A highly conserved His-His motif present in {alpha}1->3/4fucosyltransferases is required for optimal activity and functions in acceptor binding

Anne L. Sherwood, David A. Upchurch, Mark R. Stroud, William C. Davis and Eric H. Holmes1

Northwest Hospital, Molecular Medicine, Department of Cell Surface Biochemistry, 21720 23rd Drive SE, Suite 101, Bothell, WA 98021, USA

Received on April 29, 2002; revised on June 12, 2002; accepted on June 13, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1->3/4Fucosyltransferases (FucTs) from several species contain a highly conserved His-His motif adjacent to an enzyme region correlating with the ability to catalyze fucose transfer to type 1 chain acceptors. Site-directed mutagenesis has been employed to analyze structure–function relationships of this His-His motif in human FucT-IV. The results indicate that most changes of His113 and His114 and nearby residues of FucT-IV reduced the specific activity of the enzymes. Analysis of acceptor properties demonstrated close similarity of most mutants with wild-type FucT-IV, whereas an apparent preference for the H-type II acceptor was observed for the His114 mutants. Kinetic studies demonstrated that mutants of His114 had a substantially increased Km for acceptor compared to other enzymes tested. The dramatic increase in acceptor Km for the His114 mutants, particularly for the nonfucosylated acceptor, suggests that this His-His motif is involved in acceptor binding and perhaps interacts with GlcNAc residues of type 2 acceptors. The presence of fucose in acceptor substrates may promote more efficient substrate binding and presumably partially overcomes the weaker interaction with GlcNAc caused by the mutation.

Key words: carbohydrate binding site/fucosyltransferase/His motif/structure–function relationships


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1->3/4Fucosyltransferases (FucTs) catalyze fucose transfer on GlcNAc residues in lacto- or neolacto-series cell surface carbohydrates of glycolipids or glycoproteins. These structures, representing the Lea- and Lex-related antigens, accumulate in many human cancers (Hakomori, 1989Go; Alhadeff, 1989Go), undergo developmental regulation (Hakomori, 1989Go; Alhadeff, 1989Go; Sanders and Kerr, 1999Go; Listinsky et al., 1998Go), or function as natural ligands for leukocyte adhesion during inflammation (Sanders and Kerr, 1999Go; Listinsky et al., 1998Go; Renkonen et al., 1997Go; McEver, 1997Go). A large number of distinct {alpha}1->3/4FucTs have been identified and cloned in multiple organisms. For example, in humans, six distinct enzymes, designated FucT-III, -IV, -V, -VI, -VII, and -IX, have been cloned (Kukowska-Latallo et al., 1990Go; Goelz et al., 1990Go; Weston et al., 1992aGo,b; Koszdin and Bowen, 1992Go; Sasaki et al., 1994Go; Natsuka et al., 1994Go; Kaneko et al., 1999Go). Although each enzyme has the common property of binding the GDP-fucose donor substrate, subtle differences in acceptor specificity and tissue distribution distinguish members of this family of enzymes. Additionally, {alpha}1->3/4FucTs have been identified and cloned from other mammalian species (Nishihara et al., 1999Go; Costache et al., 1997Go; Gersten et al., 1995Go; Smith et al., 1996Go; Ozawa and Muramatsu, 1996Go; Kudo et al., 1998Go; Sajdel-Sulkowska et al., 1997Go; Zhang et al., 1999Go; Oulmouden et al., 1997Go), chicken (Lee et al., 1996Go), fish (Kageyama et al., 1999Go), invertebrates (Trottein et al., 2000Go; DeBose-Boyd et al., 1998Go), plants (Leiter et al., 1999Go), and bacteria (Rasko et al., 2000Go). Many of these enzymes share significant sequence homology and enzymatic properties with one or more human counterparts.

Most sequence homology among FucTs occurs in the C-terminal catalytic domain. Structure–function studies of amino acids in this region have identified residues involved in GDP-fucose binding (Holmes et al., 1995Go; Sherwood et al., 2000Go), catalysis (Sherwood et al., 1998Go; Vo et al., 1998Go; Britten and Bird, 1997Go), and disulfide bonding (Holmes et al., 2000Go). Greater sequence heterogeneity is found in the N-terminal cytoplasmic and transmembrane domains, and the stem region. Residues involved in aspects of acceptor binding have been reported to occur in more N-terminal portions of the protein (Legault et al., 1995Go; Dupuy et al., 1999Go; Nguyen et al., 1998Go). For example, Legault et al. (1995)Go reported that amino acids present in a segment of FucT-III spanning residues 105 through 151 were associated with the enzyme’s ability to transfer to type 1 chain structures. Substitution of residues in this region unique to FucT-VI disabled transfer to type 1 chain acceptors. Among these residues is R110 of FucT-VI (W111 in FucT-III). In a recent report, Dupuy et al. (1999)Go confirmed that residues in this position contribute in defining type 1 and 2 chain acceptor specificity. In another report, Nguyen et al. (1998)Go demonstrated that amino acids from a more N-terminal region of the protein were also involved in defining type 1 versus type 2 chain specificity. Modification of as few as two amino acids of FucT-V to the corresponding residues of FucT-III (Asn86 to His and Thr87 to Ile) increased {alpha}1->4FucT activity with both oligosaccharide and glycolipid acceptors. Despite the ability of these amino acid changes to elicit {alpha}1->4fucosyl transfer to type 1 acceptors, kinetic parameters clearly showed that other residues present in FucT-III must also be required for optimal transfer (Nguyen et al., 1998Go).

