Unité de Génétique Moléculaire Animale, UMR 1061 Université-INRA, GDR-CNRS 2590, Institut des Sciences de la Vie et de la Santé, Faculté des Sciences et Techniques, 87060 Limoges, France
Received on October 27, 2003; revised on December 30, 2003; accepted on December 30, 2003
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Abstract |
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Key words: aromatic amino acid / acceptor substrate specificity / conserved peptide motifs / Lewis fucosyltransferase / site-directed mutagenesis
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Introduction |
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The 3- and
3/4-FUTs have a putative common type II structure (Figure 1). Comparison of their peptide sequences localizes most of the amino acid differences in the amino-terminal part (variable segment), whereas numerous identities are present in the carboxy-terminal portion (constant segment). Four highly conserved sequences were described all along the peptide sequence (Figure 1). Motifs I and II are localized in the constant segment and are specific to
3- and
3/4-FUTs (Oriol et al., 1999
). In the variable segment, motif III contains the first amino acids required for the correct protein folding and catalysis (Dupuy et al., 2002
). Indeed, amino acid deletions occurring in motif III of human FUT3 and FUT5 gave inactive enzymes (Xu et al., 1996
). Conversely, deletions upstream of motif III did not alter the activity (Xu et al., 1996
), although mutations in this region disturbed the subcellular localization of the enzyme (Sousa et al., 2003
). The fourth motif, called the acceptor-binding motif (abm), intercedes in acceptor substrate binding and specificity (Dupuy et al., 1999
; Sherwood et al., 2002
).
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The present study focuses on the W/R residue that followed the conserved H doublet in the highly conserved acceptor-binding motif of vertebrate 3- and
3/4-FUTs. To determine its involvement in
1,3- and
1,4-FUT activities, we produced, by site-directed mutagenesis at this position, 34 mutated forms of human FUT3, FUT5, FUT6, and ox FUTb. Characterization of these enzymes demonstrated that significant
1,4 fucosylation requires an aromatic residue at the position of W/R residue. Conversely,
1,3 fucosylation is optimal only when an R residue is present at the candidate position.
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Results |
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Kinetic parameters of mutated enzymes
The kinetic parameters of human FUT3 and FUT5 wild-type enzymes, and some of their mutated forms that present strong activity, were determined for the nucleotide sugar donor substrate and for the best acceptor substrate (Table IV). The results showed that the apparent Km values for GDP-fucose were similar for wild-type and mutated FUT3 and FUT5 enzymes. Consequently, amino acid changes at the W position in the acceptor-binding motif did not modify the affinity of the mutated enzymes for the donor substrate. Significant changes in the Km values for acceptor substrates were observed compared to the wild-type enzymes for some FUT3 and FUT5 variants. The FUT3 mutated enzymes with W111Y or F substitutions had slightly higher Km values for H-type 1 acceptor oligosaccharide, and the W111
A change generated a 10-fold increase of the Km value for the same acceptor. In FUT5, the W124
Y mutation produced an enzyme with a Km value for H-type 2 acceptor oligosaccharide close to that of the wild type. However, the mutated FUT5 enzyme with W124
R substitution had a 10-fold greater affinity for the H-type 2 substrate. Thus compared to wild-type enzymes, the affinities of FUT3 and FUT5 variants toward their best acceptor substrates are in close correlation with their levels of activity (Figure 2, Table II, and Table IV).
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Discussion |
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In the present work, we showed that all these vertebrate enzymes are characterized by the consensus VxxHH(W/R) (D/E) acceptor-binding motif and their 1,3 or
1,3/4 activities are correlated with the presence of R or W, respectively. Although required for optimal activity, amino acids in this motif are not equally involved in the acceptor substrate binding. For example, the V and the first H residues of human FUT4
3-FUT are not directly involved in type 2 substrate binding (Sherwood et al., 2002
). This suggests flexibility in the amino acid composition of the motif. Such flexibility is observed for Caenorhabditis elegans lactosamine
3-FUT (DeBose-Boyd et al., 1998
) where the potential acceptor-binding motif is VLIAHMD (Table V). Conservation of hydrophobic amino acids underscores their crucial role, potentially in protein folding. Also, the presence in C. elegans enzyme of M at the strictly conserved position of W/R residue could reflect the ability of this enzyme to transfer fucose to a wider variety of lacto- or neolacto-series structures (DeBose-Boyd et al., 1998
), compared to vertebrate ones. For bacterial enzymes using type 1 and type 2 acceptor substrates (Ge et al., 1997
; Rasko et al., 2000
), no corresponding acceptor-binding motif is found. Ma et al. (2003)
showed that amino acids involved in acceptor substrate specificity of Helicobacter pylori enzymes are located in their carboxy-terminal domain. Moreover, the bacterial
3/4-FUTs seem unrelated in evolution to the vertebrate ones.
