A Single Amino Acid in the Hypervariable Stem Domain of
Vertebrate
1,3/1,4-Fucosyltransferases Determines the Type
1/Type 2 Transfer
CHARACTERIZATION OF ACCEPTOR SUBSTRATE SPECIFICITY OF THE LEWIS
ENZYME BY SITE-DIRECTED MUTAGENESIS*
Fabrice
Dupuy
,
Jean-Michel
Petit
,
Rosella
Mollicone§¶,
Rafael
Oriol§¶,
Raymond
Julien
, and
Abderrahman
Maftah
From the
Institut de Biotechnologie, Faculté
des Sciences, Université de Limoges, 123 Avenue Albert
Thomas, 87060 Limoges and § INSERM U504,
Université de Paris Sud XI, 94807 Villejuif Cedex, France
 |
ABSTRACT |
Alignment of 15 vertebrate
1,3-fucosyltransferases revealed one arginine conserved in all the
enzymes employing exclusively type 2 acceptor substrates. At the
equivalent position, a tryptophan was found in FUT3-encoded
Lewis
1,3/1,4-fucosyltransferase (Fuc-TIII) and
FUT5-encoded
1,3/1,4-fucosyltransferase, the only
fucosyltransferases that can also transfer fucose in
1,4-linkage.
The single amino acid substitution Trp111
Arg in
Fuc-TIII was sufficient to change the specificity of fucose transfer
from H-type 1 to H-type 2 acceptors. The additional mutation of
Asp112
Glu increased the type 2 activity of the double
mutant Fuc-TIII enzyme, but the single substitution of the acidic
residue Asp112 in Fuc-TIII by Glu decreased the activity of
the enzyme and did not interfere with H-type 1/H-type 2 specificity. In
contrast, substitution of Arg115 in bovine
futb-encoded
1,3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or
H-type 2 acceptors. However, the double mutation Arg115
Trp/Glu116
Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid
Trp/Arg seems to modulate the relative type 1/type 2 acceptor
specificity, and its presence is necessary for enzyme activity since
its substitution by the corresponding amide inactivated both Fuc-TIII
and Fuc-Tb enzymes.
 |
INTRODUCTION |
Fucosyltransferases are type II transmembrane proteins catalyzing
fucose transfer from GDP-fucose to different oligosaccharide acceptors
in
1,2-,
1,3-,
1,4-, and
1,6-linkages. The fucosylated glycoconjugates they produce are blood group and oncodevelopmental antigens (1) and are involved in tumorigenesis (2), embryogenesis (3),
normal leukocyte trafficking (4), and leukocyte extravasation in
inflammatory reactions (5-7). Since all fucosyltransferases utilize
GDP-fucose as donor substrate, their specificity depends on the
recognition of the acceptor substrate and the type of linkage formed.
Although the primary sequences of six human
1,3-fucosyltransferases are known, the amino acids involved in the recognition of different acceptor substrates as type 1 (Gal
1,3GlcNAc) and type 2 (Gal
1,4GlcNAc) disaccharides or blood group H-type 1 (Fuc
1,2Gal
1,3GlcNAc) and H-type 2 (Fuc
1,2Gal
1, 4GlcNAc)
trisaccharides are not yet known. These last trisaccharides are
better acceptors than the disaccharides because they give higher values
of fucose incorporation with a better Km and they
are unambiguous in the sense that C-2 of Gal is already substituted by
Fuc, and therefore, they can accept fucose only on C-3 or C-4 of
GlcNAc. This is particularly relevant for
Fuc-TIII1 since up to 4% of
1,2-fucosyltransferase activity has been found with this enzyme (8,
9).
Two enzymes (Fuc-TIII and Fuc-TV) are able to use both H-type 1 and
H-type 2 trisaccharide acceptors, and consequently, they have been
called
1,3/1,4-fucosyltransferases. The Lewis or Fuc-TIII enzyme is
~100 times more efficient on H-type 1 compared with H-type 2 acceptor
substrates (10). The Fuc-TV enzyme is more efficient on H-type 2 than
on H-type 1 substrates, although the relative type 1/type 2 activities
are of the same order of magnitude (10). Finally, the remaining four
1,3-fucosyltransferases (Fuc-TIV, Fuc-TVI, Fuc-TVII, and Fuc-TIX)
are able to use only type 2 acceptor substrates and are consequently
called
1,3-fucosyltransferases.
