3 Department of Chemistry and Biochemistry, 1600 Holloway Ave., San Francisco State University, San Francisco, CA 94132, and 4 Vertex Pharmaceuticals (Europe) Ltd., Abingdon, Oxfordshire OX14 4RY, United Kingdom
Received on May 26, 2004; revised on August 31, 2004; accepted on September 21, 2004
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Abstract |
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Key words: alanine scanning mutagenesis / alpha 1,3-fucosyltransferase motif / function / molecular modeling / structure
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Introduction |
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Applying the alanine scanning mutagenesis approach to an analysis of the alpha 1,3 FucT motif has shown that Ala substitution for residues within the motif with Ala results in a set of proteins that are either catalytically inactive proteins, have marginal fucosyltransferase activity, or have substantial enzymatic activity. Kinetic characterization of the mutant proteins with substantial enzymatic activity demonstrated that some of these proteins had altered Km for the donor substrate, and one had an altered Km for the acceptor substrate. Thus, not all of the amino acids within the alpha 1,3 FucT motif affect the binding of the donor substrate.
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Results |
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All studies were carried out with the catalytic domain of human FucT VI, which we refer to as wild type throughout this article. The motif's amino acids as described by Martin et al. (1997) are spread over a segment 19 amino acids in length (residues 240258) and a range of amino acids (including nonpolar, aromatic, basic, and acidic residues) are represented within the motif. Eight conserved residues occur at the N-terminus of the alpha 1,3 FucT motif, whereas four are located at the C-terminus, with four nonconserved amino acids (250253) intervening. Four amino acids (E, F, K, and Y) occur twice in the motif, whereas others occur once.
Site-directed mutagenesis and expression of human FucT VI
To evaluate the functional importance of the amino acids of the alpha 1,3 FucT motif described by Martin et al. (1997), 12 mutant proteins were created by substituting Ala independently for each of the motif's amino acids using site-directed mutagenesis. The proteins were purified and then characterized by western blot analysis as shown in Figure 2. Each mutant protein had
the same molecular weight as the wild-type enzyme (data not shown). Each mutant, except K241A, was produced at a level similar to that of FucT VI. The K241A mutant protein was produced at
one-tenth the level of wild-type enzyme (Figure 2).
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Mutant proteins with a relatively high level of enzyme activity were produced when Ala was substituted for alpha 1,3 FucT motif amino acids at the N-terminus (amino acid 240) and at the middle (amino acids 244, 246, and 249), but not at the C-terminus to the motif. The four C-terminal Ala mutants were either inactive or had 1% or less of wild-type enzyme activity, even though the substituted amino acids had a range of side chains (Y, T, E, and K). Three of the four mutants with relatively high activity were those in which Ala substituted for a nonpolar amino acid (Y, L, and F), the only exception being the substitution of Ala for Ser. In some cases, substitution of Ala for the same amino acid at two different locations within the alpha 1,3 FucT motif had substantially different effects on enzyme activity. For example, Y240A was the second most active mutant with 14% of wild-type activity, whereas Y254A was inactive. In addition, F242A had very low activity, whereas F246A had significant activity. In contrast, Ala substitution for E (247 and 257) or K (242 and 258) at the N- and C-termini of the motif resulted in proteins with very low activity. Finally, Ala substitution for two similar amino acids in the middle of the alpha 1,3 FucT motif, S249A and T256A, produced proteins with a dramatically different level (57% versus <1%, respectively) of fucosyltransferase activity.
To evaluate if Ala mutants of the alpha 1,3 FucT motif in another member of the fucosyltransferase family gave similar results to those for FucT VI, additional mutants were prepared by substituting Ala for two of the alpha 1,3 FucT motif amino acids (T257 and E258, homologous to T256 and E257) of human FucT III. The enzyme activities observed for these mutant enzymes were similar to those observed for FucT VI (i.e., < 1% of wild-type activity).