A highly conserved His-His motif is present in this N-terminal hypervariable region of most presently cloned FucTs (see Figure 1). This is adjacent to amino acids already discussed, which have been shown to influence acceptor binding properties (Legault et al., 1995Go; Dupuy et al., 1999Go; Nguyen et al., 1998Go). We conducted site-directed mutagenesis studies to change these His residues (as well as other nearby residues) present in FucT-IV to determine their impact on enzyme activity and acceptor binding properties. The results demonstrate that the inherent enzymatic activity is reduced by many of these changes, and, in particular, changes in His114 results in altered acceptor affinities.



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 1. Aligned amino acid sequences of cloned {alpha}1->3/4FucTs around a His-His motif (shown in bold) distributed in the N-terminal region. The aligned sequences are shown where they occur in the sequence compared to human FucT-III. Amino acid deletions are shown by dashes where they occur in the alignments. For clarity and ease of sequence comparison, underscored amino acids define flanking residues of amino acid insertions of variable size from 1 to 26 amino acids. The sequences of the inserted residues are not shown. The alignments shown for the enzymes from S. mansoni (Trottein et al., 2000Go), mung bean (Leiter et al., 1999Go) C. elegans (DeBose-Boyd et al., 1998Go), and H. pylori (Rasko et al., 2000Go) are based on those given in the indicated references.

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Structure–function studies among human {alpha}1->3/4FucTs have led to the identification of amino acids involved in defining carbohydrate acceptor binding specificity (Legault et al., 1995Go; Dupuy et al., 1999Go; Nguyen et al., 1998Go). In general, these residues are found in the N-terminal portions of the enzyme’s catalytic domain. A productive strategy to define functional characteristics of specific amino acids has proven to be analysis of aligned protein sequences of {alpha}1->3/4FucTs to identify highly conserved amino acids, production of site-directed mutants at these positions, and analysis of the impact of these modifications on enzymatic properties. One highly conserved motif among cloned {alpha}1->3/4 FucTs is a His-His sequence corresponding, for example, to His113 and His114 of FucT-IV (see Figure 1). Although there is generally considerable sequence heterogeneity in this region among FucTs, this His-His motif is found in most enzyme forms, including all human FucTs. To analyze the functional properties of this motif, site-directed mutagenesis was used to change each of these sites to an Asn residue individually and together, and the effect of these mutations on acceptor properties was analyzed.

Site-directed mutagenesis and analysis of expressed enzymes
Site-directed mutagenesis was performed by replacing the sequence for codons within FucT-IV as summarized in Table I. No mutation resulted in the introduction of a new potential N-linked glycosylation site into the enzyme. Sequencing on both strands of all mutants confirmed that there were no other nucleotide modifications present compared to the wild-type FucT-IV sequence (data not shown). The plasmid containing both the wild-type FucT-IV sequence and that of the mutant enzymes was composed of a truncated form of the FucT-IV sequence missing the coding sequence for the first 57 amino acids of the FucT-IV enzyme in the pPROTA vector. The expressed protein was thus a fusion protein containing the Protein A Ig binding domain fused to the FucT-IV sequences. This aided in the isolation of the expressed protein by allowing direct binding to Ig-Agarose beads in a single-step purification. The presence of the Protein A Ig binding domain in the fusion protein could also be used to visualize and quantitate the protein in a western blot. No difference in enzymatic properties has been observed between pPROTA expressed FucTs and their native, full-length parent enzymes (De Vries et al., 1995Go).


View this table:
[in this window]
[in a new window]
 
Table I. Site-directed mutant enzymes used in this study
 
Figure 2 shows results from a western blot analysis of the pPROTA expressed wild-type FucT-IV and the mutant enzymes. Bands migrating at approximately 70–80 kDa corresponding to expressed wild-type FucT-IV and mutant fusion proteins are observed. In some cases these bands are weaker due to differences in expression levels. The majority of the expressed protein for enzymes containing the H113N mutation (both the single and double mutant) correspond to protein bands migrating between 40 and 45 kDa. This was a consistent finding because similar results were observed with expressed enzymes containing the H113N mutation from multiple expression experiments. These faster-migrating bands most likely result from degradation of the expressed fusion protein through protein turnover, possibly related to improper protein folding caused by the H113N mutation. For specific activity determinations, protein concentrations were based upon the amount of the 70–80-kDa protein present for each enzyme.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2. Western blot analysis of pPROTA expressed enzymes. Lane 1, wild-type FucT-IV (30 ng); lane 2, V110A mutant (4 ng); lane 3, H113L mutant (7 ng); lane 4, H113N mutant (7 ng); lane 5, H113N/H114N double mutant (3 ng); lane 6, H114A mutant (18 ng); lane 7, H114Q mutant (15 ng); lane 8, H114N mutant (30 ng); and lane 9, K119Y mutant (40 ng). The conditions of the experiment were as described under Materials and methods using a standard volume of beaded enzyme containing the amount of pPROTA expressed enzyme shown in parentheses. The asterisk on the right indicates the position of a band corresponding to antibody heavy chain derived from the IgG-Agarose beads used to capture the pPROTA expressed protein. The migration of protein molecular weight standards (in kDa) is shown to the left.