The Fuc-TIII, Fuc-TV, and Fuc-TVI enzymes constituting the primate
Lewis subfamily of fucosyltransferases have appeared by two successive
duplications of an ancestral Lewis gene, which occurred rather late in
evolution (Fig. 1), after the great
mammalian radiation and before the separation of higher apes and man
from their common evolutionary path (10). These three enzymes share ~85% sequence identity; the main differences among them are located in the stem amino-terminal region (hypervariable region), whereas their
carboxyl-terminal regions are almost identical. Therefore, the
differences in type 1/type 2 specificity among these three enzymes are
expected to be determined by amino acid differences in their
hypervariable regions. Two different strategies have been developed to
define the amino acids involved in the relative differences of type
1/type 2 acceptor substrate specificity.

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Fig. 1.
Phylogenetic tree of the main subfamilies of
vertebrate 1,3-fucosyltransferases. All
these enzymes can use type 2 acceptor substrates; but Fuc-TIII and
Fuc-TV can use, in addition, type 1 acceptors, and they are the
consequence of the latest gene duplication event within the Lewis
subfamily of enzymes. Therefore, it is logical to assume that the
capacity to use type 1 acceptors had appeared in the common ancestor
( ) of these two enzymes. Values of 100 bootstrap replicates are
noted at each divergence point.
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Subdomain swapping of segment 62-110 of Fuc-TIII and the corresponding
segment of Fuc-TV increased the type 1 enzyme activity of Fuc-TV (11).
Eight amino acids were shown to be Fuc-TV-specific in this area.
Site-directed mutagenesis of two of them (Asn86 and
Thr87) by the corresponding His and Ile of Fuc-TIII also
increased type 1 enzyme activity (12), whereas site-directed
mutagenesis of other amino acids in the central area (13) or in the
COOH terminus (14) of Fuc-TIII and Fuc-TV did not modify the relative type 1/type 2 acceptor substrate specificity.
Lowe and co-workers (15) preferred to analyze the more clear-cut
difference between Fuc-TIII and Fuc-TVI. They divided the hypervariable
region into five subdomains and created enzyme chimeras by subdomain
swapping. Functional analysis of the chimeras showed that the type
1/type 2 specificity of human Fuc-TIII and Fuc-TVI depends on amino
acids in the segment corresponding to residues 103-153 of Fuc-TIII.
Eleven amino acids in this area were specific to Fuc-TVI and were
considered as candidates to determine whether or not this
1,3-fucosyltransferase can utilize, in addition, type 1 acceptor
substrates (15). These 11 amino acids could be reduced to four
following the cloning, expression, and peptide sequence analysis of
chimpanzee Fuc-TIII, Fuc-TV, and Fuc-TVI (10) and the bovine Fuc-Tb
enzyme (16). Furthermore, this number could be further reduced to two
amino acids specific to Fuc-TVI (Arg110 and
Glu111) and Fuc-Tb (Arg115 and
Glu116) by addition of the hamster Fuc-Th
enzyme2 to the previous
analysis.3 This study was
conducted to define, by site-directed mutagenesis, the contribution to
H-type 1/H-type 2 acceptor substrate specificity of the two amino acids
Arg115 and Glu116 of Fuc-Tb and the
corresponding residues Trp111 and Asp112 of
Fuc-TIII.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Anti-human Fuc-TIII antibody was kindly provided
by J. B. Lowe. H-type 1 (Fuc
1,2Gal
1,3GlcNAc-biotin) and
H-type 2 (Fuc
1,2Gal
1,4GlcNac-biotin) acceptors were purchased
from Syntesome (Munich, Germany).