Active Ala mutants of the alpha 1,3 FucT motif of human FucT VI have altered Km values
Because all members of the alpha 1,3 FucT family use GDP-fucose as their donor substrate but use different acceptor substrates, it has been proposed that the amino acids of the alpha 1,3 FucT motif may participate in donor substrate binding. To evaluate this possibility, donor substrate saturation studies (Figure 3) were carried out with wild-type FucT VI and with each of the four Ala mutants (Y240A, L244A, F246A, and S249A) that had significant enzyme activity. To obtain an estimate of the Km for the donor substrate of these two mutants it was necessary to use GDP-fucose concentrations of up to 0.5 mM, a concentration that is 20 times the Km for the wild-type FucT VI. The Km for GDP-fucose for all of the four Ala mutants tested were higher than that for wild-type FucT VI, and three of the four had GDP-fucose Km values that were 515 times higher than that for wild-type enzyme (Table II). Only S249A had a GDP-fucose Km value that was similar (two to three times) to that of the wild-type FucT VI.
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Discussion |
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As additional FucTs sequences were elucidated, it also became possible to identify additional, functionally important amino acids within the FucT sequence. The cloning of bacterial FucTs provided an opportunity to narrow the search for functionally important sequences because the FucT sequences from these organisms share only limited sequence identity with those from other organisms. As a result, Martin et al. (1997) identified a series of amino acids within a short sequence of all FucTs that is identical and referred to this region as the alpha 1,3 FucT motif and proposed that amino acids in this motif may be involved in donor binding. However, this hypothesis has not been tested. Cunningham and Wells (1989)
developed an alanine scanning mutagenesis approach to evaluate the functional importance of individual amino acids within a protein, and the approach has provided important information on a range of proteins. Therefore, we applied Ala scanning mutagenesis to the 12 amino acids within the alpha 1,3 FucT motif.
Ala mutants have a range of enzyme activity
The results shown in Table I and summarized in Figure 6 demonstrate that substitution of Ala for different amino acids within the alpha 1,3 FucT motif results in proteins with widely varying specific activities. Because the enzymatic activity levels of some of the mutants was more than 100-fold lower than the wild-type enzyme, it was necessary to alter the reaction conditions substantially to obtain an estimate of their activity. Thus, for those mutants with low activity it was necessary to carry out the incubation for up to five times as long, and with substantially more protein than for reactions carried out with wild-type enzyme. Therefore, the values listed in Table I are estimates at best of the true specific activities for the mutants.
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Inactive mutants
K258 is the C-terminal residue of the motif, four amino acids downstream from Y254. Substitution of Ala for either of these residues generated proteins devoid of FucT activity. Consequently, it was impossible to determine if either of these residues altered the protein's affinity for GDP-fucose. However, we previously evaluated the importance of the Lys that corresponds to K258 in FucT VI in two other FucTs (IV and VII) by mutagenesis (Sherwood et al., 1998, 2000
). In these earlier studies, we prepared mutants of FucT IV and VII in which conservative (K300R in FucT IV and K240R in FucT VII) substitutions for the lysine residue were made. The FucT IV mutant was found to have low activity, whereas the FucT VII mutant was inactive. The FucT IV K300R mutant was shown to have a 10-fold higher Km for GDP-fucose than the wild-type enzyme, demonstrating that GDP-fucose binding is altered by the Arg to Lys substitution in FucT IV. We anticipate that the homologous Lys residue in FucT VI is also involved in GDP-fucose binding and are currently testing this assumption by making the K258R mutant.
Low-activity mutants
Six of the alpha 1,3 FucT motif amino acids [(K241, F242) (E247, N248) (T256, E257)] occur in pairs and are distributed across the motif sequence, with two near the N-terminus, two near the center, and two near the C-terminus. Three of the six amino acids in this group are charged (one positively, two negatively), two are polar, whereas F242 is the only nonpolar amino acid in this group. All amino acids in this group have side chains of average size. Mutation of these amino acids to Ala produced enzymes with low activity levels (<1% compared to wild-type activity).
The first of these amino acids has been previously studied by Sherwood et al. (2000), who substituted Arg in FucT VII (homologous to K241 of FucT VI) with a Lys residue and found that the mutant enzyme had a twofold higher specific activity than wild-type enzyme and that the Km for GDP-fucose was unchanged. This demonstrated that this amino acid was important for catalysis but not involved in donor binding. Thus the homologous amino acid in FucT VI would likely have a similar role.
