©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function Analysis of Human 13Fucosyltransferases
A GDP-FUCOSE-PROTECTED, N-ETHYLMALEIMIDE-SENSITIVE SITE IN FucT-III AND FucT-V CORRESPONDS TO Ser IN FucT-IV (*)

(Received for publication, November 4, 1994; and in revised form, January 20, 1995)

Eric H. Holmes (1)(§) Zhenghai Xu (2)(¶) Anne L. Sherwood (1) Bruce A. Macher (2)

From the  (1)Pacific Northwest Research Foundation, Seattle, Washington 98122 and the (2)Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human alpha13fucosyltransferases constitute a family of closely related membrane-bound enzymes distinguished by differences in acceptor specificities and inherent protein biochemical properties. One such biochemical property is sensitivity to enzyme inactivation by sulfhydral-group modifying reagents such as N-ethylmaleimide. The basis for this property has been studied using a fusion protein of FucT-III and FucT-V composed of Protein A coupled to the catalytic domain of the enzyme. The results indicate that modification of FucT-V by 5,5`-dithiobis(2-nitrobenzoic acid) resulted in efficient enzyme inactivation that could be reversed by excess thiol reagent suggesting that the free sulfhydral group on the enzyme was required for activity. Recombinant forms of both FucT-III and FucT-V were irreversibly inactivated by N-ethylmaleimide and could be effectively protected from inactivation by GDP-fucose and GDP but not by UDP-galactose, fucose, or N-acetyllactosamine. Analysis of the distribution of Cys residues in aligned sequences of cloned human alpha13fucosyltransferases indicated one site, Cys of FucT-III and Cys of FucT-V, corresponded to the highly conservative replacement of Ser in FucT-IV, an enzyme insensitive to N-ethylmaleimide. A site-directed mutagenesis experiment was performed to replace Ser of FucT-IV with a Cys residue. The mutant FucT-IV enzyme was active; however, the K for GDP-fucose was increased about 3-fold compared to the native enzyme to 28 ± 3 µM. This enzyme was N-ethylmaleimide sensitive and could be partially protected by GDP-fucose but not N-acetyllactosamine. These results support the importance of Ser of FucT-IV in donor substrate binding and strongly suggest analogous Cys residues are the GDP-fucose protectable, N-ethylmaleimide-sensitive sites present in FucT-III and -V.


INTRODUCTION

Fucosylated glycoconjugates function as blood group and developmental antigens, and they have been associated with tumorigenesis (for review, see (1, 2, 3, 4) ). More recently, alpha13fucosylated glycoconjugates have been identified as ligands for proteins (i.e. selectins) that mediate normal leukocyte trafficking and leukocyte extravasation in inflammatory reactions(5) . Thus, there is a significant interest in the regulation of the expression of fucosylated structures.

The final step in the synthesis of these antigens is catalyzed by a group of enzymes known as fucosyltransferases. These enzymes facilitate the transfer of fucose from GDP-fucose to one or more acceptor substrates. A number of forms of human alpha13fucosyltransferases (FucTs) (^1)have been distinguished in various tissues on the basis of acceptor substrate specificity and other biochemical properties(6, 7, 8, 9) . Among the properties that have been used to distinguish various FucTs is an enzyme's sensitivity to inactivation by N-ethylmaleimide (NEM)(8, 9, 10, 11, 12, 13, 14) . Some preliminary studies of the NEM sensitivity of recombinant enzymes have also been performed(15) . NEM is known to chemically modify proteins by forming a covalent linkage with the sulfhydral group of cysteine (Cys) residues. As shown in Table 1, four human FucTs have been identified in human tissues. Two of these enzymes are inhibited by NEM treatment and two others are not.



Recently, several human FucTs have been cloned and partially characterized(16, 17, 18, 19, 20, 21, 22, 23) . Based on the available information, three cloned enzymes have been correlated with the enzymes earlier detected in human tissues. These are FucT-III, Lewis(16) , FucT-IV, myeloid(12, 17, 18) , and FucT-VI, plasma(24) . An extensive evaluation has been performed concerning the acceptor specificity of full-length and protein A chimeric forms which are missing the transmembrane domain and portions of the stem region of the enzyme for FucT-III, -IV, and -V (15) . These studies demonstrated that the Protein A chimeras retain the acceptor specificity properties of their full-length counterparts. Therefore, we have begun an investigation of other properties of these chimeric proteins. In the present study we have investigated whether NEM can inactivate these proteins and whether either substrate can block the chemical modification that leads to enzyme inactivation. The results with NEM treatment are in agreement with previous analyses of partially purified forms of FucT-III and -IV from natural sources. Thus, FucT-III was inactivated by NEM whereas FucT-IV was not. In addition, we found that FucT-V is inactivated by NEM. Interestingly, GDP-fucose, but not the acceptor substrate, provided substantial protection to FucT-III and -V from NEM inactivation. Based on the NEM inactivation results and sequence comparisons of Cys residues, we hypothesized that a single sequence difference (i.e. Cys to Ser) in FucT-III and -V versus FucT-IV accounts for the difference in NEM sensitivity of these enzymes. Site-directed mutagenesis was used to test this prediction. Enzymatic analyses of the FucT-IV mutant (SerCys) demonstrated that the single amino acid change produced an NEM-sensitive FucT-IV.