Site-directed Mutagenesis--
Cloned futb (16) with
an additional 3'-untranslated sequence (800 base pairs) that gives
better expression4 and
FUT3 (17, 18)5
were incorporated in the mammalian expression vector pcDNAI/Amp (Invitrogen, Carlsbad, CA) and used for mutagenesis. Propagation of
these vectors was achieved in the Escherichia coli XL1-Blue strain. Polymerase chain reaction-based mutagenesis (ExSite kit, Stratagene, La Jolla, CA) was used for all mutations, except for that
which generated the Arg115
Trp/Glu116
Asp changes in Fuc-Tb. The reaction was performed by incubating a
vector (0.1 pmol) with 15 pmol of each primer, one harboring the
mutation (Table I). The reaction was
cycled for 20 rounds (1 min at 94 °C, 2 min at 52-60 °C, and 1 min at 72 °C) with a highly efficient and reliable Taq
DNA polymerase (Stratagene), and then the last extension step was made
at 72 °C for 5 min. Parental DNA was removed by DpnI
restriction endonuclease treatment, and any extended bases produced
during the polymerase chain reaction amplification were eliminated by
Pfu DNA polymerase. The amplified vector with the expected
mutation was used for ligation and quick transformation of the E. coli XL1-Blue strain.
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Table I
Primers used in polymerase chain reaction to obtain each of the desired
mutations
Primer bases in boldface indicate mutations.
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To create the Arg115
Trp/Glu116
Asp
change on Fuc-Tb, two oligonucleotides (Eurogentec, Seraing,
Belgium;
5'-CGGTCCTCGTGCACCACTGGGACGTCAGCCACCGGCCCCAGATGCAGCTCCCGCCTTCCCCGC-3' (sense) and
5'-GGGGAAGGCGGGAGCTGCATCTGGGGCCGGTGGCTGACGTCCCAGTGGTGCACAGGC-3' (antisense)) harboring the Arg115
Trp/Glu116
Asp changes were hybridized and cloned in
pcDNAI/Amp-futb between Tth111I and
SacII restriction sites. All recombinant vectors were controlled by nucleotide sequencing using the dideoxy chain termination method (19).
Transfection and Expression of Recombinant
Enzymes--
Recombinant plasmids were isolated (plasmid midi kit,
QIAGEN Inc., Hilden, Germany) and used to transiently transfect COS-7 cells with SuperFect transfection reagent (QIAGEN Inc.). 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, and 1 mM dithiothreitol) for 2 h at 4 °C. The suspension
was then centrifuged (12,000 × g for 10 min) at
4 °C. Proteins in the supernatant were estimated using the Bradford
assay (Bio-Rad) with bovine serum albumin as a standard (20).
Western Blot Analysis--
For wild-type Fuc-TIII and its
mutated forms produced in COS-7 cells, 100 µg of supernatant proteins
were boiled for 3 min in 5% (v/v)
-mercaptoethanol and 0.02% (w/v)
bromphenol blue and then separated by SDS-polyacrylamide gel
electrophoresis using a Tris/Tricine-12% acrylamide gel. Proteins were
electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Immunoblotting was carried out by
chemiluminescence (chemiluminescence blotting substrate, Roche
Molecular Biochemicals, Mannheim, Germany). Primary antibody (rabbit
anti-human Fuc-TIII) was diluted 1:750, and secondary antibody
(horseradish peroxidase-conjugated mouse anti-rabbit IgG) was diluted
1:1000.
Fucosyltransferase Assay--
Fucosyltransferase assays were
incubated for 1 h at 37 °C in a 60-µl volume reaction
containing 25 mM sodium cacodylate (pH 6.5), 5 mM ATP, 20 mM MnCl2, 10 mM
-L-fucose, 3 µM
GDP-[14C]fucose (310 mCi/mmol; Amersham Pharmacia
Biotech), 0.1 mM trisaccharide acceptor, and 50 µg of
COS-7 extract proteins. The reaction was stopped by addition of 3 ml of
cold water and applied to a Waters Sep-Pak C18 reverse
chromatography cartridge. After washing with 15 ml of water, the
radiolabeled reaction product was eluted with 2 × 5 ml of ethanol
and counted with 2 volumes of biodegradable counting scintillant
(Amersham Pharmacia Biotech).
Determination of Kinetic Parameters--
The apparent
Km values for GDP-fucose of the wild-type enzyme and
mutated variants were determined using 10-250 µM GDP-fucose with 8 µM GDP-[14C]fucose in
each reaction. The experimental conditions of each assay were as
follows: 20 µg of protein, 0.3 mM H-type 1 or H-type 2 acceptors, and 30 min of incubation. The apparent Km values for H-type 1 and H-type 2 acceptors were determined with 0.05-1
mM acceptor and 250 µM GDP-fucose including 8 µM GDP[14C]-fucose; incubation time was 30 min. Kinetic parameter determinations for enzymes with a weak activity
were made with 13 µM GDP-[14C]fucose,
0.3-3 mM acceptor substrate, 40 µg of protein, and 45 min of incubation.
