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
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Abstract |
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Key words: carbohydrate binding site/fucosyltransferase/His motif/structurefunction relationships
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Introduction |
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Most sequence homology among FucTs occurs in the C-terminal catalytic domain. Structurefunction studies of amino acids in this region have identified residues involved in GDP-fucose binding (Holmes et al., 1995; Sherwood et al., 2000
), catalysis (Sherwood et al., 1998
; Vo et al., 1998
; Britten and Bird, 1997
), and disulfide bonding (Holmes et al., 2000
). 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., 1995
; Dupuy et al., 1999
; Nguyen et al., 1998
). For example, Legault et al. (1995)
reported that amino acids present in a segment of FucT-III spanning residues 105 through 151 were associated with the enzymes 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)
confirmed that residues in this position contribute in defining type 1 and 2 chain acceptor specificity. In another report, Nguyen et al. (1998)
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
1
4FucT activity with both oligosaccharide and glycolipid acceptors. Despite the ability of these amino acid changes to elicit
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., 1998
).
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., 1995; Dupuy et al., 1999
; Nguyen et al., 1998
). 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.
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Results |
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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., 1995).
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Discussion |
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To date, six human 1
3/4fucosyltransferases have been cloned (Kukowska-Latallo et al., 1990
; Goelz et al., 1990
; Weston et al., 1992a
,b; Koszdin and Bowen, 1992
; Sasaki et al., 1994
; Natsuka et al., 1994
; Kaneko et al., 1999
). 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., 1995
; Dupuy et al., 1999
; Nguyen et al., 1998
). In particular, these reports identified His76, Ile77 (Nguyen et al., 1998
), and Trp111 (Dupuy et al., 1999
) 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., 1999). This His-His motif is conserved among FucTs from mammalian (Nishihara et al., 1999
; Costache et al., 1997
; Gersten et al., 1995
; Smith et al., 1996
; Ozawa and Muramatsu, 1996
; Kudo et al., 1998
; Sajdel-Sulkowska et al., 1997
; Zhang et al., 1999
; Oulmouden et al., 1997
), chicken (Lee et al., 1996
), and fish (Kageyama et al., 1999
) species. However, FucTs from evolutionarily more distant organisms (i.e., plant [Leiter et al., 1999
], bacteria [Rasko et al., 2000
], and invertebrate species [Trottein et al., 2000
; DeBose-Boyd et al., 1998
]) 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., 1999
) or a wider variety (DeBose-Boyd et al., 1998
) 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 380-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., 1998, 2000; De Vries et al., 1995
). An approximately fourfold reduced Km was observed when the H-type 2 acceptor was used (see also De Vries et al., 1995
). 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 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 structurefunction relationships within human FucTs.
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Materials and methods |
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Cell culture
COS-7 cells were grown in tissue culture plates in Dulbeccos modified Eagles medium (DME), supplemented with 10% fetal calf serum. These were passed 1:4 every 56 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., 1993) 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., 1994). Properties of pPROTA expressed enzymes have been shown to be very similar to those of full-length enzymes (De Vries et al., 1995
). 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, 1990
). 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.
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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., 1995) or C18 columns for 8-methoxycarbonyl glycoside derivatives as acceptors (Palcic et al., 1988
).
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., 1998) 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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