A sequence motif involved in the donor substrate binding by {alpha}1,6-fucosyltransferase: the role of the conserved arginine residues

Tomoaki Takahashi2,3, Yoshitaka Ikeda2, Akihiro Tateishi2, Yukihiro Yamaguchi2, Mutsuo Ishikawa3 and Naoyuki Taniguchi1,2

2Department of Biochemistry, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan and 3Department of Obstetrics and Gynecology, Asahikawa Medical College, Nishikagura 4–5–3–11, Asahikawa, Hokkaido 078–8510, Japan

Received on September 16, 1999; revised on November 12, 1999; accepted on November 15, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1,6-Fucosyltransferase catalyzes the transfer of fucose to the innermost GlcNAc residue of an N-linked oligosaccharide. In order to identify the amino acid residue(s) which are associated with the enzyme activity and to investigate their function, we prepared a series of mutant human {alpha}1,6-fucosyltransferases in which the conserved residues in the region homologous to {alpha}1,2-fucosyltransferase had been replaced. These proteins were then characterized by kinetic analyses. The wild-type and mutant {alpha}1,6-fucosyltransferases were expressed using a baculovirus-insect cell system. The activity assay showed that replacement of Arg-365 by Ala or Lys led to a complete loss of activity while substitution of Ala or Lys for the neighboring Arg-366 decreased the activity to about 3% that of the wild type. Kinetic analyses revealed that the replacements of Arg-366 lead to an increase in the apparent Km value for both GDP-fucose and the acceptor oligosaccharide but did not markedly affect the apparent Vmax. When these mutants were inhibited by GDP in a competitive manner with respect to the donor substrate, the Ki values were found to be 50–100 times higher than the value in the wild type. On the other hand, in the inhibition by GMP, the Ki values for the mutants were very similar to that of the wild type. These findings suggest that Arg-366 contributes to the binding of GDP-fucose via an interaction with the ß-phosphoryl group of the GDP moiety of the donor, and that Arg-365 may also play an essential role in substrate binding. The results suggest that the motif common to {alpha}1,2- and {alpha}1,6-fucosyltransferases is critical for binding of the donor substrate, GDP-fucose.

Key words: fucosyltransferase/N-glycan


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1,6-Fucosyltransferase is involved in the biosynthesis of Asn-linked oligosaccharides of glycoproteins and catalyzes the transfer of fucose from GDP-fucose to the innermost GlcNAc residue of the oligosaccharide of a glycoprotein, giving rise to an {alpha}1,6-fucosylated sugar chain (Wilson et al., 1976Go; Longmore and Schachter, 1982Go; Schachter, 1986Go). Fucosyltransferases are classified, according to the linkages catalyzed, into several families which include {alpha}1,2-, {alpha}1,3- ({alpha}1,3/4-) and {alpha}1,6-fucosyltransferases. Each of these families, with the exception of {alpha}1,6, consists of several types of fucosyltranserases, which display a significant sequence homology (Costache et al., 1997Go; Breton et al., 1998Go). It is known that {alpha}1,6-fucosyltransferase is the only enzyme in this class which catalyzes the formation of {alpha}1,6-linked fucose in mammalians. However, homologous enzymes which transfer a fucose via an {alpha}1,6-linkage have been reported in other organisms (Stacey et al., 1994Go; Mergaert et al., 1996Go).

{alpha}1,6-Fucosyltransferase is widely distributed in mammalian tissues, as evidenced by activity assay and Northern hybridization (Miyoshi et al., 1997Go). While oligosaccharides that contain {alpha}1,6-fucose residues are frequently found in the N-glycans of a variety of glycoproteins, this structure is not found in the serum proteins biosynthesized by the liver, probably because of the very low activity of the enzyme in hepatocytes (Campion et al., 1989Go; Yamashita et al., 1989Go; Noda et al., 1998aGo). Nevertheless, {alpha}1,6-fucosyltransferase activity is increased in the liver for cases of certain diseased states such as hepatocellular carcinomas, and, as a result, the content of {alpha}1,6-fucose residues in the Asn-linked oligosaccharides of the serum proteins, produced in the liver, are significantly elevated in such cases (Aoyagi et al., 1985Go, 1993a,b; Hutchinson et al., 1991Go; Ohno et al., 1992Go; Noda et al., 1998bGo). It has been proposed that this structural alteration, which is associated with carcinogenesis, could be of value in the differential diagnosis of the malignant diseases.