A naturally occurring FucT VI mutant, E247K, which changes the functional group at this position from a negatively charged to a positively charged one, has been shown to be inactive (Mollicone et al., 1994). We show that a mutation from glutamic acid to the nonpolar alanine produces a protein with minimal enzyme activity.
High-activity mutants
Four mutants, Y240A, L244A, F246A, and S249A, had activity levels of 14%, 7%, 7%, and 59%, respectively, of the wild-type specific activity. Except for S249, the amino acids in this group are nonpolar and are part of a hydrophobic cluster of six amino acids within the motif (Y240, F242, Y243, L244, F246, and L250). Mutating the three nonpolar amino acids, Y240, L244, and F246 to Ala maintains their hydrophobic character. It is possible that the main requirement for these amino acids is that their side chains are hydrophobic and the specific side chain is a result of evolutionary optimization. It is interesting to note that although our L244A mutant has less than one-tenth of wild-type activity, the naturally occurring mutant FucT VI L244V, described by Elmgren et al. (2000), has a specific activity one-third that of the wild-type enzyme.
Substrate affinity
The four mutants (Y240A, L244A, F246A, and S249A) with the highest enzyme activity were kinetically analyzed. Our results demonstrate that three of these mutants, Y240A, L244A, and F246A, have acceptor substrate affinities that are similar to the wild-type enzyme. Thus, substituting Ala for any of these three nonpolar residues did not have a measurable effect on acceptor substrate binding. Elmgren et al. (2000) also found that the naturally occurring mutant L244V did not have an altered affinity for acceptor substrate.
In contrast, substituting Ala for the only polar residue among this group, S249, resulted in a protein with a much lower affinity for the two acceptor substrates, LacNAc-C8 and H-type II-C8. Although we were unable to obtain an accurate Km for LacNAc-C8, we were able to determine that the mutant had a 10-fold lower affinity for H-type II-C8. Some FucTs have a higher affinity for H-type II-C8 than for LacNAc-C8, and this is also the case for the S249A mutant. Thus, substituting Ala for S249 does not result in an altered acceptor substrate preference but results in an altered affinity for acceptor substrates in general. Therefore, other amino acids must account for the increased affinity of FucTs for H-type II-C8 versus LacNAc-C8.
Each of the four highly active mutants had a lower affinity for GDP-fucose compared with wild-type enzyme. All three mutants (Y240A, L244A, F246A) in which Ala was substituted for a nonpolar amino acid had large (515 times wild type) changes in Km for GDP-fucose, with the L244A having the largest change relative to the wild-type enzyme. Interestingly, Elmgren et al. (2000) found that the naturally occurring L244V mutation in FucT VI did not change the affinity of the protein for GDP-fucose. Given the similarity of the amino acid substitutions (i.e., Ala or Val for Leu) in the two mutant forms of FucT VI, it is surprising that the mutants would vary substantially in their affinity for GDP-fucose.
In contrast to the three other highly active mutants, S249A had only a modest ( three times wild-type) reduction in affinity for GDP-fucose. Because this mutant has a dramatically altered Km for acceptor and our donor saturation studies were carried out with LacNAc-C8 as the acceptor substrate, the observed change in affinity for GDP-fucose must be considered only an estimated value.
Alpha 1,3 FucT motif predicted structure
To obtain a better insight in the 3D arrangement of mutated residues in the alpha 1,3 FucT motif, the secondary structures of FucT III and FucT VI were predicted using the Holley and Karplus (1989) method implemented in Quanta 2000. An
-helical secondary structure is predicted for residues T236N248 and I255W267 of FucT VI, whereas the connecting residues 249254 are predicted to have a random coil conformation. Similarly,
-helices are predicted for residues T237N249 and I256W268 in FucT III with the surrounding residues predicted to be random coil.