 
Effect on enzyme specific activity and acceptor substrate specificity
Table II shows the effect of the mutations on enzyme-specific activity utilizing the disaccharide LacNAc as the acceptor. Although the specific activity for the K119Y mutant was very similar to wild-type FucT-IV, other mutants had reduced specific activities of from 3- to 3500-fold compared with wild-type FucT-IV. In particular, broad changes in specific activities for His114 mutants were observed with the H114Q, H114A, and H114N mutants having values 3-, 17-, and 80-fold lower, respectively, compared with wild-type FucT-IV. The H113L mutant and the H113N/H114N double mutant were found to be inactive with LacNAc as the acceptor substrate.


View this table:
[in this window]
[in a new window]
 
Table II. Specific activities and Km values for wild-type FucT-IV and site-directed mutants
 
To further characterize the active mutants, a range of acceptor substrates were used. This analysis utilized synthetic 8-methoxycarbonyl derivatives of acceptor oligosaccharides because many of these free carbohydrates are not commercially available. The results are shown in Table III and are reported relative to the activity observed with the 8-methoxycarbonyl derivative of LacNAc. A standard acceptor concentration was used in each case based on optimized concentrations for wild-type FucT-IV. In these experiments, wild-type FucT-IV and mutants at positions 110, 113, and 119 behaved very similarly for each acceptor substrate tested. Similar results in comparison to wild-type FucT-IV were also observed for the His114 mutants with all acceptor oligosaccharides tested except for the H-type 2 acceptor. Relative transfer to the H-type 2 acceptor was increased approximately two- to threefold compared to wild-type FucT-IV for these mutants, indicating an inherent preference for an acceptor with a terminal {alpha}1->2-linked fucose for the His114 mutant enzyme. Wild-type FucT-IV is known to transfer fucose to sialylated acceptors poorly, and no difference was observed in the relative transfer to the sialylated acceptor by either His mutant. No significant fucose transfer to type 1 chain acceptors occurred with any enzyme tested.


View this table:
[in this window]
[in a new window]
 
Table III. Fucose transfer to oligosaccharide acceptors catalyzed by wild-type FucT-IV and site-directed mutants
 
Analysis of enzyme kinetic parameters
The apparent Km values for wild-type FucT-IV and mutant enzymes for GDP-fucose donor and various acceptor oligosaccharides were determined as shown in Table II. The results demonstrate that none of the mutant enzymes had a significantly altered Km for GDP-fucose compared to wild-type FucT-IV. In contrast, mutants of His114 had significantly higher Km values for both LacNAc and H-type 2 acceptors when compared with wild-type FucT-IV, as well as mutants at other nearby sites. Interestingly, the increase in Km was not of the same magnitude for both acceptor substrates. Wild-type FucT-IV and mutants at positions 110, 113, and 119 had Km values for LacNAc in generally the 1 mM range, which was reduced to approximately 0.25 mM for the H-type 2 acceptor. A much larger proportionate difference was observed with the His114 mutants tested. Km values for LacNAc varied from 5 to 11.6 mM and 0.6 to 1.1 mM for H-type 2. Thus the His114 mutant enzymes have an increased Km for both acceptor substrates compared to wild-type FucT-IV, but the relative (fucosylated versus nonfucosylated substrate) effect on acceptor substrate binding is different. This suggests that mutations of His114 differentially alter the acceptor substrate binding site. Therefore, this amino acid may be closer to the portion of the enzyme that binds the GlcNAc rather than the Gal residue of type 2 acceptors. In contrast to the alterations in acceptor substrate Km values, no significant differences were found in Vmax when either LacNAc or H-type 2 was used as an acceptor. Due to the very low activity obtained with the H113N enzyme, limited kinetic studies were possible. The results obtained indicate that the H113N mutant has a Km for LacNAc similar to that observed for the wild-type FucT-IV enzyme. The results obtained with the two His mutants were different even though the two amino acids are adjacent to each other in the linear sequence.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosyltransferases catalyze the biosynthesis of cell surface carbohydrates composed of multiple sugar moieties in discrete sequences and linkages. A surprising observation is that families of enzymes capable of catalyzing the same reaction are commonly found in a single organism. Examples include families of ß1->3- and ß1->4galactosyltransferases (Amado et al., 1999Go), {alpha}2->3sialyltransferases (Natsuka and Lowe, 1994Go; Kapitonov and Yu, 1999Go), and {alpha}1->3/4FucTs (Oriol et al., 1999Go). Although we do not understand why there are so many forms of these enzymes expressed in one organism, the availability of the multiple forms provides opportunities to obtain useful information about how the substrate specificity is encoded into the protein.