In order to investigate the molecular basis for oligosaccharide alteration associated with the hepatocarcinogesis, we have purified {alpha}1,6-fucosyltransferase from pig brain and a human gastric cancer cell line, cloned their cDNAs, and analyzed the genomic structure of the human enzyme (Uozumi et al., 1996aGo; Yanagidani et al., 1997Go; Yamaguchi et al., 1999Go). Structural analyses of the cDNA clones indicates that the enzyme is a type II membrane protein, and that the domain structure of the enzyme is similar to those of other classes of fucosyltransferases, as well as other glycosyltransferases. However, when the complete amino acid sequence was compared with those of the other fucosyltransferases, no remarkable homology was found among them. Only a small region in the {alpha}1,6-fucosyltransferase was found to be significantly homologous to a portion of {alpha}1,2-fucosyltransferase, based on a comparison of their sequences (Breton et al., 1998Go). This suggests that the motif would be involved in functional properties which are common to the {alpha}1,2- and {alpha}1,6-fucosyltransferases.

Of the numerous fucosyltransferases, the catalytic properties of the {alpha}1,3- or {alpha}1,3/4-fucosyltransferase involved in the biosynthesis of Lewis antigens have been intensively investigated in terms of kinetic properties and catalytic mechanism (Murray et al., 1996Go, 1997; Nguyen et al., 1998Go; Sherwood et al., 1998Go; Vo et al., 1998Go). On the other hand, although Glick et al. purified the {alpha}1,6-fucosyltransferase from cultured skin fibroblasts and characterized some of the kinetic properties of the enzyme (Voynow et al., 1991Go), the detailed catalytic mechanism remains unclear. Moreover, amino acid residues that are involved or essential for activity have not yet been identified. Information, as obtained by chemical modification studies or site-directed mutagenesis, would be highly desirable for the elucidation of the catalytic mechanism of the enzyme.

In this study, the residues conserved in the homologous motifs between {alpha}1,2- and {alpha}1,6-fucosyltransferases were selected as likely candidate residues which might play an essential or important role in the function of the enzyme, and then prepared mutant {alpha}1,6-fucosyltransferases in which these residues were replaced. These mutants, as well as the wild-type enzyme, were produced using a baculovirus–insect cell expression system, and were characterized by kinetic analyses to examine the role of the identified residues.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Comparison of amino acid sequences of {alpha}1,2- and {alpha}1,6-fucosyltransferases
In order to identify the candidate amino acid residues which are essential for the activity of {alpha}1,6-fucosyltransferase, the entire amino acid sequences of human {alpha}1,2- and {alpha}1,6-fucosyltransferases were compared by dotplot analysis (Figure 1A). The analysis showed several small homologous regions, with the most significant being in residues 361–370 in the {alpha}1,6-fucosyltransferase. When this portion of the amino acid sequences from the {alpha}1,6-fucosyltransferases were aligned with the corresponding regions of other {alpha}1,6-fucosyltransferases from different species, as well as various {alpha}1,2-fucosyltransferases, as shown in Figure 1B, the alignment showed that the enzymes have perfectly conserved histidine and arginine residues, which correspond to His-363 and Arg-365 in the human {alpha}1,6-fucosyltransferase. Thus, mutant enzymes of human {alpha}1,6-fucosyltransferase in which these conserved residues had been replaced were prepared by the site-directed mutagenesis. The mutants with replacements at another arginine residue, Arg-366, were also prepared because this residue is conserved among the mammalian enzymes. The mutants examined in this study are summarized in Figure 1C.



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Fig. 1. Comparison of amino acid sequences from {alpha}1,2- and {alpha}1,6-fucosyltransferases. (A) Dotplot of the sequences from the {alpha}1,2- and {alpha}1,6-fucosyltransferases. The amino acid sequences of human {alpha}1,2- and {alpha}1,6-fucosyltransferases were compared by a window size of 15 residues and 40% of the identity using the computer software program, Align. The numbers beside the matrix indicate the residue number of each enzyme. Diagonal plots show the homologous regions which satisfy the above conditions. The sequences of the most homologous region are given outside the matrix. (B) Amino acid sequence alignment of the homologous regions of human {alpha}1,2- and {alpha}1,6-fucosyltransferases. The amino acid sequences in the homologous region of {alpha}1,6-fucosyltransferases and {alpha}1,2-fucosyltransferases from various species were aligned by Clustal. The amino acid residues which are conserved in all mammalians are indicated by shaded boxes. The basic amino acids conserved and subjected to mutational analysis are indicated by the arrowheads. (C) The amino acid sequences of the {alpha}1,6-fucosyltransferase mutants. The mutated sequences of the region examined in this study are listed. Bold letters are substituted amino acids for the wild-type sequence.