Sequence Analysis--
Multiple alignments were performed with
ClustalW version 1.7 (21), and the phylogenetic tree was made by
neighbor joining with Seaview and Phylo_win
(22).6
 |
RESULTS |
Peptide Alignments of
1,3/4-Fucosyltransferases--
All
previous attempts to define the amino acids involved in the
determination of the type 1/type 2 acceptor substrate specificity of
Fuc-TIII were performed within the Lewis subfamily of enzymes, comparing either Fuc-TIII and Fuc-TV (11, 12) or Fuc-TIII and Fuc-TVI
(10, 15). These studies led to the conclusion that the acceptor
specificity probably resides in the hypervariable stem region.
In this study, we have extended the peptide sequence comparisons of
this hypervariable region to all other
1,3-fucosyltransferase subfamilies of enzymes: myeloid (Fuc-TIV), leukocyte (Fuc-TVII), and
brain (Fuc-TIX). This new multi-alignment showed that three amino acids
are invariant in this region (Val-X-X-His-His),
and only one amino acid (boldface Trp in Table
II) appears to be specific for enzymes
using both type 1 and type 2 acceptors. This amino acid is replaced by
Arg (boldface in Table II) in all the enzymes using only type 2 acceptors and is always followed by an acidic residue (boldface Asp or
Glu in Table II).
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Table II
Alignment of the peptide sequences of the known vertebrate 1,3 and
1,3/1,4-fucosyltransferases around the two candidate amino acids
WD and RE or RD for the
determination of type 1 and type 2 acceptor substrate specificity
Asterisks identify the four previously defined potential candidate
amino acids based on the multiple alignment of the Lewis subfamily of
enzymes (10). Hyphens indicate amino acids identical to the human
Fuc-TIII enzyme.
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Preparation of Fuc-TIII and Fuc-Tb Mutants--
Site-directed
mutagenesis was used to obtain the Fuc-TIII variants harboring the
single amino acid changes Trp111
Arg,
Asp112
Glu, and Asp112
Asn, and the
double mutant Trp111
Arg/Asp112
Glu. In
parallel, Fuc-Tb variants were constructed with the amino acid changes
Arg115
Trp, Glu116
Asp, and
Glu116
Gln and the double mutant Arg115
Trp/Glu116
Asp. Sequencing of the complete open reading
frame confirmed that no other changes were present in the nucleotide
sequence of these constructions.
The levels of expression of all the recombinant Fuc-TIII enzymes in
COS-7 cells were evaluated by Western blotting, and as shown in Fig.
2, some variations occurred.
Nevertheless, the enzyme assays were conducted on crude protein
extracts from transfected COS-7 cells.

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Fig. 2.
Western blot analysis of wild-type and
mutated Fuc-TIII enzymes expressed in COS-7 cells. Lane
1, negative control, 100 µg of proteins from COS-7 cells
transfected with pcDNAI/Amp; lane 2, Fuc-TIII;
lane 3, Fuc-TIII with the Asp112 Glu
mutation; lane 4, Fuc-TIII with the Asp112 Asn mutation; lane 5, Fuc-TIII with the Trp111
Arg mutation); lane 6, Fuc-TIII with the
Trp111 Arg/Asp112 Glu mutations.
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Acceptor Substrate Specificity of Fuc-TIII Mutants--
For each
Fuc-TIII mutant, fucose transfer reactions were conducted with H-type 1 and H-type 2 acceptors. Under the experimental conditions used,
Fuc-TIII had a very weak activity with the H-type 2 acceptor compared
with that obtained with the H-type 1 acceptor (Table
III).
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Table III
Enzyme activities of wild-type Fuc-TIII and Fuc-Tb and the mutated
variants with H-type 1 and H-type 2 acceptor substrates
Activities were measured in the presence of 3 µM
GDP-[14C] fucose and 0.1 mM acceptor substrate
(H-type 1 or H-type 2).