To date, six human {alpha}1->3/4fucosyltransferases have been cloned (Kukowska-Latallo et al., 1990Go; Goelz et al., 1990Go; Weston et al., 1992aGo,b; Koszdin and Bowen, 1992Go; Sasaki et al., 1994Go; Natsuka et al., 1994Go; Kaneko et al., 1999Go). These enzymes have relatively high sequence homology, particularly in the C-terminal catalytic domain. They differ in their tissue distribution and acceptor specificity properties. Previous reports have mapped amino acids involved in acceptor specificity properties, particularly the ability to catalyze fucose transfer to type 1 chain acceptors to amino acids present in the N-terminal hypervariable region (spanning amino acids 76 through 151) of FucT-III (Legault et al., 1995Go; Dupuy et al., 1999Go; Nguyen et al., 1998Go). In particular, these reports identified His76, Ile77 (Nguyen et al., 1998Go), and Trp111 (Dupuy et al., 1999Go) as being associated with transfer to type 1 chain acceptors. In addition to variable amino acids that correlate with a specific catalytic property, highly conserved amino acids are also found in this region of the protein and likely also play a critical role in acceptor binding.

One example is a highly conserved His-His motif that is immediately proximal to the W/R position, which correlates with type 1/type 2 chain transfer specificity (Dupuy et al., 1999Go). This His-His motif is conserved among FucTs from mammalian (Nishihara et al., 1999Go; Costache et al., 1997Go; Gersten et al., 1995Go; Smith et al., 1996Go; Ozawa and Muramatsu, 1996Go; Kudo et al., 1998Go; Sajdel-Sulkowska et al., 1997Go; Zhang et al., 1999Go; Oulmouden et al., 1997Go), chicken (Lee et al., 1996Go), and fish (Kageyama et al., 1999Go) species. However, FucTs from evolutionarily more distant organisms (i.e., plant [Leiter et al., 1999Go], bacteria [Rasko et al., 2000Go], and invertebrate species [Trottein et al., 2000Go; DeBose-Boyd et al., 1998Go]) do not have this motif. Studies of the acceptor specificities of enzymes that do not contain the His-His motif are incomplete; however, the evidence that is available indicates that at least some of these enzymes catalyze fucose transfer to either different (Leiter et al., 1999Go) or a wider variety (DeBose-Boyd et al., 1998Go) of acceptor carbohydrate structures than lacto- or neolacto-series structures.

The results presented demonstrate these His residues in human FucT-IV are important for optimal activity. Modification of His113 resulted in either inactive or weakly active enzymes; in some cases the mutant enzyme was partially degraded, possibly due to improper protein folding. Mutants of His114 also had reduced specific activity compared to wild-type FucT-IV, although to a lesser extent of 3–80-fold. A double mutant H113N/H114N was catalytically inactive with LacNAc. Other mutants at surrounding positions had varying effects on specific activity but had kinetic parameters very similar to the wild-type enzyme. Interestingly, analysis of acceptor specificity properties of the active mutants revealed that when normalized to the activity with LacNAc as the acceptor, all had very similar acceptor properties to the wild-type enzyme except for mutants of His114, which displayed a two- to threefold increased preference for the H-type 2 acceptor compared to the wild-type and other enzymes. Kinetic analyses of these mutant enzymes revealed that all mutant enzymes had a Km for GDP-fucose similar to the wild-type enzyme. Apparent Km values for LacNAc for mutants other than His114 were determined to be equal to that of wild-type FucT-IV. In contrast, significant differences in the Km for acceptors were found between wild-type FucT-IV and the His114 mutants.

Although a contribution of His114 to maintaining proper protein structure cannot be ruled out, changes in this site dramatically altered the ratio of the Kms for the two acceptors, leading to the likelihood that this position is directly involved in acceptor binding. Saturation of wild-type FucT-IV, as well as mutants in positions 110, 113, and 119, with LacNAc resulted in an apparent Km for the acceptor of approximately 1 mM, similar to other published reports (Sherwood et al., 1998Go, 2000; De Vries et al., 1995Go). An approximately fourfold reduced Km was observed when the H-type 2 acceptor was used (see also De Vries et al., 1995Go). Increased Km values were observed for both acceptors with the His114 mutants. The apparent Km for LacNAc varied between 5 and 11.6 mM for the His114 mutants versus values between 0.6 and 1.1 mM for the H-type 2 acceptor. Although both Kms are increased, the significantly lower Km for H-type 2 gives rise to the increased relative activity with this acceptor for the His114 mutant enzymes. The altered relative binding of a fucosylated versus a nonfucosylated acceptor suggests that this region may be an element of an acceptor binding pocket that interacts with GlcNAc residues of type 2 acceptors. Presumably, the presence of a fucose residue on the terminal Gal of the acceptor partially overcomes the reduced acceptor binding caused by weaker interactions with the GlcNAc residue.