 
Expression of the wild type and mutants of human {alpha}1,6-fucosyltransferase in insect cells
The mutant {alpha}1,6-fucosyltransferases were expressed using a baculovirus–insect cell system, since {alpha}1,6-fucosyltransferase activity was not detected in Sf21 cells, the insect cells used in this study. Expression of the wild-type and mutant enzymes were verified by immunoblot analysis of cell homogenates of the baculovirus-infected Sf21 cells using a specific antibody (Figure 2). The expression levels of mutants were very similar to that of the wild type, and, as a result, these comparable expression levels allowed us to compare the activities of the wild type and mutants using cell homogenates.



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Fig. 2. Immunoblot analysis of the wild-type and mutant human {alpha}1,6-fucosyltransferases expressed in the baculovirus-infected insect cells. Noninfected or infected-insect cell homogenates (0.1 µg of proteins) were subjected to SDS-PAGE. The proteins were resolved on a 10% gel and transferred to a PVDF membrane. The protein bands of the {alpha}1,6-fucosyltransferases ({alpha}1,6FucT) were visualized as described under Materials and methods.

 
Enzyme activities of the wild-type and mutant {alpha}1,6-fucosyltransferases
A high activity was found in the wild-type and H363A mutant enzymes, while significantly lower activities were detected in the Arg-366-substituted mutants (Table I). However, the mutants with substitutions at Arg-365 exhibited no activity, as well as control noninfected cells (Figure 3). Even when the homogenate which was added to the assay mixture was 100 times more concentrated, no activity was detected in the mutants. These results suggest that Arg-365 is required for enzyme activity, and that the neighboring arginine, Arg-365, also plays an important role in the activity even though it is not essential. Interestingly, the replacement of Arg-365 by Lys also resulted in a complete loss of activity even though a positive charge was retained after the substitution. This indicates the absolute requirement of the guanidino group of the arginine. It was also found that His-363 plays no significant role in catalysis in spite of the fact that it is perfectly conserved.


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Table I. Specific activities of the wild-type and mutant {alpha}1,6-fucosyltransferases expressed in the baculovirus-infected Sf21 cells
 


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Fig. 3. HPLC elution profiles of the fluorescence-labeled acceptor substrate and the product by {alpha}1,6-fucosyltransferase. The large peak which eluted at 20 min shows the unreacted PABA-labeled oligosaccharide. The substrate panel represents the elution pattern of the substrate without the reaction. The peak at 30 min in the wild-type panel, indicated by an arrow head, is the product. Asterisks show contaminated fluorescent substances.

 
Kinetic analysis of the wild-type and mutant {alpha}1,6-fucosyltransferases
The R366A and R366K mutants, which retained activity, were subjected to kinetic analysis in order to explore the role of the arginine residue (Figure 4, Table II). The kinetic analysis showed that the replacement of Arg-366 led to the distinct kinetic properties from that of the wild type. Apparent Km values for both GDP-fucose as a donor and the oligosaccharide-Asn-PABA as an acceptor were increased by the replacements when the parameters were determined at the fixed concentration of either of the donor or acceptor. Since apparent Vmax determined by relatively high concentration of GDP-fucose and various acceptor concentrations was not markedly changed by the replacements, it is suggested that the arginine residue would be involved in the binding of the substrates rather than the catalysis. In addition, since essentially no difference in the apparent parameters was observed between the R366A and R366K mutants, it seems unlikely that lysine can take the place of the arginine. When the kinetic analysis by varying the concentrations of both donor and acceptor substrates was carried out to assess the effects of the substitutions on the enzymatic properties by comparison of true Km values, it was found that the mutants follow the distinct reaction mechanism from that of the wild type; a ping-pong Bi-Bi mechanism for the wild type and a sequential mechanism for both mutants (data not shown). Since Km values based on the distinct reaction mechanisms have different meanings, the Km of the mutants could not be compared with that of the wild type in order to quantitatively argue the change in the binding affinity.



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Fig. 4. Kinetic analysis of the wild-type and mutant {alpha}1,6-fucosyltranserases. (A) The activity was determined using variable concentrations of the donor substrate, GDP-fucose in the presence of a fixed concentration of acceptor (5 µM agalacto biantennary-Asn-PABA). (B) Assayed with variable concentrations of the acceptor and a fixed concentration of the donor, 500 µM GDP-fucose. The symbols used are solid circles for the wild type, open circles for R366A, and solid triangles for R366K. Velocities were normalized for a maximal velocity for each enzyme.