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In this study, we demonstrate that the ability of FUT3 enzymes to use type 1 substrates, and consequently their capacity to transfer fucose with 1,4 linkage, involves the aromatic character of the W residue in their acceptor-binding domain. Indeed, only enzymes with an aromatic residue (W, Y, or F) at the candidate position had significant
1,4 activity and high affinities for GDP-Fuc donor and H-type 1 acceptor substrates. Alternatively, we showed that the optimal
1,3 activity of Lewis FUTs could depend on the presence of R residue in the acceptor-binding motif. FUT3 improved its
1,3 activity only when W111 was substituted by R (Figure 2). In the same way, the only increase in
1,3 activity and in affinity for H-type 2 acceptor of FUT5 was observed when W124 was replaced by R (Tables II and IV). Moreover,
1,3 activities of FUT6 and FUTb decreased when their R residue was substituted by basic or charged ones.
The correlation between acceptor substrate specificity and amino acid composition of the acceptor-binding motif is less obvious for FUT5 enzymes. They show W in their acceptor-binding motif but preferentially use H-type 2 trisaccharide substrates (Costache et al., 1997b). The mutated FUT5 with another aromatic residue (F or Y) at the candidate position had a lower
1,3 activity compared with the wild-type enzyme, whereas other substitutions (W124
A/V) allow the conservation of the original
1,3 activity. It is worth noticing that, as for W111
Y/F substitutions in FUT3, the W124
Y substitution in FUT5 gave an enzyme with around 50% of its wild-type
1,4 activity. We concluded that the type 1/type 2 substrate recognition by FUT5 enzymes involves amino acids of the acceptor-binding motif and one or several other residues of the catalytic domain. This hypothesis could also explain the lack of FUT6
1,3/4 activities, when R110 was substituted by W.
The type 1 and 2 disaccharides have different molecular topographies. The minimum energy conformations of type 1 and 2 structures invert the relative orientation of Gal and GlcNAc residues of disaccharides by approximately 180°. The key polar groups of lacto- and neolacto-series acceptor substrates are hydroxyl groups at C-3 or C-4 of GlcNAc and C-6 of Gal (De Vries et al., 1997; Du and Hindsgaul, 1996
; Gosselin and Palcic, 1996
). Because C-6 of Gal is necessary for activity whatever the acceptor substrate is, a correctly positioned Gal residue appears to be essential for enzyme activity. The active site of Lewis FUTs could consecutively form a pocket, which preferentially binds the GlcNAc residue of type 1 or type 2 acceptor substrates. It has been proposed that recognition of carbohydrate acceptors by glycosyltransferases occurred through hydrogen bonds and stacking of the sugar ring with aromatic amino acids (Hindsgaul et al., 1991
; Matsui et al., 1994
; Vyas et al., 1991
). Although W/R residue could also be involved in the protein folding, we hypothesize that the binding of type 2 acceptor substrates by
3-FUTs would partially rely on a hydrogen bond between the R residue of their acceptor-binding domain and a key polar group of the GlcNAc residue. Conversely, the presence of W residue in the acceptor-binding domain of
3/4-FUTs could allow the recognition of type 1 and type 2 substrates by the stacking of the acceptor GlcNAc ring with the cycle of W residue. The particular affinities of FUT5 enzymes toward type 1/type 2 substrates could be explained by a crucial interaction between a residue in FUT5 enzymes, which remains to be identified, and perhaps a reactive group of the acceptor substrate.