Human {alpha}1->3/4FucTs contain five highly conserved His residues. The data presented in this report demonstrate an involvement in acceptor binding for certain of these residues. Other studies we are conducting address the involvement of His residue(s) in GDP-fucose binding. Future reports will address these results to provide a broader perspective of structure–function relationships within human FucTs.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
COS-7 cells were obtained from the American Type Cell Collection (Rockville, MD). N-acetyllactosamine (LacNAc), 2'-fucosyl-N-acetyllactosamine (H-type 2 trisaccharide), GDP-fucose, rabbit IgG-agarose beads, and DEAE-Dextran were obtained from Sigma (St. Louis, MO). Plasmid pCR2.1 TOPO was from Invitrogen (San Diego, CA), and pPROTA and pPROTA-FucT-IV (long form, amino acids 58–405) plasmids were received from Dr. Bruce A. Macher (San Francisco State University, San Francisco, CA). GDP-[14C]fucose (283 mCi/mmol) and [35S]dATP were obtained from Dupont NEN (Boston, MA). DNA sequencing of initial constructs was done using the Sequenase Version 2.0 DNA sequencing kit or the Sequenase polymerase chain reaction (PCR) product sequencing kit obtained from U.S. Biochemical (Cleveland, OH). Automated DNA sequencing of final pPROTA constructs was performed at the San Francisco State University Sequencing Facility, by Thetagen (Seattle, WA), or by GeneMed Synthesis (San Francisco, CA). PCR primers were obtained from Oligos, Etc. (Wilsonville, OR), Integrated DNA Technologies (Coralville, IA), or were made on a Beckman Oligo 1000 Synthesizer. All other reagents were of the highest quality commercially available.

Cell culture
COS-7 cells were grown in tissue culture plates in Dulbecco’s modified Eagle’s medium (DME), supplemented with 10% fetal calf serum. These were passed 1:4 every 5–6 days.

Transfection of FucT constructs into COS-7 cells for enzyme expression
COS-7 cells were transfected by the DEAE dextran technique (Ausubel et al., 1993Go) with the pPROTA Fuc T-IV constructs: chimeric, truncated wild-type FucT-IV, V110A, H113L, H113N, H113N/H114N, H114A, H114Q, H114N, and K119Y. Three to five days posttransfection, secreted fusion proteins were adsorbed to rabbit IgG-Sepharose beads (Sigma), washed extensively with phosphate buffered saline (PBS), and stored in PBS containing 0.02% NaN3 for use in these experiments.

Site-directed mutagenesis
Parental FucT coding sequences for site directed mutagenesis were truncated catalytic domain forms of the native FucT-IV in the pPROTA vector (Henion et al., 1994Go). Properties of pPROTA expressed enzymes have been shown to be very similar to those of full-length enzymes (De Vries et al., 1995Go). Site-directed mutant enzymes used in this study are shown in Table I. The flanking and mutagenic primers used in forming the FucT-IV V110A, H113L, H113N, H113N/H114N, H114A, H114Q, H114N, and K119Y mutants described in this study are shown in Table IV. Site-directed mutagenesis of FucT-IV in pPROTA was conducted as follows via recombinant PCR (Higuchi, 1990Go). The forward flanking primers used for the three FucT-IV mutants contained an EcoR1 site and nucleotides flanking the truncated form of each enzyme. The reverse flanking primers contained nucleotides flanking the C-terminal end of each enzyme, a STOP codon, and an EcoR1 site for the H113N/H114N double mutant, or a SalI site for the other single mutants. The SalI site was included in the single-mutant constructs to provide flexibility in cloning into an alternate pPROTA expression vector with a more diverse polylinker, if needed. This site was not used.


View this table:
[in this window]
[in a new window]
 
Table IV. Primers used for recombinant PCR mutagenesis
 
In the first step, a specific mutation was introduced by using a pair of mutagenic primers and a pair of opposite end flanking primers. For the H114N mutant, for example, one PCR mixture included the forward flanking primer and H114N reverse primer to generate the N-terminal half of the mutated FucT-IV; another PCR mixture included H114N forward and reverse flanking primers to generate the C-terminal half of the mutated FucT-IV. The PCR products were gel purified and used together with only the flanking primers for the second-step PCR mixture. The resulting PCR product for each FucT-IV mutant was a 1053-bp DNA fragment encompassing the truncated FucT-IV (Pro58 to Arg405) catalytic domain. These PCR products were ligated into Invitrogen pCR2.1 TOPO vector for amplification. The 1053-bp DNA fragments were excised with EcoR1 and subcloned into pPROTA. The correct orientation for these constructs was established by either HindIII/MluI or HindIII/EcoNI double digestion. Each mutation was confirmed by DNA sequencing using an Applied Biosystems Model 373A DNA sequencing system.