EXPERIMENTAL PROCEDURES

Materials

N-Acetyllactosamine (LacNAc), UDP-galactose, fucose, and GDP were obtained from Sigma. The type 2 H-oligosaccharide 8-methoxycarbonyloctyl glycoside was provided by Dr. Ole Hindsgaul, Edmonton, Alberta. GDP-[^14C]fucose (283 mCi/mmol) and [S]dATP were obtained from DuPont NEN. Unlabeled GDP-fucose was prepared by the method of Ginsburg(25) . DNA sequencing was done using the Sequenase Version 2.0 DNA sequencing kit obtained from United States Biochemical Corp., Cleveland, OH. All other reagents were of the highest purity commercially available.

Enzymes

All FucTs were expressed as Protein A fusion proteins. The following constructs were used: FucT-III containing amino acids 52-361; FucT-IV containing amino acids 58-405; FucT-V containing amino acids 65-374. The procedures used to obtain the fusion proteins have been described in detail(26) . Briefly, plasmids (pPROTA) containing the coding regions described above were transfected into COS-7 cells, and the secreted fusion proteins were purified using IgG-agarose beads. The beads were washed with PBS (8.1 mM Na(2)HPO(4), 1.5 mM KH(2)PO(4), 137 mM NaCl, 2.7 mM KCl, pH 7.4) and used as a source of enzyme. The cDNAs encoding these enzyme constructs have been completely sequenced and confirmed to be identical to previously published alpha13fucosyltransferase sequences(15) .

Methods

alpha13Fucosyltransferase Assays

alpha13Fucosyltransferase enzyme activities were determined in reaction mixtures composed of 1 µmol of HEPES buffer, pH 7.2, 6 nmol of GDP-[^14C]fucose (12,000 counts/min/nmol), 0.4 µmol of LacNAc, 2 µmol of NaCl, 0.125 µmol of MnCl(2), 10 µg of bovine serum albumin, 0.01 µmol of 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 37 °C and stopped by the addition of 180 µl of deionized water and immediately placed onto a 0.2-ml resin bed volume column of Dowex-1X8 formate form (200-400 mesh) which had previously been extensively washed with deionized water. The reaction tube was washed with 200 µl of deionized water and added to the column followed by a final wash of the column by an additional 600 µl of deionized water. The combined passthrough and washes of the Dowex-1 column, total volume of 1 ml, was vortexed and an aliquot, 0.5 ml, was counted in a liquid scintillation counter. In each case a control assay was conducted wherein LacNAc was excluded from the reaction mixture to serve as a blank.

Enzyme assays using type 2 H-oligosaccharide 8-methoxycarbonyloctyl glycoside as an acceptor were conducted as described previously(27) .

Treatment of Chimeric alpha13Fucosyltransferase Enzymes with Sulfhydral Reagents

DTE Treatment

Chimeric enzyme bound to IgG-agarose beads was suspended in a buffer composed of 50 mM HEPES buffer, pH 7.2, 150 mM NaCl and was supplemented with DTE to yield final concentrations varying from 0 to 5 mM. The treatment was conducted for 30 min at room temperature prior to assay to determine the effect on enzyme activity.

DTNB Treatment

Chimeric enzyme bound to IgG-agarose beads was suspended in a buffer composed of 50 mM HEPES buffer, pH 7.2, 150 mM NaCl, 1 mM DTE. Duplicate treatments of enzyme supplemented with DTNB final concentrations varying from 0 to 4 mM were conducted for 30 min at room temperature. The final volume of each treatment was 9 µl. At the end of this period 1 µl of 0.5 M DTE was added to one of the duplicate treatments for each concentration of DTNB, and 1 µl of water was added to the other. These samples were then allowed to stand at room temperature for 10 min. The activity present in each enzyme treatment was then determined.