 

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Table II. Kinetic parameters for the wild type and Arg-366-substituted mutants
 
Inhibition studies by GMP and GDP
{alpha}1,2- and {alpha}1,6-Fucosyltransferases do not share a common oligosaccharide as their acceptor substrates, but utilize the common donor substrate, GDP-fucose. Therefore, it is more likely that the effects of the amino acid replacements at Arg-366 are primarily due to an impairment in the binding of the donor substrate but not of the acceptors. Hence, to further examine the roles of the arginine residue in interactions with the donor, the effects of its nucleotide moieties on the activity were investigated in terms of the competitive inhibition against the donor substrate. Both GDP and GMP inhibited the wild-type and the mutants with replaced Arg-366 in a competitive manner, as found in another class of fucosyltransferase (Murray et al., 1996Go) (Figures 5, 6). The Ki values for these nucleotides were determined using variable concentrations of the donor and the inhibitors in the presence of a fixed concentration of the acceptor (Table III). Ki values for GDP in mutants R366A and R366K, were much higher, as compared to that in the wild type. Even in the case of substitution with Lys, the increase in Ki value was similar in magnitude to that in the replacement by Ala, which is consistent with the results for the apparent Km values for the donor. In contrast to inhibition by GDP, these amino acid replacements lead to no apparent change in Ki values for GMP. These findings indicate that Arg-366 does not contribute to the binding of GMP but is greatly involved in GDP binding. This suggests that the arginine residue serves to bind the donor substrate via interaction with the ß-phosphate of the substrate.



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Fig. 5. Inhibition of the wild-type and Arg-366-substituted mutant enzymes by GDP. Left panels, double reciprocal plots obtained with variable concentrations of GDP-fucose and 5 µM acceptor. The numbers in the panels indicate the inhibitor (GDP) concentration expressed in µM. Right panels, re-plots of Km/Vmax for each concentration of GDP as the function of GDP. Ki values for GDP were determined from the x-axis intercept.

 


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Fig. 6. Inhibition of the wild-type and mutant enzymes by GMP. Left panels, double reciprocal plots obtained with variable concentrations of GDP-fucose and 5 µM acceptor. The numbers in the panels indicate the inhibitor (GDP) concentration expressed in mM. Right panels, re-plots of Km/Vmax for each concentration of GMP as the function of GMP. Ki values for GMP were determined from the x-axis intercept.

 

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Table III . Ki values for GDP and GMP in the wild-type and Arg-366-substituted mutants
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study, Arg-365 has been identified as an essential residue for the activity of {alpha}1,6-fucosyltransferase, and it is also shown that the neighboring arginine residue, Arg-366, would play an important role in the binding of the substrates. Furthermore, it is suggested that Arg-366 interacts with the ß-phosphoryl group of GDP-fucose. It seems more likely that the essential residue, Arg-365, also functions at least as a substrate-binding residue in a similar manner to the neighboring arginine, even though the role(s) of the arginine could not be explored by the kinetic analyses because of the complete loss of activity in its mutants.

Although it is entirely possible that the catalytic mechanism of {alpha}1,6-fucosyltransferases is similar to that of {alpha}1,2-fucosyltransferase and involves these conserved residues, it was found that Arg-366, a conserved residue, is not a significant participant in the catalysis of the enzyme. {alpha}1,6-Fucosyltransferase shares only a common donor substrate, GDP-fucose, with {alpha}1,2-fucosyltransferase, but not a common acceptor. Therefore, it can reasonably be argued that the residues play a significant role, primarily in interactions with the donor, rather than with acceptors.

The nature of the interaction of the arginine residues of {alpha}1,6-fucosyltransferase with the nucleotide sugar is not clearly known. In general, enzymes which bind nucleotides, such as, for example, kinases, appear to frequently contain the positively charged amino acid residues, Lys and Arg in the nucleotide-binding site, and these residues interact directly with diphosphate or triphosphate groups of the substrates in an electrostatic manner. Furthermore in some enzymes or proteins, even a mutation which retains a positive charge, Arg-to-Lys or Lys-to-Arg, at such a residue, interaction with the nucleotide leads to the abolition of binding (Shen et al., 1991Go; Li et al., 1995Go; Chan and Gill, 1996Go; Tohgo et al., 1997Go; Kazuta et al., 1998Go). Thus, an electrostatic interaction would be the most likely interaction between the arginine residue identified in the enzyme and the nucleotide sugar. The character specific to arginine such as the capability of forming two hydrogen bonds, the bulkiness of the guanidino group, and a wider distribution of the positive charge may be of critical importance for interaction of the enzyme with the donor substrate.