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The recombinant Fuc-TIII enzyme sharing the single mutation
Trp111
Arg lost its type 1 acceptor activity and
acquired type 2 acceptor activity. The double mutation
Trp111
Arg/Asp112
Glu conferred to the
recombinant enzyme higher activity (~2-fold increase with the H-type
2 acceptor), which became equivalent to the activity obtained with the
native Fuc-TIII enzyme using the H-type 1 acceptor. The single
conservative substitution of Fuc-TIII (Asp112
Glu)
decreased by about half the activity of the enzyme without changing its
relative type 1/type 2 acceptor substrate specificity, suggesting that
the nature of the acidic residue (Asp or Glu) does not by itself
determine the H-type 1/H-type 2 transfer activity, although this acidic
residue can help to modulate the relative rates of enzyme activities.
Acceptor Substrate Specificity of Fuc-Tb Mutants--
The native
bovine
1,3-fucosyltransferase Fuc-Tb utilizes exclusively type 2 acceptors (16). Any single substitution of the Fuc-Tb enzyme at each of
the candidate amino acids (Arg115
Trp or
Glu116
Asp) generated an inactive enzyme with both
acceptor substrates (Table III). Only a very small increase in activity
on the H-type 1 acceptor was observed from 7 ± 0.5 to 11 ± 0.5 pmol/h/mg of protein with the double mutation Arg115
Trp/Glu116
Asp, which also conserved a weak
activity (58 ± 3 pmol/h/mg of protein) on the H-type 2 acceptor.
Involvement of Acidic Residue Asp112 of Fuc-TIII and
Glu116 of Fuc-Tb in Enzyme Activity--
In all
1,3-fucosyltransferases listed in Table II, the candidate amino acid
involved in H-type 1/H-type 2 specificity is followed by an acidic
residue (Asp or Glu). Aspartic acid is found in enzymes able to
transfer fucose either on the H-type 1 or H-type 2 acceptor (Fuc-TIII
or Fuc-TV), but also in enzymes acting only on the H-type 2 acceptor
(Fuc-TIV, and Fuc-TIX), whereas Glu is present exclusively in
1,3-fucosyltransferases such as Fuc-TVI, Fuc-TVII, Fuc-Tb, and
Fuc-Th.
The presence of Asp or Glu in wild-type Fuc-TIII or its variants seems
to modulate enzyme efficiency (Table III). However, in Fuc-Tb, only the
presence of Glu, as in the native enzyme, was able to confer activity
to the protein. Indeed, the Glu116
Asp change was
associated with enzyme activity loss, whatever the substrate acceptor.
To define more precisely the requirement of the acidic residue for
enzyme activity, Asp112 of Fuc-TIII was substituted by the
corresponding amide Asn. The Fuc-TIII mutant (Asp112
Asn) became inactive with both acceptors (Table III). The same kind of
result was obtained with modified Fuc-Tb (Glu116
Gln),
which was also unable to transfer fucose on either of the two acceptor
substrates (Table III).
Kinetic Parameters of Native and Mutated Enzymes--
For Fuc-TIII
variants, the apparent Km values for GDP-fucose (~ 30 µM) were similar to that of the parental enzyme (Table
IV). Therefore, amino acid changes in
enzyme variants did not modify the affinity of the Fuc-TIII variants
for the donor substrate. However, significant changes in the
Km values for the H-type 1 and H-type 2 acceptor
substrates were observed for native Fuc-TIII compared with the Fuc-TIII
mutants. As expected, native Fuc-TIII and Fuc-TIII with the
Asp112
Glu mutation had better Km
values for H-type 1 than for H-type 2 acceptors, whereas the two
Fuc-TIII mutants with the Trp111
Arg change had a good
affinity for the H-type 2 acceptor and a poor affinity for the H-type 1 acceptor. It also appeared that the nature of the acidic residue (Asp
or Glu) adjacent to the candidate amino acid (Trp or Arg) had a
significant effect on the kinetics of the recombinant enzyme. The
Trp-Asp association found in wild-type Fuc-TIII is favorable for H-type
1 activity, whereas the Arg-Glu association found in double mutant
Fuc-TIII is convenient for H-type 2 activity (Table IV).