NEM Treatment

The effect of NEM on enzyme activity was determined using aliquots of the beaded chimeric enzyme incubated in treatments containing 0.5 mmol of HEPES buffer, pH 7.2, 0.15 mmol of NaCl, 0.05 mmol of MnCl(2), 4 nmol of DTE, and varying amounts of NEM in a total volume of 16 µl. The treatment was conducted for 30 min at room temperature and stopped by the addition of 1 µl of 0.5 M DTE prior to assay of the remaining activity. In some experiments the inactivation reaction mixture was supplemented with varying concentrations of possible protecting agents, UDP-galactose, GDP, fucose, LacNAc, or GDP-fucose. In incubations using either acceptor LacNAc or the donor GDP-fucose, the amount of each substrate in the final assay reaction mixture was adjusted to account for what was present in the enzyme aliquot.

Site-directed Mutagenesis of FucT-IV

A FucT-IV mutant containing SerCys was prepared by in vitro site-directed mutagenesis of concatamerized FucT-IV DNA constructs as described previously in detail(28) . The primers used to create the single amino acid mutation were AGTCGCCCTCGCACTGCCC (upstream primer corresponding to nucleotides 518-536 of FucT-IV, with a substitution of C for G at nucleotide 533) and CGAAGTTCATCCAAACCCAGCG (downstream primer corresponding to the reverse complement of nucleotides 517-496 of FucT-IV). The final PCR product was digested with EcoRI and ligated into the vector pPROTA.

The nucleotide base replacement in FucT-IV was confirmed by DNA sequence analysis. The DNA sequence was determined by the dideoxynucleotide chain termination method(29) . Sequence was obtained from the FucT-IV mutant (pPROTA-1) clone, spanning nucleotides 487-628 (the region containing the putative mutation site), on both strands and from the FucT-IV long form wild type clone, spanning nucleotides 455-642, on the forward 5`3` strand only, for comparison.

Primers were made on a Beckman 1000 Oligo Synthesizer (forward primer: 5`-ACT GCC GAG GAG GTG GAT CTG-3`, 21-mer, 5` position 400; and reverse primer: 5`-TTT CCT GGA CAG TGG CGG GG-3`, 20-mer, 3` position 665). Five pmol of primer was annealed to 5 µg of alkaline-denatured double-stranded template and labeled according to standard procedures.


RESULTS

Effect of Sulfhydral Reagents on alpha13Fucosyltransferase Activity

The effect of addition of DTE on activity of a recombinant chimeric alpha13fucosyltransferase enzyme was tested. The enzyme was composed of Protein A fused to the catalytic domain of human FucT-V and bound to IgG-agarose beads. As shown in Fig. 1, preincubation of the beaded enzyme with increasing concentrations of DTE resulted in activation of the enzyme which reached a plateau corresponding to a 2.5-fold increase in activity over the untreated enzyme. The results suggest an essential sulfhydral is present in this enzyme and that it was partially oxidized in the isolated enzyme. Based upon this information, enzymes were routinely supplemented with 1 mM DTE to stabilize and maintain maximal enzyme activity.


Figure 1: Effect of increasing DTE concentrations on chimeric FucT-V activity. Chimeric FucT-V bound to IgG-agarose beads was preincubated with the indicated final concentration of DTE for 30 min at room temperature prior to assay of activity. alpha13Fucosyltransferase assays were conducted as described under ``Experimental Procedures.''



Reversible Inactivation of Chimeric FucT-V with DTNB

To confirm the reversible nature of oxidation of this site, the effect of increasing concentrations of DTNB on enzyme activity was tested. DTNB is a modifier of protein sulfhydral groups which can be reversed in the presence of excess sulfhydral reagent. In this experiment duplicate reaction mixtures containing the enzyme were incubated in the presence of increasing concentrations of DTNB followed by a subsequent addition of excess DTE or an equal volume of water. The activity remaining was then determined. The results shown in Fig. 2indicate that DTNB effectively inactivated the enzyme yielding over 90% inactivation at 0.5 mM DTNB. Addition of excess DTE prior to assay resulted in about a 75% recovery of activity at all concentrations of DTNB tested indicating that enzyme activity correlates with the reversible oxidation of this protein group.


Figure 2: Reversible inhibition of chimeric FucT-V by DTNB. The chimeric FucT-V enzyme on IgG-agarose beads was incubated for 30 min at room temperature in the presence of the indicated final concentration of DTNB. Duplicate samples were treated with DTNB, one was made 50 mM in DTE (times), and the other was supplemented with the equivalent volume of water (bullet). After a 10-min incubation at room temperature, the activity remaining in each reaction mixture was determined. Control activity in the absence of DTNB corresponded to transfer of 312 pmol/h.



Inactivation of Chimeric FucT-III and FucT-V Enzymes by NEM

The effect of NEM treatment on activity of the chimeric FucT-III and FucT-V enzymes bound to IgG-agarose beads was tested as shown in Fig. 3. In both cases the enzyme was efficiently inactivated as the concentration of NEM was increased. Maximal loss of enzyme activity occurred at final NEM concentrations above 3 mM. In contrast to the results with DTNB, it was not possible to restore activity with excess DTE, consistent with the nature of the irreversible modification which results from NEM treatment. These results are similar to those previously reported for native, full-length Lewis enzyme(8, 9, 10) .