In many glycosyltransferases requiring a metal ion such as Mn2+ for reaction, the metal ion is believed to allow the diphosphate moiety of the nucleotide sugar to coordinate and thus facilitate the binding to the enzymes (Powell and Brew, 1976aGo,b; Andree and Berliner, 1980Go; Boeggeman et al., 1995Go). In addition, the divalent metal ion might possibly play a role, even as an electrostatic catalyst, stabilizing the negative charge which develops on the cleavage of the donor nucleotide. On the other hand, {alpha}1,6-fucosyltransferase, {alpha}1,2-fucosyltransferase, and certain other glycosyltransferases do not require a divalent cation for reaction, and thus it is conceivable that a positively charged residue, probably arginine in this case, substitutes for the divalent metal. The absolute requirement of Arg-365 could be explained by its involvement both in substrate binding and catalysis, and, in this case, the residue might possibly serve as a substitute for the divalent metal ion. A more detailed characterization of the Arg-365-substituted mutant of {alpha}1,6-fucosyltransferase may contribute to our understanding of the catalytic mechanism of divalent metal-independent glycosyltransferases.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Restriction endonuclease and DNA modifying enzymes were purchased from Takara, TOYOBO and New England Biolabs. Oligonucleotide primers were synthesized by Greiner Japan. Other common chemicals were from Wako pure chemicals or Nacalai tesque.

Construction of transfer plasmids
Human {alpha}1,6-fucosyltransferase cDNA, which was previously cloned in our laboratory (Yanagidani et al., 1997Go), was excised from the pBluescript, and inserted into a transfer vector, pVL1393, by BamHI and EcoRI sites. The resultant plasmids were purified by a Qiagen plasmid purification kit, and then subjected to the transfection experiments.

Site-directed mutagenesis
Site-directed mutagenesis was carried out according to Kunkel (Kunkel, 1985Go), as described previously (Ikeda et al., 1995aGo). A 0.5 kb fragment obtained by digestion of human {alpha}1,6-fucosyltransferase cDNA with HindIII was subcloned into pBluescript KS+, and the resulting plasmid was used for transformation of CJ236 (dut , ung). The uracil-substituted single stranded DNA was prepared by infection of the transformed CJ236 with helper phage M13K07. This template was used with oligonucleotide primers to replace the conserved residues. The primers used in this study were 5'-GTTATTGGAGTGGCCGTCAGACGCAC-3' for replacement of His-363 by Ala (designated as H363A), 5'-GTCCATGTCGCGCGCACAGAC-3' for Arg-365 by Ala (R365A), 5'-GTCCATGTCAGAGCCACAGACAAAG-3' for Arg-366 by Ala (R366A), 5'-GGAGTCCATGTCGCGGCCACAGACAAAGTG-3' for the double replacement of Arg-365 by Ala and Arg-366 by Ala (R365A/R366A), 5'-GGAGTCCATGTCAAGCGCACAGACAAAGTG-3' for Arg-365 by Lys (R365K), 5'-GGAGTCCATGTGCGCAAGACAGACAAAGTG-3' for Arg-366 by Lys (R366K) and 5'-GTTATTGGAGTACATGTCAAAAAGACAGACAAAGTG-3' for the double substitution of Lys for Arg-365 and Arg-366. The resulting mutations were verified by dideoxy sequencing using a DNA sequencer (Applied Biosystems, model 373A), as were the entire sequences which had been subjected to mutagenesis. The corresponding region of the wild-type {alpha}1,6-fucosyltransferase cDNA was replaced by each mutant sequence. The transfer plasmids for these mutant enzymes were constructed in a manner similar to that of the wild-type enzyme, and used for transfection.

Cell culture and general manipulation of viruses
Spodoptera frugiperda (Sf) 21 cells were maintained at 27°C in Grace’s insect media (GIBCO-BRL) supplemented with 10% fetal bovine serum, 3.33 g/l yeastolate, 3.33 g/l lactalbumin hydrolysate, and 100 mg/l kanamycin. Recombinant viruses were manipulated as described (Piwnica-Worms, 1987Go).

Preparation of recombinant viruses
The purified transfer plasmids containing the wild-type or mutant {alpha}1,6-fucosyltransferase (1 µg) were cotransfected into 5 x 105 Sf21 cells with 10 ng of BaculoGold DNA (PharMingen). Transfection experiments were carried out by the Lipofectin (GIBCO-BRL) method (Felgner et al., 1987Go), as described previously (Ikeda et al., 1995bGo,c). Media containing the recombinant viruses generated by homologous recombinations were collected 6 days after transfection. The recombinant viruses were further amplified to more than 5 x 107 plaque forming units/ml prior to use.

Electrophoresis and immunoblot analysis
SDS-PAGE analysis was carried out on 10% gels, according to Laemmli (Laemmli, 1970Go). The separated proteins were transferred onto PVDF membrane, and the resultant blot was blocked by 5% skim milk. The membrane was reacted with the anti-peptide antibody specific to porcine and human {alpha}1,6-fucosyltransferases, followed by reaction with a horseradish peroxidase–conjugated anti-rabbit IgG-antibody. The reactive bands were visualized by an ECL kit (Amersham).

