(Received for publication, June 20, 1996, and in revised form, December 3, 1996)
From the Only one bovine gene, corresponding to the human
cluster of genes FUT3-FUT5-FUT6, was found by Southern blot
analysis. The cognate bovine 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 Previous histochemical data having revealed the absence of
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.
The gene described represents the first bovine
fucosyltransferase gene. It will be designated futb and the
cognate 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
Primers used to synthesize the 459-bp probe and to check futb
transcripts
Institut de Biotechnologie,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
(1,3)-fucosyltransferase shares 67.3, 69.0, and 69.3% amino acid sequence identities with human FUC-T3,
FUC-T5, and FUC-T6 enzymes, respectively. As revealed by protein
sequence alignment, potential sites for asparagine-linked glycosylation and conserved cysteines, the bovine enzyme is an intermediate between
FUC-T3, FUC-T5, and FUC-T6 human enzymes. Transfected into COS-7 cells,
the bovine gene induced the synthesis of an
(1,3)-fucosyltransferase
enzyme with type 2 substrate acceptor pattern specificity and induced
expression of fucosylated type 2 epitopes (Lex and
sialyl-Lex), but not of type 1 structures (Lea
or sialyl-Lea), suggesting that it has an acceptor
specificity similar to the human plasma FUC-T6. However, no enzyme
activity was detected in bovine plasma. Gene transcripts are detected
on tissues such as bovine liver, kidney, lung, and brain. The type 2 sialyl-Lex epitope was found in renal macula densa and
biliary ducts, and Lex and Ley epitopes were
detected on the brush border of epithelial cells of small and large
intestine, suggesting a tissue distribution closer to human FUC-T3, but
fucosylated type 1 structures (Lea, Leb, or
sialyl-Lea) were not detected at all in any bovine tissue.
Analysis of genetic distances on a combined phylogenetic tree of
fucosyltransferase genes suggests that the bovine gene is the
orthologous homologue of the ancestor of human genes constituting the
present FUT3-FUT5-FUT6 cluster.
(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).
(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 (Lex and
sialyl-Lex) on COS-7 cells transfected with
futb, and (iii) the presence of fucosylated type 2 epitopes
(Lex, Ley, and sialyl-Lex) on
normal bovine tissues.
Nomenclature
(1,3)-fucosyltransferase enzyme Futb.
(Stratagene), and sequenced.
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.
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).
DNA Sequence Analysis
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.).
Southern Blot Analysis of Bovine Genomic DNABovine 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.
Reverse Transcriptase-PCR Detection of futb TranscriptsAll 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)30N
1N-3
(N
1 = 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.
Fucosyltransferase Enzyme AssayTransfected 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).
Synthetic Oligosaccharide AcceptorsTrisaccharide acceptor substrates with the 8-methoxycarbonyloctyl aglycone, R = (CH2)8COOCH3, were obtained from Chembiomed (Alberta Research Council, Edmonton, Alberta, Canada).
The Gal-type 2, Gal
1-3Gal
1-4GlcNAc
-R was synthesized by
incubating 10 mg of Gal
1-4GlcNAc
-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.
Tissue Immunofluorescence StainingRoutine 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.
Mouse Monoclonal AntibodiesAnti-Lex were obtained from Sigma (CD15), Valbiotech (SSEA1), and Chembiomed (82H5); anti-Lea (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-Lex was from Valbiotech (KM93) and P. I. Terasaki (UCLA) (TT19A6); anti-Ley (75.12) was from P. Avner (Pasteur Institute, Paris, France); anti-Lea (7LE), anti-Leb (2.25LE), and anti-sialyl-Lea (19.9) were from J. Bara (INSERM U55, St. Antoine Hospital, Paris, France).
Molecular PhylogenyTwelve 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.
|
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).
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).
Southern Blot Analyses of Bovine Genomic DNAFUT3
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.
Identification of mRNA Transcripts of futb
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.
Expression of Bovine futb and Human FUT3, FUT5, and FUT6 Genes in COS-7 Cells
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.
|
|
Both analyses demonstrate that pcDNAI/Amp-futb can
determine transient expression of type 2 (Lex and
sialyl-Lex) but not type 1 (Lea or
sialyl-Lea) 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.
Tissue Enzyme Distribution
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-Lex, 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-Lex (Table V).
|
All vascular endothelium and bronchial epithelium of lung were strongly stained with GSI-B4 (Table V).
Type 2-fucosylated (Lex and Ley) epitopes were detected only on the brush border of epithelial cells of small (Fig. 7G) and large intestine (Fig. 7H), whereas fucosylated type 1 (Lea, Leb, sialyl-Lea) was not detected at all in any of the cow intestinal sections studied (Table V). Presence of Lex 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).
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 Gal1-3Gal
1-4GlcNAc
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 Lex detected on bovine
tissues may be secondary to bovine Lex epitopes being built
on this
Gal-type 2 acceptor. The resulting
Gal tetrasaccharide
epitope Gal
1-3Gal
1-4(Fuc
1-3)GlcNAc is not well recognized
by anti-Lex reagents, because the terminal
Gal masks the
Lex 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-Lex 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 (Lex, Ley, sialyl-Ley) under control of FUT1 (H), FUT4, FUT6, and FUT7 genes, whereas exocrine cells produce mainly type 1 ABH and Lewis structures (Lea, Leb, sialyl-Lea) under control of FUT2 (Se) and FUT3 (Le) genes (58). The Lex epitope is mainly found on neutrophiles, brain (26), and epithelial cells of kidney proximal convoluted tubules (34, 59). Sialyl-Lex is also found on renal proximal convoluted tubules and on monocytes and on hepatocytes (60). Lea and Leb are found on biliary and pancreatic ducts (61), on bronchial epithelium, and on surface digestive epithelium, whereas Lex and Ley 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 Lex-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
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X87810[GenBank].
We are grateful to Dr. P. F. Gallet for expert technical assistance and helpful discussions.