A Single Amino Acid in the Hypervariable Stem Domain of Vertebrate alpha 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 DupuyDagger , Jean-Michel PetitDagger , Rosella Mollicone§, Rafael Oriol§, Raymond JulienDagger , and Abderrahman MaftahDagger parallel

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alignment of 15 vertebrate alpha 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 alpha 1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha 1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha 1,4-linkage. The single amino acid substitution Trp111 right-arrow 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 right-arrow 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 alpha 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 right-arrow Trp/Glu116 right-arrow 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fucosyltransferases are type II transmembrane proteins catalyzing fucose transfer from GDP-fucose to different oligosaccharide acceptors in alpha 1,2-, alpha 1,3-, alpha 1,4-, and alpha 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 alpha 1,3-fucosyltransferases are known, the amino acids involved in the recognition of different acceptor substrates as type 1 (Galbeta 1,3GlcNAc) and type 2 (Galbeta 1,4GlcNAc) disaccharides or blood group H-type 1 (Fucalpha 1,2Galbeta 1,3GlcNAc) and H-type 2 (Fucalpha 1,2Galbeta 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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.

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 alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Anti-human Fuc-TIII antibody was kindly provided by J. B. Lowe. H-type 1 (Fuc alpha 1,2Galbeta 1,3GlcNAc-biotin) and H-type 2 (Fuc alpha 1,2Galbeta 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 right-arrow Trp/Glu116 right-arrow 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.

To create the Arg115 right-arrow Trp/Glu116 right-arrow Asp change on Fuc-Tb, two oligonucleotides (Eurogentec, Seraing, Belgium; 5'-CGGTCCTCGTGCACCACTGGGACGTCAGCCACCGGCCCCAGATGCAGCTCCCGCCTTCCCCGC-3' (sense) and 5'-GGGGAAGGCGGGAGCTGCATCTGGGGCCGGTGGCTGACGTCCCAGTGGTGCACAGGC-3' (antisense)) harboring the Arg115 right-arrow Trp/Glu116 right-arrow 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) beta -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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide Alignments of alpha 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 alpha 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 alpha 1,3 and alpha 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.

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 right-arrow Arg, Asp112 right-arrow Glu, and Asp112 right-arrow Asn, and the double mutant Trp111 right-arrow Arg/Asp112 right-arrow Glu. In parallel, Fuc-Tb variants were constructed with the amino acid changes Arg115 right-arrow Trp, Glu116 right-arrow Asp, and Glu116 right-arrow Gln and the double mutant Arg115 right-arrow Trp/Glu116 right-arrow 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 right-arrow Glu mutation; lane 4, Fuc-TIII with the Asp112 right-arrow Asn mutation; lane 5, Fuc-TIII with the Trp111 right-arrow Arg mutation); lane 6, Fuc-TIII with the Trp111 right-arrow Arg/Asp112 right-arrow Glu mutations.

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).

The recombinant Fuc-TIII enzyme sharing the single mutation Trp111 right-arrow Arg lost its type 1 acceptor activity and acquired type 2 acceptor activity. The double mutation Trp111 right-arrow Arg/Asp112 right-arrow 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 right-arrow 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 alpha 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 right-arrow Trp or Glu116 right-arrow 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 right-arrow Trp/Glu116 right-arrow 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 alpha 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 alpha 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 right-arrow 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 right-arrow Asn) became inactive with both acceptors (Table III). The same kind of result was obtained with modified Fuc-Tb (Glu116 right-arrow 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 right-arrow 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 right-arrow 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).

                              
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Table IV
Kinetic parameters of Fuc-TIII and its mutated variants
See "Experimental Procedures" for details.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 1,3-fucosyltransferases. A single amino acid, Arg in alpha 1,3-fucosyltransferases and Trp in alpha 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 alpha 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 alpha 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 GalNAcbeta 1,4GlcNAcbeta 1-R to generate GalNAcbeta 1,4(Fucalpha 1,3)GlcNAcbeta 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 alpha 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 right-arrow Asn in Fuc-TIII or Glu116 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow His and Thr87 right-arrow 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.

parallel 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 alpha 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 alpha 1,3/1,4-fucosyltransferase; Fuc-TIV, FUT4-encoded myeloid alpha 1,3-fucosyltransferase; Fuc-TV, FUT5-encoded alpha 1,3/1,4-fucosyltransferase; Fuc-TVI, FUT6-encoded plasma alpha 1,3-fucosyltransferase; Fuc-TVII, FUT7-encoded leukocyte alpha 1,3-fucosyltransferase; Fuc-TIX, FUT9-encoded brain alpha 1,3-fucosyltransferase; Fuc-Tb, bovine futb-encoded alpha 1,3-fucosyltransferase; Fuc-Th, hamster futh-encoded alpha 1,3-fucosyltransferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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