Activity assay
{alpha}1,6-Fucosyltransferase activity was assayed using a fluorescence-labeled sugar chain substrate, according to the method of Uozumi (Uozumi et al., 1996bGo). An agalacto-biantennary sugar chain labeled with pyridylaminobutylamine was used as an acceptor substrate. Cell homogenates were incubated at 37°C with 5 µM of the acceptor substrate and 0.5 mM GDP-fucose as a donor in 0.1 M MES-NaOH, 1% Triton X-100 (pH 7.0). The reactions were terminated by boiling after an appropriate reaction time, and the mixtures were centrifuged at 10,000 x g in a microcentrifuge for 10 min. The resulting supernatants were injected to a reversed phase HPLC equipped with TSKgel, ODS 80TM (4.6 x 150 mm). The product and the substrate were separated isocratically with 20 mM ammonium acetate buffer (pH 4.0) containing 0.15% n-butanol. Fluorescence of the column elute was detected with fluorescence detector (Shimazu, model RF-10AXL) at excitation and emission wavelengths of 320 nm and 400 nm, respectively.

Kinetic analysis and inhibition study with nucleotides
Kinetic analysis was carried out under essentially the same conditions as used above with the exception of the concentration of the substrates. When the kinetic parameters for GDP-fucose were assessed, the concentration of the fluorescence-labeled acceptor, 5 µM, was used with variable concentrations of the donor. For the determination of parameters for the acceptor substrate, the concentration of GDP-fucose was fixed at 0.5 mM. Apparent kinetic parameters for these substrates were obtained by double reciprocal plots. Ki values for GDP and GMP in competition with the donor substrate were determined in the activity assay using various concentrations of the donor and fixed concentration (5 µM) of the acceptor.

Protein determination
Protein content was determined according to the method of Bradford using bovine serum albumin as a standard (Bradford, 1976Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Milton S.Feather for correcting the manuscript. This research was supported, in part, by Grants-in-Aid for Scientific Research on Priority Area No. 10178104 from the Ministry of Education, Science, Sports and Culture of Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
PABA, 4-(2-pyridylamino)butylamine; GDP-fucose, guanosinediphospho-fucopyranoside; GDP, guanosine diphosphate; GMP, guanosine monophosphate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Andree,P.J. and Berliner,L.J. (1980) Metal ion and substrate binding to bovine galactosyltransferase. Biochemistry, 19, 929–934.[ISI][Medline]

Aoyagi,Y., Isemura,M., Yoshizawa,Z., Suzuki,Y., Sekine,C., Ono,T. and Ichida,F. (1985) Fucosylation of serum alpha-fetoprotein in patients with primary hepatocellular carcinoma. Biochim. Biophys. Acta, 830, 217–233.[ISI][Medline]

Aoyagi,Y., Suzuki,Y., Igarashi,A., Saitoh,M., Oguro,T., Yokota,S., Mori,T., Suda,T., Isemura,M. and Asakura,H. (1993a) Carbohydrate structures of human alpha-fetoprotein of patients with hepatocellular carcinoma: presence of fucosylated and non-fucosylated triantennary glycans. Br. J. Cancer, 67, 486–492.[ISI][Medline]

Aoyagi,Y., Suzuki,Y., Igarashi,A., Yokota,S., Mori,T., Suda,T., Naitou,A., Isemura,M. and Asakura,H. (1993b) Highly enhanced fucosylation of {alpha}-fetoprotein in patients with germ cell tumor. Cancer, 72, 615–618.[ISI][Medline]

Boeggeman,E.E., Balaji,P.V. and Qasba,P.K. (1995) Functional domains of bovine beta-1,4 galactosyltransferase. Glycoconj. J., 12, 865–878.[ISI][Medline]

Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[ISI][Medline]

Breton,C., Oriol,R. and Imberty,A. (1998) Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology, 8, 87–94.[Abstract/Free Full Text]

Campion,B., Leger,D., Wieruszeski,J.M., Montreuil,J. and Spik,G. (1989) Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinoma cell line Hep G2. Eur. J. Biochem. 184, 405–413.[Abstract]

Chan,C.L. and Gill,G.N. (1996) Mutational analysis of the nucleotide binding site of the epidermal growth factor receptor and v-Src protein-tyrosine kinases. J. Biol. Chem., 271, 22619–22623.[Abstract/Free Full Text]

Costache,M., Apoil,P.A., Cailleau,A., Elmgren,A., Larson,G., Henry,S., Blancher,A., Iordachescu,D., Oriol,R. and Mollicone,R. (1997) Evolution of fucosyltransferase genes in vertebrates. J. Biol. Chem., 272, 29721–29728.[Abstract/Free Full Text]

