          a   +           H UEAIb     ±         ±     ± +  Gal GSI-B4c ++ + +++ +   +++ ± +++ +++ ++ + +++ Human blood group A A 013   +++ +++   ++   ++ + +++ +++ +++ +++ a Mainly negative, but epithelial cells of the macula densa in the juxtaglomerulus apparatus were positive ± (Fig. 7D). b UEAI, isolectin 1 of U. europaeus, reacting mainly with the H-type 2 epitope (Fuc1-2 Gal 1-4GlcNAc). c GSI-B4, isolectin I-B4 cf G. simplicifolia reacting mainly with the Gal epitope (Gal1-3Gal).

All vascular endothelium and bronchial epithelium of lung were strongly stained with GSI-B4 (Table V).

Type 2-fucosylated (Le^x and Le^y) epitopes were detected only on the brush border of epithelial cells of small (Fig. 7G) and large intestine (Fig. 7H), whereas fucosylated type 1 (Le^a, Le^b, sialyl-Le^a) was not detected at all in any of the cow intestinal sections studied (Table V). Presence of Le^x epitope has also been reported on bovine pituitary hormones (54).

Human blood group A and/or H epitopes were not present on bovine red cells nor on vascular endothelium but were found in exocrine epithelial cells of pancreas (Fig. 7B), renal distal convoluted tubules, biliary ducts, lung, small intestine, and colon (Fig. 7F and Table V).

DISCUSSION

The bovine futb gene is similar in its structure to FUT3, FUT5, and FUT6 human genes, and the corresponding cognate enzymes are nearly identical in their COOH-terminal regions (catalytic domain).

Some amino acids in the subdomains 4 and 5 play an important role in determining the efficiency with which human FUC-T3, FUC-T5, or FUC-T6 use type 1 and type 2 acceptor substrates (51, 55). The bovine enzyme presents, in this region (positions 115-155), a greater homology to the human FUC-T6 enzyme, in good agreement with the exclusive type 2 acceptor substrate specificity of both enzymes. Since the main acceptor chain present in bovine tissue is the Gal1-3Gal1-4GlcNAc epitope, which is absent from human tissues, but is in vitro an acceptor for human (56), mouse (29), and a bovine lymph node (53) (1,3)-fucosyltransferases, a special effort was made to add this acceptor to the present study. This trisaccharide with the same hydrophobic aglycone tail as the other three acceptors was synthesized, but it was not a better acceptor for the bovine enzyme as compared with the other acceptors tested (Table III). Therefore, the quantitative difference in fucose incorporation between bovine and human gene products cannot be ascribed to a specific preference of this bovine enzyme for the Gal-type 2 acceptor substrate.

Large amounts of Gal epitopes are present in all bovine tissues, suggesting that the low amounts of Le^x detected on bovine tissues may be secondary to bovine Le^x epitopes being built on this Gal-type 2 acceptor. The resulting Gal tetrasaccharide epitope Gal1-3Gal1-4(Fuc1-3)GlcNAc is not well recognized by anti-Le^x reagents, because the terminal Gal masks the Le^x epitope (57). Furthermore, the Gal-type 2 epitope is a good acceptor for the bovine lymph node (1,3)-fucosyltransferase (53). However, preliminary histochemical results suggest that the coffee bean -galactosidase digestion removes the Gal epitope from tissue sections but does not increase the staining of anti-Le^x on calf tissue.³ The question now is to know if the (1,3)-fucosyltransferase is effectively expressed in the bovine tissues that we have probed. To overcome this difficulty, we looked for futb transcripts in mRNA extracts from some bovine tissues. Reverse transcriptase-PCR analysis (Fig. 5) showed that futb is transcribed in liver, kidney, brain, and lung.

To control that the lower expression of enzyme activity of futb cannot be ascribed to an incorrectly folded mRNA transcript lacking its 5-untranslated region, we also transfected COS-7 cells with a pcDNAI/Amp construct containing the futb coding sequence plus the 5-untranslated region of a bovine kidney transcript. A high increase of enzyme activity (about 10-fold) was observed with this DNA construct suggesting that the expression of futb is under a tight control of 5-untranslated regions.⁴

Fucosylated glycoconjugate epitopes are synthesized in two compartments in man. Mesodermal cells produce mainly type 2 ABH and Lewis structures (Le^x, Le^y, sialyl-Le^y) under control of FUT1 (H), FUT4, FUT6, and FUT7 genes, whereas exocrine cells produce mainly type 1 ABH and Lewis structures (Le^a, Le^b, sialyl-Le^a) under control of FUT2 (Se) and FUT3 (Le) genes (58). The Le^x epitope is mainly found on neutrophiles, brain (26), and epithelial cells of kidney proximal convoluted tubules (34, 59). Sialyl-Le^x is also found on renal proximal convoluted tubules and on monocytes and on hepatocytes (60). Le^a and Le^b are found on biliary and pancreatic ducts (61), on bronchial epithelium, and on surface digestive epithelium, whereas Le^x and Le^y are mainly found on deep glands of digestive mucosae (62). This distribution of glycoconjugate epitopes on human tissues is different from the immunofluorescent pattern found in bovine tissues (Table V and Fig. 7). It suggests that different glycoconjugate epitopes have been selected by different species, in different tissues, and it illustrates that type 2 Le^x-related structures are poorly represented in bovine tissues.

Despite using the same GDP-fucose donor substrate, having the same type II transmembrane topology, a similar location of the catalytic domain in the COOH terminus, a similar size (Table II), and a common three-dimensional folding (63), less than 20% of sequence identity was found between the two main families of -2- and -3-fucosyltransferase enzymes (63). The present phylogenetic analysis is compatible with a low degree of homology, since the largest genetic distance detected in the Phylip matrix corresponds to these two main families of -2- and -3-fucosyltransferases. Consequently, the root of the tree is located between these two families of fucosyltransferases (Fig. 6) (20).

The position of futb on the phylogenetic tree suggests that the duplication event, at the origin of this gene, occurred before the duplication events that originated FUT3, FUT5, and FUT6 human genes. Therefore, it is not surprising that, by Southern blot, whatever the stringency conditions, only one hybridization signal is obtained on bovine genomic DNA (Fig. 4, A-C), regardless of restriction enzymes and human or bovine origin of the probes used. Alternatively, the presence of three hybridization bands on human genomic DNA with the bovine probe confirms that the three human genes have a high degree of sequence homology and cross-hybridize with the bovine probe.

In addition, the futb gene that we propose to be the orthologous homologue of the ancestor of the human fucosyltransferase cluster of genes FUT3-FUT5-FUT6 is located on the bovine chromosome 7 (36), which is, by comparative mapping, homologous of the human chromosome 19, bearing the FUT3-FUT5-FUT6 cluster.

In good agreement with the present phylogenetic evolutionary model, we have recently cloned three different chimpanzee genes, each with about 98% sequence identity with the corresponding human FUT3, FUT5, and FUT6 genes, suggesting that the divergency of chimpanzee and human species occurred after the duplication events which originated the present FUT3-FUT5-FUT6 cluster of genes.⁵