A 3D model of the alpha 1,3 FucT motif was built using the Protein Design module in Quanta using standard -helical backbone geometry for the helical regions. The resulting model is depicted in Figure 7. The two predicted
-helices place the hydrophobic residues whose mutation minimally affected the enzymatic activity of FucT VI (Y240, L244, and F246) on one side of the N-terminal
-helix. S249 is located in the random coil region directly following the first helix. The concentration of hydrophobic residues on one side of this helix suggests that they may form a portion of the hydrophobic core of FucT VI and function to anchor the helix to the hydrophobic core of the enzyme. This is consistent with the observation that the mutation of these residues to a smaller but still hydrophobic Ala residue does not affect enzyme activity as substantially as other Ala substitutions within the motif. The generally more polar residues K241, F242, E247, and N248, whose mutation resulted in proteins with very low enzymatic activity, cluster on the opposite side of the helix. Their more polar nature and the substantial effect of their mutation on enzymatic activity suggest that these residues are solvent-exposed and possibly in direct contact with the substrates or products.
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The mutational results do not provide any insight into the relative spatial orientation of the two -helices with respect to one another. The presence of a Pro at position 252, however, suggests the presence of some form of turn between the two helices, bringing them in close proximity. A similar Pro residue is found in FucT III at position 253.
The 3D model also reveals that other amino acids (S238 and A245 in the first -helix and I255, N262, and A266 in the second
-helix) are located on the same face as the amino acids that dramatically reduce FucT activity. Their placement spatially adjacent to residues now known to be important for enzyme activity makes them interesting candidates for future mutational studies.
Other conserved sequences within alpha 1,3 FucT sequences
Since Martin et al. (1997) proposed an alpha 1,3 FucT motif, FucT sequences from several other species have been identified. Based on an alignment of the FucT sequences, Oriol et al. (1999)
have identified two highly conserved regions. One referred to as "highly conserved sequence II of alpha-3-fucosyltransferases" is shown in Figure 1. This region includes the amino acids of the alpha 1,3 FucT motif, but extends further up- and downstream and includes one additional invariant residue (a proline residue near the C-terminus) and several other residues that are found in the FucT sequences of most species. These amino acids, as well as those found in the "highly conserved sequence I of alpha-3-fucosyltransferases" are also important candidates for analysis by mutagenesis (Oriol et al., 1999
).
Finally, Dupuy et al. (2004) have recently used mutagenesis analysis to evaluate what they referred to as the acceptor-binding motif, VxxHH(W/R)(D/E), of FucTs. They have demonstrated that one of the key residues affecting acceptor substrate specificity (i.e., alpha 1,3 versus alpha 1,4) is the W/R residue of this motif. Holmes and co-workers (Sherwood et al., 2002
) have shown that the His-His residues of this motif also alter acceptor substrate binding. Thus, amino acids within the alpha 1,3 FucT motif, the acceptor binding motif, and possibly region I described by Oriol et al. (1999)
would appear to work in concert to regulate the acceptor and donor substrate binding in FucTs.
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Materials and methods |
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Truncated, wild-type FucT VI template
A truncated, wild-type FucT VI in the pPROTA vector was created and used as a template to generate the alanine mutants described in this study. The template encodes the wild-type catalytic domain (amino acid 40 to the C-terminus) of FucT VI with XmaI and XbaI restriction enzyme sites introducing silent mutations at nucleotides 611 and 789, respectively. This construct was used for cassette mutagenesis to produce the alanine mutants.