Felgner,P.L., Gadek,T.R., Holm,M., Roman,R., Chan,H.W., Wenz,M., Northrop,J.R., Ringold,G.M. and Danielsen,M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA, 84, 7413–7417.[Abstract]

Hutchinson,W.L., Du,M.Q., Johnson,P.J. and Williams,R. (1991) Fucosyltransferases: differential plasma and tissue alterations in hepatocellular carcinoma and cirrhosis. Hepatology, 13, 683–688.[ISI][Medline]

Ikeda,Y., Fujii,J., Taniguchi,N. and Meister,A. (1995a) Human gamma-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit. J. Biol. Chem., 270, 12471–12475.[Abstract/Free Full Text]

Ikeda,Y., Fujii,J., Taniguchi,N. and Meister,A. (1995b) Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain. Proc. Natl Acad. Sci. USA 92, 126–130.[Abstract]

Ikeda,Y., Fujii,J. anderson,M.E., Taniguchi,N. and Meister,A. (1995c) Involvement of Ser-451 and Ser-452 in the catalysis of human gamma-glutamyl transpeptidase. J. Biol. Chem., 270, 22223–22228.[Abstract/Free Full Text]

Kazuta,Y., Tokunaga,E., Aramaki,E. and Kondo,H. (1998) Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue. FEBS Lett., 427, 377–380.[ISI][Medline]

Kunkel,T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA, 82, 488–492.[Abstract]

Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Li,W., Yu,J.C., Shin,D.Y. and Pierce,J.H. (1995) Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J. Biol. Chem., 270, 8311–8318.[Abstract/Free Full Text]

Longmore,G.D. and Schachter,H. (1982) Product-identification and substrate-specificity studies of the GDP-L-fucose:2-acetamido-2-deoxy-ß-D-glucoside (FUC goes to Asn-linked GlcNAc) 6-{alpha}-L-fucosyltransferase in a Golgi-rich fraction from porcine liver. Carbohydr. Res., 100, 365–392.[ISI][Medline]

Mergaert,P., D’Haeze,W., Fernandez, Lopez,M., Geelen,D., Goethals,K., Prome,J.C., Van-Montagu,M. and Holsters,M. (1996) Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolK, nodZ as well as noeC and/or downstream genes. Mol. Microbiol., 21, 409–419.[ISI][Medline]

Miyoshi,E., Uozumi,N., Noda,K., Hayashi,N., Hori,M., Taniguchi,N. Expression of {alpha}1-6 fucosyltransferase in rat tissues and human cancer cell lines. (1997) Int. J. Cancer, 72, 1117–1121.[ISI][Medline]

Murray,B.W., Takayama,S., Schultz,J. and Wong,C.H. (1996) Mechanism and specificity of human {alpha}-1,3-fucosyltransferase V. Biochemistry, 35, 11183–11195.[ISI][Medline]

Murray,B.W., Wittmann,V., Burkart,M.D., Hung,S.C. and Wong,C.H. (1997) Mechanism of human {alpha}-1,3-fucosyltransferase V: glycosidic cleavage occurs prior to nucleophilic attack. Biochemistry 36, 823–831.[ISI][Medline]

Nguyen,A.T., Holmes,E.H., Whitaker,J.M., Ho,S., Shetterly,S. and Macher,B.A. (1998) Human {alpha}1,3/4-fucosyltransferases. I. Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis. J. Biol. Chem., 273, 25244–25249.[Abstract/Free Full Text]

Noda,K., Miyoshi,E., Uozumi,N., Gao, CX., Suzuki,K., Hayashi,N., Hori,M., Taniguchi,N. (1998b) High expression of {alpha}-1–6 fucosyltransferase during rat hepatocarcinogenesis. Int. J. Cancer, 75, 444–450.[ISI][Medline]

Noda,K., Miyoshi,E., Uozumi,N., Yanagidani,S., Ikeda,Y., Gao,C., Suzuki,K., Yoshihara,H., Yoshikawa,M., Kawano,K., Hayashi,N., Hori,M. and Taniguchi,N. (1998a) Gene expression of alpha1-6 fucosyltransferase in human hepatoma tissues: a possible implication for increased fucosylation of {alpha}-fetoprotein. Hepatology, 28, 944–952.[ISI][Medline]

Ohno,M., Nishikawa,A., Kouketsu,M., Taga,H., Endo,Y., Hada,T., Higashino,K. and Taniguchi,N. (1992) Enzymatic basis of sugar structures of {alpha}-fetoprotein in hepatoma and hepatoblastoma cell lines: correlation with activities of {alpha} 1–6 fucosyltransferase and N-acetylglucosaminyltransferases III and V. Int. J. Cancer, 51, 315–317.[ISI][Medline]

Piwnica-Worms,H. (1987) In Ausubel,F.M., Brent,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (eds.), Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York, pp. 16.8.1–16.11.7.