Enzyme assays
FucT-IV enzyme activities utilizing oligosaccharide acceptors were determined in standard reaction mixtures composed of 1 µmol HEPES buffer, pH 7.2, 6 nmol GDP-[14C]fucose (15,000 cpm/nmol), 0.4 µmol LacNAc or 0.072 µmol of 8-methoxycarbonyloctyl glycoside derivatives, 2 µmol NaCl, 0.125 µmol MnCl2, 10 µg bovine serum albumin, 0.01 µmol ATP, and chimeric enzyme bound to IgG-Agarose beads in a total volume of 0.02 ml. The reaction mixture was incubated for 1 h at room temperature and stopped and quantitated as described previously utilizing Dowex-1 for oligosaccharide acceptors (De Vries et al., 1995Go) or C18 columns for 8-methoxycarbonyl glycoside derivatives as acceptors (Palcic et al., 1988Go).

Assays conducted with the mutant enzymes were modified to increase sensitivity of the assay. This was accomplished by increased specific activity of GDP-[14C]fucose (30,000 cpm/nmol), longer assay times up to 4 h, and increased amounts of beaded enzyme in the 0.02 ml reaction mixture. These modifications resulted in assays with increased sensitivity of detection to allow activity determinations for enzymes with low inherent activity. All assays were linear with respect to both time and protein concentration over the assay times used.

Western blot analysis of pPROTA expressed enzymes
The pPROTA expressed recombinant FucT enzymes were separated on 12% Tris-glycine polyacrylamide gels, transferred to nitrocellulose membranes, and probed as described previously (Nguyen et al., 1998Go) to determine protein concentration of the expressed enzyme. In all cases, protein quantitation from western blot data for the purposes of specific activity determinations was based on the amount of intact fusion protein migrating between 70 and 80 kDa present in the expressed protein bound to IgG-Agarose beads.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Monica Palcic (University of Alberta, Canada) for the 8-methoxycarbonylactyl glycosides for substrate specificity studies, Ms. Stephanie Ho (San Francisco State University) for her assistance in the western blot analyses of pPROTA expressed enzymes, and Dr. Bruce Macher (San Francisco State University) for critical reading of the manuscript. This study was supported by research grant CA70740 from the National Cancer Institute to E.H.H.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FucT, fucosyltransferase; PBS, phosphate buffered saline; PCR, polymerase chain reaction.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: eholmes@nwbio.com Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Alhadeff, J.A. (1989) Malignant cell glycoproteins and glycolipids. CRC Crit. Rev. ncol./Hematol., 9, 37–107.

Amado, M., Almeida, R., Scheientek, T., and Clausen, H. (1999) Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim. Biophys. Acta, 1473, 35–53.[ISI][Medline]

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) (1993) Current protocols in molecular biology. Wiley, New York

Britten, C.J. and Bird, M.I. (1997) Chemical modification of an {alpha}3-fucosyltransferase. Definition of amino acid residues essential for enzyme activity. Biochim. Biophys. Acta, 1334, 57–64.[ISI][Medline]

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]

DeBose-Boyd, R.A., Nyame, A.K., and Cummings, R.D. (1998) Molecular cloning and characterization of an {alpha}1, 3-fucosyltransferase, CEFT-1, from Caenorhabditis elegans. Glycobiology, 8, 905–917.[Abstract/Free Full Text]

De Vries, T., Srnka, C.A., Palcic, M.M., Swiedler, S.L., van den Eijnden, D.H., and Macher, B.A. (1995) Acceptor specificity of different length constructs of {alpha}1, 3/4fucosyltransferases. Replacement if the stem region and the transmembrane domain if fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity. J. Biol. Chem., 270, 8712–8722.[Abstract/Free Full Text]

Dupuy, F., Petit, J.M., Mollicone, R., Oriol, R., Julien, R., and Maftah, A. (1999) A single amino acid in the hypervariable stem domain of vertebrate {alpha}1, 3/4-fucosyltransferases determines the type 1/type 2 transfer: characterization of acceptor substrate specificity of the Lewis enzyme by site-directed mutagenesis. J. Biol. Chem., 274, 12257–12262.[Abstract/Free Full Text]

Gersten, K.M., Natsuka, S., Trinchera, M., Petryniak, B., Kelly, R.J., Hiraiwa, N., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., and Lowe, J.B. (1995) Molecular cloning, expression, chromosomal assignment, and tissue specific expression of a murine {alpha}1, 3-fucosyltransferase locus corresponding to the human ELAM-1 ligand fucosyltransferase. J. Biol. Chem., 270, 25047–25056.[Abstract/Free Full Text]

Goelz, S.E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) ELFT: a gene that directs the expression of an ELAM-1 ligand. Cell, 63, 1349–1356.[ISI][Medline]

Hakomori, S. (1989) Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res., 52, 257–331.[ISI][Medline]

Henion, T.R., Macher, B.A., Anaraki, F., and Galili, U. (1994) Defining the minimal size of catalytically active primate {alpha}1, 3galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology, 4, 193–201.[Abstract]

Higuchi, R. (1990) Recombinant PCR. In: PCR protocols: a guide to methods and applications. Academic Press, New York, pp. 177–183.