 |
DISCUSSION |
Fucosyltransferases have a common domain structure including a
short NH2-terminal cytoplasmic tail, a signal anchor
domain, a stem region, and a globular COOH-terminal catalytic domain. Truncation at the COOH terminus of one or more amino acids induces a
dramatic loss of enzyme activity, whereas truncation at the NH2 terminus of as much as 61 amino acids for Fuc-TIII or
75 amino acids for Fuc-TV does not alter the enzyme activity (11, 23). First one (24, 25) and then two (26) peptide conserved motifs were
described in the COOH-terminal catalytic domain of all the
1,3-fucosyltransferases, and they are presumed to be involved in
GDP-fucose binding (26, 27), whereas the acceptor substrate-binding domain has been tentatively ascribed to a portion of the hypervariable region comprised between either positions 62 and 110 (11) or positions
103 and 153 (15) of Fuc-TIII.
Based on our previous results (10), a short peptide segment
corresponding to the sequence Pro101-Leu121 of
Fuc-TIII was used for peptide sequence alignment of all known vertebrate
1,3-fucosyltransferases. A single amino acid, Arg in
1,3-fucosyltransferases and Trp in
1,3/1,4-fucosyltransferases, was expected to contribute to the type 1/type 2 acceptor specificity. By site-directed mutagenesis, we have now demonstrated that the single
substitution of Trp111 by Arg conferred to the recombinant
Lewis enzyme the ability to use efficiently H-type 2 instead of H-type
1 acceptor substrate. This newly acquired activity increased when, in
addition, Asp112 was replaced by Glu. The involvement of
Arg in fucose transfer was confirmed by the loss of Fuc-Tb activity
when Trp substituted for Arg115.
Several invertebrate and bacterial
1,3-fucosyltransferases have been
recently found (reviewed in Ref. 28). Two Helicobacter pylori enzymes have been cloned and expressed, and they both use type 2 acceptors (24, 25) and have the similarly charged amino acid Lys
instead of Arg in the candidate position. A Caenorhabditis elegans
1,3-fucosyltransferase has also been cloned and
expressed (29), and it has Gln in the candidate position, but it can
transfer Fuc onto acceptors as GalNAc
1,4GlcNAc
1-R to generate
GalNAc
1,4(Fuc
1,3)GlcNAc
1-R. These nematode
oligosaccharides are different from all known type 1 or type 2 oligosaccharides of the lacto or neolacto series. The remaining
invertebrate and bacterial putative
1,3-fucosyltransferases have
only been defined by sequence homology to vertebrate enzymes, and
nothing is known about their enzyme activity or about the structure of
the acceptor substrates used. Nevertheless, some of them did not have
either Trp or Arg in the corresponding candidate position of the
putative acceptor-binding domain, suggesting that as in the case of
C. elegans, other acceptors might be used by these enzymes.
However, the overall structure of the acceptor substrate-binding domain
is, in general, preserved with a conserved His residue before and a
carboxylic group (Asp or Glu) after the candidate position.
Previous work by Hindsgaul et al. (30) suggested that
oligosaccharide-reactive acceptor hydroxyl groups are involved in a
critical hydrogen bond donor interaction with the glycosyltransferases. The main key polar groups on the oligosaccharide acceptors have been
identified as the reactive hydroxyls at C-3 or C-4 of GlcNAc and C-6 of
Gal (31, 32). On the enzyme side, different amino acids can be involved
in this hydrogen bond, but typical hydrogen bond acceptors are His and
carboxylates (26), as those found in the conserved amino acid positions
flanking the candidate Arg or Trp residue for the definition of the
type 1/type 2 enzyme activity. Therefore, these His, Glu, and Asp
residues can be considered as candidates for the formation of hydrogen
bonds with the acceptor oligosaccharide. Based on recent studies, it
seems reasonable to suggest that the active-site base in the protein
may be a carboxylate anion (33, 34). In another study, Britten and Bird
(35) investigated the amino acids essential for the activity of Fuc-TVI through chemical modification, and they concluded that the
substrate-binding site of the enzyme possesses His residue(s) that are
essential for enzyme activity.