Genes encoding FUTs with 1,4 activity arose by duplications of an
3-FUT ancestor gene (for review, see Javaud et al., 2003
). Among the different events that conduced to the appearance of
3/4-FUTs, the switch of the basic R to the aromatic W residue in the acceptor-binding motif was certainly a crucial point. The presence of W residue in
3/4-FUTs could be explained by a single point mutation changing R codon in W codon (CGG
TGG). The new data presented in this study gave support to our model of Lewis gene evolution in Primates (Dupuy et al., 2002
), which proposed that
1,4 fucosylation appeared first in FUT3 and latterly in FUT5 independently. The differences between wild-type and mutated FUT3 and FUT5 enzymes concerning their proper
1,3/4 activities could then be explained by an independent process conferring the
1,4 function. Sequence comparison of a broad panel of Lewis enzymes is now needed to point out the amino acids, other than the W in the acceptor-binding motif, conferring the original
1,4 fucosylation in the primate lineage.
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Materials and methods |
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Peptide alignment of 3-,
3/4-, and
4-FUTs
Alignments were performed with DIALIGN (http://bioweb.pasteur.fr/seqanal/interfaces/dialign2.html) and refined with further manual adjustments using the ED program (Philippe, 1993) of the MUST 2000 package (http://sorex.snv.jussieu.fr/must2000.html).
Site-directed mutagenesis
The different FUT coding sequences were inserted into the mammalian expression vector pcDNA1/Amp (Invitrogen) and directly used for polymerase chain reactionbased mutagenesis. All mutations were performed with the QuickChange Kit (Stratagene, La Jolla, CA). The primers used for mutagenesis are summarized in the Table VI. The open reading frames containing mutations were completely checked as described.
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FUT assays
Blood group H-type 1 (Fuc1,2Galß1,3GlcNAc-sp-biotin) and H-type 2 (Fuc
1,2Galß1,4GlcNAc-sp-biotin) trisaccharide acceptor substrates were purchased from Syntesome (Munich, Germany). FUT assays were performed in 60 µl volume containing 0.1 mM acceptor substrate, 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, Little Chalfont, U.K.) and 20 µg protein extracts from transfected COS-7 cells. Activities were measured as previously described (Dupuy et al., 1999
): the reaction was stopped by addition of 3 ml cold water, and the reaction mixture was then applied to a conditioned Sep-Pack C18 reverse chromatography cartridge (Waters Millipore, Bedford MA). Unreacted GDP-[14C]-fucose was washed off with water. The radiolabeled reaction product was eluted with ethanol and counted with Biodegradable Counting Scintillant (Amersham Pharmacia Biotech) in a liquid scintillation beta counter (Liquid scintillation analyzer, Tri-Carb-2100TR, Packard, USA).
Western blot analysis
Thirty micrograms of soluble proteins from COS-7 cells were boiled for 3 min after the addition of ß-mercaptoethanol (5% v/v) and bromophenol blue (0.02% w/v). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was carried out on Tris/Tricine-10.5% sodium dodecyl sulfatepolyacrylamide gel. Separated proteins were electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The immunoblots were processed by chemiluminescence detection (Chemiluminescence Blotting Substrate [POD], Roche Molecular Biochemicals, Mannheim, Germany). Anti-human FUT3 and anti-ox FUTb antibodies were produced in rabbit using truncated proteins of FUT3 (P45T361) and FUTb (R35Q365), which were obtained in Escherichia coli BL21 (DE3). The blot was first incubated with rabbit anti-human FUT3 antibodies (4 µg/ml) for recombinant FUT3, FUT5, and FUT6 enzymes or rabbit anti-ox FUTb antibodies (2 µg/ml) for recombinant FUTb, then with the secondary antibody, a pig anti-rabbit IgG conjugated to horseradish peroxidase (dilution 1:1000) (Dako, Denmark).
Kinetic constant determination
The apparent Km values for GDP-fucose were determined using 10200 µM GDP-fucose including 8 µM GDP-[14C]fucose in each reaction, 2040 µg proteins, and 2 mM H-type 1 or H-type 2 acceptors. The incubation time was between 30 and 90 min. The apparent Km values for H-type 1 and H-type 2 acceptors were determined with 0.053 mM acceptor and 200 µM GDP-fucose, including 8 µM GDP[14C]-fucose and incubation times of 3090 min.
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Acknowledgements |
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Footnotes |
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