Figure 3: Inactivation of chimeric FucT-III and FucT-V by NEM. The enzymes were treated with the indicated final concentrations of NEM as described under ``Experimental Procedures'' prior to assay for remaining activity. Panel A, FucT-III; panel B, FucT-V. Control activity in the absence of NEM was 139 pmol/h for FucT-III and 127 pmol/h for FucT-V.



Protection of FucT-III and FucT-V Chimeric Enzymes by Substrates from Inactivation by NEM

Protection experiments were conducted in the presence 3 mM NEM containing increasing concentrations of reaction substrates or analogs. The results of these experiments are shown in Fig. 4. Both FucT-III and FucT-V were effectively protected from inactivation by increasing concentrations of the donor GDP-fucose. In these experiments the enzymes were 70-80% inactivated by NEM alone. Increasing amounts of GDP-fucose protected 75-80% of the original activity. In experiments in which NEM alone reduced enzyme activity to about 50%, there was almost complete protection of the enzyme by GDP-fucose concentrations greater than or equal to 300 µM (results not shown). Protection was also observed with GDP, although to a lesser extent, about 50-60% of the original activity. This may be due to reduced affinity of both enzymes for GDP compared to GDP-fucose, or result from inhibition in the assay since in this experiment the GDP was not removed from the enzyme source prior to the enzyme assay. In contrast to the results for GDP and GDP-fucose, no protection was observed with increasing concentrations of the sugar nucleotide UDP-galactose, fucose, or with the acceptor LacNAc. Thus, the results suggest that NEM inactivation is due to reaction with a catalytically essential sulfhydral group on the enzyme that is associated with binding the donor substrate GDP-fucose. Further, the observation that protection is also observed with GDP but not fucose suggests that this group may be associated with binding of the nucleotide portion of the molecule.


Figure 4: Protection of chimeric FucT-III and FucT-V toward inactivation by NEM. The experiment was conducted as described under ``Experimental Procedures'' using varying concentrations of nucleotides GDP-fucose (bullet), GDP (circle), UDP-galactose (times), or saccharides LacNAc (+), and fucose (). Panel A, FucT-III; panel B, FucT-V. Control activity in the absence of NEM was 141 and 596 pmol/h for FucT-III and FucT-V, respectively.



Analysis of the Distribution of Cys Residues in Human alpha13Fucosyltransferases and Site-directed Mutagenesis of Chimeric FucT-IV

Human fucosyltransferases contain 6-8 Cys residues depending on enzyme form. These are generally enriched in the N-terminal portion of the enzyme, a region of the enzyme with considerable sequence heterogeneity among enzyme forms. Fig. 5shows the distribution of Cys residues in portions of human FucTs similar to protein regions present in the chimeric enzymes used in this study. There is a considerable degree of homology in the distribution of Cys residues in the aligned sequences shown. Slight differences are seen in the more N-terminal portions of the alignments. The exact alignment of Cys in FucT-IV and -VII near the N-terminal region is difficult to assign due to the substantial sequence heterogeneity in this region. Examination of the aligned sequences indicates that one site, containing conserved Cys residues in FucT-III, -V, and -VI (enzymes subject to NEM inactivation), is replaced by a Ser in the NEM-insensitive FucT-IV. These residues are Cys in FucT-III, Cys in FucT-V, Cys in FucT-VI, and Ser in FucT-IV. This site is in a region of all enzyme forms with few Cys residues (i.e. in a region between Cys and Cys of FucT-IV). Interestingly, the corresponding site in FucT-VII is Thr. Thus, there is a highly conservative amino acid substitution from a CysSer at this site in FucT-IV which correlates with the status of NEM sensitivity. This has led us to postulate that the corresponding site on FucT-III, -V, and -VI is associated with the observed NEM inactivation of these enzyme forms.


Figure 5: Alignment of sequences of cloned human alpha13fucosyltransferases showing distribution of Cys residues. Dashes shown in the figure represent either amino acids other than Cys or insertions to allow best sequence alignment. The sequences shown start at the following residues for each enzyme: FucT-III (56), -IV (58), -V (67), -VI (53), and -VII (58). The residue numbers of Cys residues are shown in each enzyme. One site representing Cys of FucT-III, Cys of FucT-V, and Cys of FucT-VI has highly conservative amino acid substitutions with Ser of FucT-IV and Thr of FucT-VII. These conservative substitutions are also shown.