Alanine scanning mutagenesis
The QuikChange Site-Directed Mutagenesis Kit was used to generate the alanine point mutations. Twelve alanine mutations were created using the following upper primers: Y240A (5'-G ATG GAG ACG CTG TCT CGA GCC AAG TTC TAT CTG-3'); K241A (5'-G ATG GAG ACG CTC TCG AGG TAC GCG TTC TAT CTG G-3'); F242A (5'-CTG TCC CGG TAC AAG GCC TAT CTG GCC TTC GAA AAC TCC TTG CAC-3'); L244A (5'-AC AAG TTC TAT GCG GCC TTC GAA AAC TCC TTG CAC-3'); F246A (5'-G TCC CGG TAC AAG TTC TAT CTG GCC GCC GAG AAT TCC TTG CAC-3'); E247A (5'-C TAT CTG GCC TTC GCG AAT TCC TTG CAC CCC GAC-3'); N248A (5'-C TAT CTG GCC TTC GAA GCC TCC TTG CAC CCC GAC TAC ATC-3'); S249A (5'-C TAT CTG GCC TTC GAA AAC GCC TTG CAC CCC GAC TAC ATC-3'); Y254A (5'-G AAC TCC TTG CAC CCC GAC GCC ATC ACC GAG AAG CTT TGG AGG AAC-3'); T256A (5'-GAC TAC ATC GCC GAG AAG CTT TGG AGG AAC G-3'); E257A (5'-CC GAC TAC ATC ACC GCG AAG CTT TGG AGG AAC-3'); and K258A (5'-C TAC ATC ACC GAA GCG CTG TGG AGG AAC-3'). Lower primers were of the same length as and complementary to the upper primers. Following polymerase chain reaction, the mixture was treated with DpnI to digest the template DNA. To excise the cassette, the mutant constructs were double-digested with XmaI and XbaI and the 178-bp DNA products were gel purified and subcloned into the truncated, wild-type FucT VI-pPROTA construct from which the XmaI and XbaI cassette had been removed. Each resulting alanine mutation and the truncated wild-type were propagated in the JM109 strain of Escherichia coli. The nucleotide sequence of each mutant construct was confirmed by completely sequencing both strands of the coding region (Microchemical Core Facility at San Diego State University).
Enzyme expression
The truncated, wild-type construct and the alanine mutant constructs were transfected into 90% confluent COS-7 cells using the lipofectamine method and grown in Dulbecco's modified Eagle medium containing 10% fetal calf serum. The enzymes were expressed as soluble proteins that have an N-terminal protein A, IgG binding domain (De Vries et al., 1995). The protein AFucT VI chimeric, soluble proteins were harvested on day 2 and 3 posttransfection and purified from the cell culture media using IgG-agarose affinity beads.
Quantitative western blot analysis
Expressed chimeric enzymes were detected and quantified by western blot analysis (Xu et al., 1996). Briefly, the proteins were separated on 420% TrisHCl polyacrylamide gels and transferred to nitrocellulose membranes. The protein AIgG binding domain of the chimeric proteins was detected by incubating the blot with anti-goat IgGalkaline phosphatase conjugate and developing the blot with 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium. Quantification of the amount of FucT was accomplished by comparing the band intensities of samples to those obtained for known quantities of IgG.
FucT assays
The enzyme activities of wild-type and mutant FucT enzymes were determined as previously described (Nguyen et al., 1998) using soluble enzyme in media or IgG-agarose bound enzyme. The standard reaction mixture contained 50 mM MOPS-NaOH, pH 6.5; 40 mM MnCl2, 0.05% bovine serum albumin, 3.0 nmol GDP-fucose, 0.02 µCi GDP-[3H]-fucose, 20 nmol acceptor substrate, and varying volumes of enzyme (total volume of 20 µl). For kinetic studies, the donor or acceptor concentrations were varied. The reactions were incubated for time periods ranging from 15 min to 20 h and stopped by dilution with 380 µl water. The reaction products were separated from substrate by reverse-phase chromatography (Sep-Pak C18) and quantified as described previously (Xu et al., 1996
). The results obtained were analyzed by fitting the initial rate data to the Michaelis-Menten equation using nonlinear regression analysis (KaleidaGraph 3.51, Synergy Software, Reading, PA). When Km values were determined for Ala mutants, the concentration of the second substrate was added at a level that is known to be saturating for wild-type FucT VI.
Secondary structure prediction and molecular modeling
The secondary structures of FucT III and FucT VI were predicted using the Holley-Karplus method (Holley and Karplus, 1989) implemented in Quanta 2000 (Accelrys, San Diego, CA). On the basis of the predicted secondary structure, 3D models of the FucT motifs were built using the Protein Design module in Quanta 2000 using standard
-helical backbone geometry for the helical regions and an extended conformation for loop regions.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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