It has been suggested, in the case of the oligosaccharyltransferase,
that the divalent cation cofactor might be in close proximity to the
acceptor oligosaccharide substrate (36). A similar mechanism could
occur in fucosyltransferases, and it was recently suggested that
Mn2+ could interact with a carboxylate anion (Glu or Asp)
in the fucosyltransferase on one side and with the oligosaccharide
acceptor substrate on the other side (37). The complete loss of enzyme
activity by the substitution Asp112
Asn in Fuc-TIII or
Glu116
Gln in Fuc-Tb suggests that this carboxylate
group is necessary for enzyme activity, and therefore, it constitutes a
possible candidate for the active-site carboxylate anion.
The Arg115
Trp substitution in Fuc-Tb was not
sufficient to change the specificity of the enzyme toward type 1 acceptors even with the additional mutation Glu116
Asp.
Therefore, Trp seems to contribute to a type 1 activity, but it is not
sufficient by itself; and even more than the two tested candidates, Trp
and Asp, are probably necessary to change the Fuc-Tb specificity. This
is in accordance with the fact that only a very small increase in the
number of cells producing Lea and sialyl-Lea
antigens was observed in cells transfected by a Fuc-TVI chimera containing Fuc-TIII subdomains 4 and 5 (positions 103-153) (15). In
another work (12), it was shown that two other amino acid changes in
Fuc-TV (Asn86
His and Thr87
Ile)
increased the type 1 activity of the recombinant enzyme.
The molecular phylogeny of fucosyltransferase genes suggested that the
duplication events at the origin of the bovine futb (16) and
primate FUT6 genes occurred before the duplication that
produced the primate FUT3 and FUT5 genes (Fig.
1). Hence, it seems that the common ancestor of FUT3 and
FUT5 genes has acquired the capacity to use type 1 acceptors
without loss of the ability to use type 2 acceptors. This would help to
explain the change in enzyme specificity by a single amino acid
substitution in Fuc-TIII, whereas more complex changes are expected in
Fuc-Tb or Fuc-TVI to generate a strong type 1 acceptor activity because
other independent mutations might have occurred in Fuc-TVI and/or
Fuc-Tb since their earlier divergence from the common evolutionary path
(Fig. 1).
 |
ACKNOWLEDGEMENTS |
We thank P. F. Gallet, M. P. Laforet, and M. Marenda for assistance in sequencing and transfection experiments.
 |
FOOTNOTES |
*
This work was performed in the frame of the French Network
"GT-rec" partially supported by Ministère de l'Education
Nationale de la Recherche et de la Technologie Grant ACC SV 9514111 and CNRS Program Physique et Chimie du Vivant and by Concerted Action 3026PL950004 and Shared Cost Xenotransplantation IO4-CT97-2242 from
Immunology Bio/Technology Program DG XII, the European Union, and the
Conseil Régional du Limousin.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Full-time investigator of CNRS.
To whom correspondence and reprint requests should be
addressed. Tel.: 33-(0)5-55-45-76-84; Fax: 33-(0)5-55-45-76-53; E-mail: maftah{at}unilim.fr.
2
Both Fuc-Tb and Fuc-Th are orthologous
homologous to the ancestor of the human Fuc-TIII, Fuc-TV, and Fuc-TVI enzymes.
3
R. Mollicone and R. Oriol, unpublished results.
4
A. Wierinckx, personal communication.
5
FUT3 to FUT7 and
FUT9 are the Genome Data Base names of the six human
1,3-fucosyltransferase genes.
6
These programs are available on the server from
Cis Infobiogen (E-mail: bioinfo{at}infobiogen.fr; WEB:
http://www.infobiogen.fr/).
 |
ABBREVIATIONS |
The abbreviations used are:
Fuc-TIII, FUT3-encoded Lewis
1,3/1,4-fucosyltransferase;
Fuc-TIV, FUT4-encoded myeloid
1,3-fucosyltransferase;
Fuc-TV, FUT5-encoded
1,3/1,4-fucosyltransferase;
Fuc-TVI, FUT6-encoded plasma
1,3-fucosyltransferase;
Fuc-TVII, FUT7-encoded leukocyte
1,3-fucosyltransferase;
Fuc-TIX, FUT9-encoded brain
1,3-fucosyltransferase;
Fuc-Tb, bovine
futb-encoded
1,3-fucosyltransferase;
Fuc-Th, hamster
futh-encoded
1,3-fucosyltransferase;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
 |
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