One way to test this hypothesis is to perform site-directed mutagenesis on FucT-IV to replace Ser with a Cys residue and determine if the enzyme retains activity and if so, whether the enzyme has become NEM-sensitive. Fig. 6shows a portion of the nucleotide sequence of the chimeric FucT-IV construct corresponding to an area of the full-length FucT-IV coding sequence between nucleotides about 500 and 575 which spans the coding sequence for Ser. Site-directed mutagenesis was employed to replace deoxycytosine at position 533 with deoxyguanosine. DNA sequencing confirmed that the only difference between the native FucT-IV and the FucT-IV mutant was a single base change (transversion) at nucleotide 533, resulting in an amino acid change from serine (TCC) to cysteine (TGC) at this position. This mutation was clearly observed on both strands in the FucT-IV mutant. No other nucleotide substitutions in the coding sequence for this enzyme were detected.


Figure 6: DNA sequence comparison of native and mutant FucT-IV enzyme coding sequences. A PCR site-directed mutagenesis experiment was conducted to replace SerCys in FucT-IV. A portion of the nucleotide sequence corresponding to the coding sequence of the full-length FucT-IV from approximately nucleotide 500-575 is shown. The arrow indicates the replacement of a deoxycytosine in the native coding sequence with deoxyguanosine in the mutant construct at nucleotide 533. The mutation was confirmed on both strands in the FucT-IV mutant. Sequence from the forward strand only on both templates is shown.



Effect of NEM on Chimeric FucT-IV Cys Mutant Enzyme

The FucT-IV Cys mutant enzyme construct was expressed in COS cells and the secreted enzyme purified by binding to IgG-agarose beads. Initial tests indicated that the mutant enzyme retained activity with acceptors LacNAc and Fucalpha12Galbeta14GlcNAcbeta-O-(CH(2))(8)-COOCH(3). Consequently, its sensitivity toward NEM inactivation was tested as shown in Fig. 7. The mutant enzyme was efficiently inactivated by NEM as its concentration was increased. In contrast, the native FucT-IV enzyme was confirmed to be insensitive to NEM treatment. The mutant FucT-IV enzyme was, in fact, highly sensitive to NEM inactivation. Concentrations of NEM exceeding the molar amounts of exogenously added sulfhydral groups from DTE by as little as 0.1 mM final concentration resulted in essentially complete inactivation of the mutant enzyme.


Figure 7: Inactivation of native and Cys mutant chimeric FucT-IV by NEM. The enzymes were treated with the indicated final concentrations of NEM as described under ``Experimental Procedures'' prior to assay for remaining activity. bullet, mutant FT-IV; times, native FT-IV. Control activity in the absence of NEM was 196 pmol/h for native FucT-IV and 372 pmol/h for Cys mutant FucT-IV.



Substrate Protection of Mutant FucT-IV from NEM Inactivation

Fig. 8shows the effect of increasing concentrations of GDP-fucose and LacNAc on inactivation of the FucT-IV Cys mutant enzyme by NEM. In this experiment a final NEM concentration of 0.625 mM was used in reaction mixtures which contained enzyme with exogenous sulfhydral groups from DTE (added to stabilize the enzyme) at a final concentration of 0.5 mM. Moderate protection of enzyme activity up to about 25% of the original activity was observed under these conditions as the GDP-fucose concentration was increased up to 480 µM. In contrast, no protection was observed with LacNAc up to 40 mM final concentration. These results are very similar to those found with FucT-III and FucT-V (Fig. 4) except that the level of GDP-fucose protection was much less for the FucT-IV mutant enzyme. Despite the differences in the relative amounts of protection by GDP-fucose for each of these enzymes, the inherent capability of GDP-fucose to afford protection of the mutant FucT-IV enzyme toward NEM inactivation indicates that these are similar functional sites in the differing enzyme forms. The quantitative differences in the amount of GDP-fucose protection could possibly be explained by a higher NEM reactivity of the Cys residue in the mutant FucT-IV compared to the analogous sites in FucT-III and FucT-V. This is consistent with protection studies of FucT-V conducted in the presence of higher final concentrations of NEM (4 mM) which yielded very similar degrees of enzyme protection observed for the FucT-IV mutant enzyme (results not shown). In addition, weaker affinity of GDP-fucose binding to the mutant enzyme compared to the other enzyme forms may also be responsible for the differing protection results obtained.


Figure 8: Protection of Cys mutant chimeric FucT-IV toward inactivation by NEM. The experiment was conducted as described under ``Experimental Procedures'' using varying concentrations of GDP-fucose (bullet) and LacNAc (times). Control activity in the absence of NEM was 358 pmol/h.