Holmes, E.H., Xu, Z., Sherwood, A.L., and Macher, B.A. (1995) Structure-function analysis of human {alpha}1, 2fucosyltransferases: a GDP-fucose- protected, N-ethylmaleimide-sensitive site in FucT-III and FucT-V corresponds to Ser 178 in FucT-IV. J. Biol. Chem., 270, 8145–8151.[Abstract/Free Full Text]

Holmes, E.H., Yen, T.Y., Thomas, S., Joshi, R., Nguyen, A., Long, T., Julien, R., Gallet, F., Maftah, A., and Macher, B.A. (2000) Human {alpha}1, 3/4-fucosyltransferases: II. Characterization of highly conserved cysteine residues and N-linked glycosylation sites. J. Biol. Chem., 275, 24237–24245.[Abstract/Free Full Text]

Kageyama, N., Natsuka, S., and Hase, S. (1999) Molecular cloning and characterization of two zebrafish {alpha}1, 3-fucosyltransferase genes developmentally regulated in embryogenesis. J. Biochem., 125, 838–845.[Abstract]

Kaneko, M., Kudo, T., Iwasaki, H., Ikehara, Y., Nishihara, S., Nakagawa, S., Sasaki, K., Shiina, T., Inoko, H., Saitou, N., and Narimatsu, H. (1999) {alpha}1, 3-Fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse. Molecular cloning, characterization and tissue distribution of human Fuc-TIX. FEBS Lett., 452, 237–242.[CrossRef][ISI][Medline]

Kapitonov, D. and Yu, R.K. (1999) Conserved domains of glycosyltransferses. Glycobiology, 9, 961–978.[Abstract/Free Full Text]

Koszdin, K.L. and Bowen, B.R. (1992) The cloning and expression of a human {alpha}1, 3fucosyltransferase capable of forming the E-selectin ligand. Biochem. Biophys. Res. Commun., 187, 152–157.[ISI][Medline]

Kudo, T., Ikehara, Y., Togayachi, A., Kaneko, M., Hiraga, T., Sasaki, K., and Narimatsu, H. (1998) Expression cloning and characterization of a novel murine {alpha}1, 3-fucosyltransferase, mFuc-TIX, that synthesizes the Lewis x (CD15) epitope in brain and kidney. J. Biol. Chem., 273, 26729–26738.[Abstract/Free Full Text]

Kukowska-Latallo, J., Larsen, R.D., Nair, R.P., and Lowe, J.B. (1990) A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group {alpha}1, 3/4-fucosyltransferase. Genes Dev., 4, 1288–1303.[Abstract]

Lee, K.P., Carlsom, L.M., Woodcock, J.B., Ramachandra, N., Schultz, T.L., Davis, T.A., Lowe, J.B., Thompson, C.B., and Larsen, R.D. (1996) Molecular cloning and characterization of CFT1, a developmentally regulated avian {alpha}1, 3-fucosyltransferase gene. J. Biol. Chem., 271, 32960–32967.[Abstract/Free Full Text]

Legault, D.J., Kelly, R.J., Natsuka, Y., and Lowe, J.B. (1995) Human {alpha}1, 3/4-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J. Biol. Chem., 270, 20987–20996.[Abstract/Free Full Text]

Leiter, H., Mucha, J., Staudacher, E., Grimm, R., Glossl, J., and Altmann, F. (1999) Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc {alpha}1, 3-fucosyltransferase from mung beans. J. Biol. Chem., 274, 21830–21839.[Abstract/Free Full Text]

Listinsky, J.J., Siegal, G.P., and Listinsky, C.M. (1998) {alpha}-L-fucose: a potentially critical molecule in pathologic processes including neoplasia. Am. J. Clin. Pathol., 110, 425–440.[ISI][Medline]

McEver, R.P. (1997) Selectin-carbohydrate interactions during inflammation and metastasis. Glycoconj. J., 14, 585–591.[CrossRef][ISI][Medline]

Natsuka, S. and Lowe, J.B. (1994) Enzymes involved in mammalian oligosaccharide biosynthesis. Curr. Opin. Struct. Biol., 4, 683–691.[ISI]

Natsuka, S., Gersten, K.M., Zenita, K., Kannagi, R., and Lowe, J.B. (1994) Molecular cloning of a cDNA encoding a novel human leukocyte {alpha}1, 3fucosyltransferase capable of synthesizing the sialyl Lewis x determinant. J. Biol. Chem., 269, 16789–16794.[Abstract/Free Full Text]

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

Nishihara, S., Iwasaki, H., Kaneko, M., Tawada, A., Ito, M., and Narimatsu, H. (1999) {alpha}1, 3-Fucosyltransferase 9 (FUT9; Fuc-TIX) preferentially fucosylates the distal GlcNAc residue of polylactosamine chain while the other four {alpha}1, 3FUT members preferentially fucosylate the inner GlcNAc residue. FEBS Lett., 462, 289–294.[CrossRef][ISI][Medline]