Powell,J.T. and Brew,K. (1976a) Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative. Biochemistry, 15, 3499–3505.[ISI][Medline]

Powell,J.T. and Brew,K. (1976b) Metal ion activation of galactosyltransferase. J. Biol. Chem., 251, 3645–3652.[Abstract]

Schachter,H. (1986) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem. Cell Biol.., 64, 163–181.[ISI][Medline]

Shen,J.B., Orozco,E.M. Jr. and Ogren,W.L. (1991) Expression of the two isoforms of spinach ribulose 1,5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain. J. Biol. Chem., 266, 8963–8968.[Abstract/Free Full Text]

Sherwood,A.L., Nguyen,A.T., Whitaker,J.M., Macher,B.A., Stroud,M.R. and Holmes,E.H. (1998) Human {alpha}1,3/4-fucosyltransferases. III. A Lys/Arg residue located within the {alpha}1,3-FucT motif is required for activity but not substrate binding. J. Biol. Chem., 273, 25256–25260.[Abstract/Free Full Text]

Stacey,G., Luka,S., Sanjuan,J., Banfalvi,Z., Nieuwkoop,A.J., Chun,J.Y., Forsberg,L.S. and Carlson,R. (1994) nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum. J. Bacteriol., 176, 620–633.[Abstract]

Tohgo,A., Munakata,H., Takasawa,S., Nata,K., Akiyama,T., Hayashi,N. and Okamoto,H. (1997) Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase. J. Biol. Chem., 272, 3879–3882.[Abstract/Free Full Text]

Uozumi,N., Yanagidani,S., Miyoshi,E., Ihara,Y., Sakuma,T., Gao,C.X., Teshima,T., Fujii,S., Shiba,T. and Taniguchi,N. (1996a) Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-ß-D-glucosaminide {alpha}1->6fucosyltransferase. J. Biol. Chem., 271, 27810–27817.[Abstract/Free Full Text]

Uozumi,N., Teshima,T., Yamamoto,T., Nishikawa,A., Gao,Y.E., Miyoshi,E., Gao,C.X., Noda,K., Islam,K.N., Ihara,Y., Fujii,S., Shiba,T. and Taniguchi,N. (1996b) A fluorescent assay method for GDP-L-Fuc:N-acetyl-ß-D-glucosaminide {alpha}1–6fucosyltransferase activity, involving high performance liquid chromatography. J. Biochem., 120, 385–392.[Abstract]

Vo,L., Lee,S., Marcinko,M.C., Holmes,E.H. and Macher,B.A. (1998) Human {alpha}1,3/4-fucosyltransferases. II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties. J. Biol. Chem., 273, 25250–25255.[Abstract/Free Full Text]

Voynow,J.A., Kaiser,R.S., Scanlin,T.F. and Glick,M.C. (1991) Purification and characterization of GDP-L-fucose-N-acetyl beta-D-glucosaminide {alpha} 1-6fucosyltransferase from cultured human skin fibroblasts. Requirement of a specific biantennary oligosaccharide as substrate. J. Biol. Chem., 266, 21572–72157.[Abstract/Free Full Text]

Wilson,J.R., Williams,D. and Schachter,H. (1976) The control of glycoprotein synthesis: N-acetylglucosamine linkage to a mannose residue as a signal for the attachment of L-fucose to the asparagine-linked N-acetylglucosamine residue of glycopeptide from {alpha}1-acid glycoprotein. Biochem. Biophys. Res. Commun., 72, 909–916.[ISI][Medline]

Yamaguchi,Y., Fujii,J., Inoue,S., Uozumi,N., Yanagidani,S., Ikeda,Y., Egashira,M., Miyoshi,O., Niikawa,N. and Taniguchi,N. (1999) Mapping of the {alpha}-1,6-fucosyltransferase gene, FUT8, to human chromosome 14q24.3. Cytogenet. Cell Genet., 84, 58–60.[ISI][Medline]

Yamashita,K., Koide. N., Endo. T., Iwaki. Y. and Kobata. A. (1989) Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. J. Biol. Chem. 264, 2415–2423.[Abstract/Free Full Text]

Yanagidani,S., Uozumi,N., Ihara,Y., Miyoshi,E., Yamaguchi,N. and Taniguchi,N. (1997) Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-ß-D-glucosaminide:{alpha}1-6 fucosyltransferase ({alpha}1-6 FucT) from human gastric cancer MKN45 cells. J. Biochem., 121, 626–632.[Abstract]