Table 2shows a comparison of the K(m) values for GDP-fucose for the native and mutant chimeric FucT enzymes utilized in this study. The K(m) for GDP-fucose using 40 mM LacNAc as the acceptor was tested with the FucT-IV mutant and determined to be 28 ± 3 µM. This is in contrast to a K(m) of 9 ± 2 µM for the normal chimeric FucT-III, -IV, and -V enzymes used in this study(15) . The 3-fold higher K(m) for GDP-fucose of the mutant FucT-IV enzyme indicates a substantially lower affinity for the substrate compared to the other enzymes. Thus, the weaker protection provided by GDP-fucose is most likely a difference in GDP-fucose binding affinity of the mutant FucT-IV enzyme.




DISCUSSION

Multiple, distinct alpha13fucosyltransferases exist in the human genome. Five of these forms have been cloned(16, 17, 18, 19, 20, 21, 22, 23) . It has been known for several years that some enzyme forms can be inactivated by NEM indicating the presence of one or more catalytically essential sulfhydral groups in the enzyme(8, 9, 10) . Other enzyme forms, e.g. FT-IV and the enzyme from NCI-H69 cells(13) , are insensitive toward inactivation by sulfhydral group modifying reagents. Until now this observation has only been used as a criteria for distinguishing alpha13fucosyltransferase enzymes. The results presented in this paper extend this observation to show that NEM inactivation is due to modification of a GDP-fucose-protected site on the enzyme. The results further indicate that modification of this site is reversible indicating the free sulfhydral group on the enzyme is required for activity.

The enzymes used in this study were recombinant chimeric enzyme forms composed of Protein A fused to the catalytic domain of the enzyme. These enzyme forms retain acceptor specificity properties of the full-length enzyme(15) . The results indicate that similar NEM concentrations were effective in inactivating the appropriate enzyme forms as was described previously for native, full-length enzymes (8, 9, 10) . Further, FucT-IV which is NEM resistant as a full-length enzyme remained so as a fusion protein. Thus, the NEM-sensitive site is associated with the catalytic domain and not the transmembrane or stem regions of the protein.

Analysis of the distribution of Cys residues in aligned sequences of the cloned alpha13fucosyltransferase enzymes indicates considerable homology of its distribution, particularly with FucT-III, -V, and -VI enzyme forms which are all sensitive to NEM inactivation. This is consistent with the localization of these three forms to chromosome 19 (19, 20, 30) and the close proximity of FucT-III and FucT-VI which map only 13 kilobases apart(31) . Their high homology, similar properties, and chromosomal localization are consistent with their formation by gene duplication. Careful examination of the distribution of Cys residues between enzyme forms indicates one site, Cys of FucT-III or Cys of FucT-V, corresponds to a highly conservative replacement of Ser in the NEM-insensitive FucT-IV enzyme. This site in FucT-IV was chosen for site-directed mutagenesis to replace the Ser with Cys. Analysis of the mutant FucT-IV enzyme indicated that not only did it retain activity, it had also acquired a GDP-fucose protectable NEM sensitivity confirming the importance of this site in the protein. Thus, this site in the aligned sequences of these enzymes is most probably associated with either binding of the nucleotide donor GDP-fucose or it participates in transfer of the fucose moiety to the acceptor carbohydrate. It is also of interest that the newly cloned FucT-VII, which is highly specific for sialylated acceptors, has a Thr residue in the corresponding site, Thr(22, 23) . Although no data are presently available to answer the question, it can be anticipated that FucT-VII will prove to be insensitive to sulfhydral reagents such as NEM.

An alternate perspective of these results suggests that replacement of Ser with a Cys residue in FucT-IV results in formation of an enzyme that coincidentally is inactivated by NEM but in a way independent of GDP-fucose protection. This is based upon the proportionately lower extent of GDP-fucose protection of the mutant FucT-IV enzyme (Fig. 8) compared to either FucT-III or -V (Fig. 4). However, GDP-fucose has the inherent capability of providing protection of the mutant FucT-IV enzyme. Further, the extent of protection observed for FucT-V is dependent on the NEM concentration used. These results, and the homology of the Cys residues in these enzymes, strongly suggest that the only reasonable conclusion is that this site in these enzymes is associated with GDP-fucose binding or catalysis. Direct chemical analysis of the amino acid sequence surrounding NEM-modified Cys residues associated with inactivation of FucT-III and -V will be required to provide an absolute answer. These experiments are being pursued.

Despite the availability of cloned enzymes and significant amounts of sequence data for human alpha13fucosyltransferases, there are little data presently available to define protein sites which are involved in substrate binding and catalysis. A recent report has presented evidence to suggest that a basic group in at least two human alpha13fucosyltransferases participates in acceptor binding and catalysis(32) . A GDP-fucose protectable, pyridoxal-5-P modifiable Lys residue has been shown to be present in the alpha13fucosyltransferase from human lung small cell carcinoma NCI-H69 cells(33) . Further evidence indicates that a Lys group with this property is also present in FucT-III, -IV, and -V. (^2)It is necessary to obtain further information of this type on this family of enzymes to provide an understanding of protein function, what mediates acceptor specificity, and why so many similar enzymes are encoded in the human genome.