Oriol, R., Mollicone, R., Cailleau, A., Balanzino, L., and Breton, C. (1999) Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria. Glycobiology, 9, 323–334.[Abstract/Free Full Text]

Oulmouden, A., Wierinckx, A., Petit, J.M., Costache, M., Palcic, M.M., Mollicone, R., Oriol, R., and Julien, R. (1997) Molecular cloning and expression of a bovine {alpha}1, 3-fucosyltransferase gene homologous to a putative ancestor gene of the human FUT3-FUT5-FUT6 cluster. J. Biol. Chem., 272, 8764–8773.[Abstract/Free Full Text]

Ozawa, M. and Muramatsu, T. (1996) Molecular cloning and expression of a mouse {alpha}1, 3-fucosyltransferase gene that shows homology with the human {alpha}1, 3-fucosyltransferase IV gene. J. Biochem., 119, 302–308.[Abstract]

Palcic, M.M., Heerze, L.D., Pierce, M., and Hindsgaul, O. (1988) The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays. Glycoconj. J., 5, 49–63.[ISI]

Rasko, D.A., Wang, G., Palcic, M.M., and Taylor, D.E. (2000) Cloning and characterization of the {alpha}1, 3/4-fucosyltransferase of Helicobacter pylori. J. Biol. Chem., 275, 4988–4994.[Abstract/Free Full Text]

Renkonen, R., Mattila, P., Majuri, M.L., Räbinä, J., Toppila, S., Renkonen, J., Hirvas, L., Niittymäki, J., Turunen, J.P., Renkonen, O., and Paavonen, T. (1997) In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands. Glycoconj. J., 14, 593–600.[CrossRef][ISI][Medline]

Sajdel-Sulkowska, E.M., Smith, F.I., Wiederschain, G., and McLuer, R.H. (1997) Cloning of a rat {alpha}1, 3-fucosyltransferase gene: A member of the fucosyltransferase IV family. Glycoconj. J., 14, 249–258.[CrossRef][ISI][Medline]

Sanders, D.S. and Kerr, M.A. (1999) Lewis blood group and CEA related antigens; coexpressed cell–cell adhesion molecules with roles in the biological progression and dissemination of tumours. Mol. Pathol., 52, 174–178.[Abstract]

Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) Expression cloning of a novel {alpha}1, 3fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x determinant in leukocytes. J. Biol. Chem., 269, 14730–14737.[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->3fucosyltransferases: III. A Lys/Arg residue located within the {alpha}1->3FucT motif is required for activity but not substrate binding. J. Biol. Chem., 273, 25256–25260.[Abstract/Free Full Text]

Sherwood, A.L., Davis, W.C., Ho, S., Macher, B.A., Stroud, M.R., Upchurch, D.A., and Holmes, E.H. (2000) A GDP-fucose protected, pyridoxal-5'-phosphate/NaBH4 sensitive Lys residue common to human FucT’s corresponds to Lys300 in FucT-IV. Biochem. Biophys. Res. Commun., 273, 870–876[CrossRef][ISI][Medline]

Smith, P.L., Gersten, K.M., Petryniak, B., Kelly, R.J., Rogers, C., Natsuka, Y., Alford, J.A., Scheidegger, E.P., Natsuka, S., and Lowe, J.B. (1996) Expression of the {alpha}1, 3-fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands. J. Biol. Chem., 271, 8250–8259.[Abstract/Free Full Text]

Trottein, F., Mollicone, R., Fontaine, J., de Mendonca, R., Piller, F., Pierce, R., Oriol, R., and Capron, M. (2000) Molecular cloning of a putative {alpha}3-fucosyltransferase from Schistosoma mansoni. Mol. Biochem. Parisitol., 107, 279–287[CrossRef][ISI][Medline]

Vo, L., Lee, S., Marcinko, M.C., Holmes, E.H., and Macher, B.A. (1998) Human {alpha}1->3fucosyltransferases: 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]

Weston, B.W., Nair, R.P., Larsen, R.D., and Lowe, J.B. (1992a) Isolation of a novel human {alpha}1, 3- fucosyltransferase gene and molecular comparison to the human Lewis blood group {alpha}1, 3/4-fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities. J. Biol. Chem., 267, 4152–4160.[Abstract/Free Full Text]

Weston, B.W., Smith, P.L., Kelly, R.J., and Lowe, J.B. (1992b) Molecular cloning of a fourth member of a human {alpha}1, 3fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x and difucosyl sialyl Lewis x epitopes. J. Biol. Chem., 267, 24575–24584.[Abstract/Free Full Text]

Zhang, A., Potvin, B., Zaiman, A., Chen, W., Kumar, R., Phillips, L., and Stanley, P. (1999) The gain-of-function chinese hamster ovary mutant LEC11B expresses one of two chinese hamster FUT6 genes due to the loss of a negative regulatory factor. J. Biol. Chem., 274, 10439–10450. [Abstract/Free Full Text]