Recent progress has been made in describing elements of the Lewis FucT-III enzyme which are required for activity(34, 35, 36, 44) . These studies have focused on a basis for understanding the Lewis negative phenotype and have identified specific amino acid replacements which occur in the catalytic domain of the enzyme and result in loss of activity. No information is available to date to describe the potential importance of these amino acids and protein segments in substrate binding or catalysis. It should be possible to gain considerable information through a systematic analysis of these protein sites using a strategy similar to that employed in this study (i.e. the combined use of chemical modification and molecular biological analyses).

There are a few examples of progress in understanding protein groups involved in catalysis of glycosyltransferase enzymes. Amino acid substitutions in the histo-blood group A and B transferase enzymes have been shown to define sugar nucleotide donor specificity of UDP-GalNAc versus UDP-galactose(37) . More progress has been made in studies of beta14galactosyltransferase(38, 39, 40, 41, 42, 43) . For example, a region of the enzyme between Lys and Lys was suggested to be involved in UDP-galactose binding(38) . Further, an analogous finding to that reported in the present paper indicated binding of UDP derivatives protected a single Cys residue in beta14galactosyltransferase from modification by thiol group-specific reagents(40, 41, 42) . It has been suggested that Cys is this essential residue associated with UDP-galactose binding(43) . This is in the same region as essential Lys residues involved in UDP-galactose binding. Thus, the Cys and Lys residues identified as important in UDP-galactose binding are nearby in the linear sequence of the enzyme. The proximity of the essential Cys residue reported in this paper to Lys residues also involved in GDP-fucose binding (33) for human alpha13fucosyltransferases is unknown, and it will require further study to understand their relationship.

The results presented in this paper were obtained by a combination of classic biochemical analyses to identify protein functional groups with a molecular biological approach to study specific protein sites. Given the large amount of protein sequence data available for the family of human alpha13fucosyltransferases, this multidisciplinary approach will be utilized further to identify and analyze other protein sites involved in substrate binding and catalysis. These studies will likely have broad benefit in understanding functional aspects of glycosyltransferases, in general, and alpha13fucosyltransferases in particular.


FOOTNOTES

*
This investigation was supported by Grant CA41521 (to E. H. H.) and Grant CA32826 (to B. A. M.) from the National Cancer Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Pacific Northwest Research Foundation, 720 Broadway, Seattle, WA 98122.

Present address: Protein Design Labs, Inc., 2375 Garcia Ave., Mountain View, CA 94043.

(^1)
The abbreviations used are: FucTs, alpha13fucosyltransferases; PBS, phosphate-buffered saline, DTE, dithioerythritol; DTNB, 5,5`-dithiobis(2-nitrobenzoic acid); NEM, N-ethylmaleimide; LacNAc, Galbeta14GlcNAc; type 2 H-oligosaccharide, Fucalpha12Galbeta14GlcNAc.

(^2)
E. H. Holmes and B. A. Macher, unpublished observations.


REFERENCES

  1. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hakomori, S. (1984) Annu. Rev. Immunol. 2, 103-126 [CrossRef][Medline] [Order article via Infotrieve]
  3. Hakomori, S. (1989) Adv. Cancer Res. 52, 257-331 [Medline] [Order article via Infotrieve]
  4. Alhadeff, J. A. (1989) CRC Crit. Rev. Oncol. Hematol.9, 37-107
  5. Rosen, S. D., and Bertozzi, C. R. (1994) Curr. Opin. Cell Biol. 6, 663-673 [Medline] [Order article via Infotrieve]
  6. Macher, B. A., Holmes, E. H., Swiedler, S. J., Stults, C. L. M., and Srnka, C. A. (1991) Glycobiology 1, 577-584 [Medline] [Order article via Infotrieve]
  7. de Vries, T., and van den Eijnden, D. H. (1992) Histochem. J. 24, 761-770 [Medline] [Order article via Infotrieve]
  8. Mollicone, R., Gibaud, A., Francois, A., Ratcliffe, M., and Oriol, R. (1990) Eur. J. Biochem. 191, 169-176 [Abstract]
  9. Stroup, G. B., Anumula, K. R., Kline, T. F., and Caltabiano, M. M. (1990) Cancer Res. 50, 6787-6792 [Abstract]
  10. Prieels, J.-P., Monnom, D., Dolmans, M., Beyer, T. A., and Hill, R. L. (1981) J. Biol. Chem. 256, 10456-10463 [Abstract/Free Full Text]
  11. Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622 [Abstract/Free Full Text]
  12. Kumar, R., Potvin, B., Muller, W. A., and Stanley, P. (1991) J. Biol. Chem. 266, 21777-21783 [Abstract/Free Full Text]
  13. Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627 [Abstract/Free Full Text]
  14. Foster, C. S., Gillies, D. R. B., and Glick, M. C. (1991) J. Biol. Chem. 266, 3526-3531 [Abstract/Free Full Text]
  15. de Vries, T., Srnka, C. A., Palcic, M. M., Swiedler, S. J., van den Eijnden, D. H., and Macher, B. A. (1995) J. Biol. Chem ., in press
  16. Kukowska-Latallo, J., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes &Dev 4, 1288-1303
  17. Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356 [Medline] [Order article via Infotrieve]
  18. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R. J., and Ernst, L. K. (1991) J. Biol. Chem. 266, 17467-17477 [Abstract/Free Full Text]
  19. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992) J. Biol. Chem. 267, 4152-4160 [Abstract/Free Full Text]
  20. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem. 267, 24575-24584 [Abstract/Free Full Text]
  21. Koszdin, K. L., and Bowen, B. R. (1992) Biochem. Biophys. Res. Commun. 187, 152-157 [Medline] [Order article via Infotrieve]
  22. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730-14737 [Abstract/Free Full Text]
  23. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269, 16789-16794 [Abstract/Free Full Text]
  24. Mollicone, R., Reguigne, I., Fletcher, A., Aziz, A., Rustam, M., Weston, B. W., Kelly, R. J., Lowe, J. B., and Oriol, R. (1994) J. Biol. Chem. 269, 12662-12671 [Abstract/Free Full Text]
  25. Ginsburg, V. (1966) Methods Enzymol. 8, 293-295
  26. Henion, T. R., Macher, B. A., Anaraki, F., and Galili, U. (1994) Glycobiology 4, 193-202 [Abstract]
  27. Palcic, M. M., Heerze, L. D., Pierce, M., and Hindsgaul, O. (1988) Glycoconjugate J. 5, 49-63
  28. Heda, D. H., Henion, T. R., and Galili, U. (1992) Nucleic Acids Res. 20, 5241-5242 [Medline] [Order article via Infotrieve]
  29. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  30. Ball, S. P., Tongue, N., Gibaud, A., LePendu, J., Mollicone, R., Gerard, G., and Oriol, R. (1991) Ann. Hum. Genet.55, 225-233 [Medline] [Order article via Infotrieve]
  31. Nishihara, S., Nakazato, M., Kudo, T., Kimura, H., Ando, T., and Narimatsu, H. (1993) Biochem. Biophys. Res. Commun. 190, 42-46 [CrossRef][Medline] [Order article via Infotrieve]
  32. Hindsgaul, O., Kaur, K. J., Srivastava, G., Blaszczyk-Thurin, M., Crawley, S. C., Heerze, L. D., and Palcic, M. M. (1991) J. Biol. Chem. 266, 17858-17862 [Abstract/Free Full Text]
  33. Holmes, E. H. (1992) Arch. Biochem. Biophys. 296, 562-568 [Medline] [Order article via Infotrieve]
  34. Nishihara, S., Yazawa, S., Iwasaki, H., Nakazato, M., Kudo, T., Ando, T., and Narimatsu, H. (1993) Biochem. Biophys. Res. Commun. 196, 624-631 [CrossRef][Medline] [Order article via Infotrieve]
  35. Koda, Y., Kimura, H., and Mekada, E. (1993) Blood 82, 2915-2919 [Abstract]
  36. Mollicone, R., Reguigne, I., Kelly, R. J., Fletcher, A., Watt, J., Chatfield, S., Aziz, A., Cameron, H. S., Weston, B. W., Lowe, J. B., and Oriol, R. (1994) J. Biol. Chem. 269, 12662-12671 [Abstract/Free Full Text]
  37. Yamamoto, F., and Hakomori, S. (1990) J. Biol. Chem. 265, 19257-19262 [Abstract/Free Full Text]
  38. Yadav, S., and Brew, K. (1990) J. Biol. Chem. 265, 14163-14169 [Abstract/Free Full Text]
  39. Aoki, D., Lee, N., Yamaguchi, N., Dubois, C., and Fukuda, M. N. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4319-4323 [Abstract/Free Full Text]
  40. Kitchen, B. J., and Andrews, P. (1974) Biochem. J.141, 173-178 [Medline] [Order article via Infotrieve]
  41. O'Keeffe, E. T., Hill, R. L., and Bell, J. E. (1980) Biochemistry 19, 4954-4962 [Medline] [Order article via Infotrieve]
  42. Magee, S. C., and Ebner, K. E. (1974) J. Biol. Chem. 249, 6992-6998 [Abstract/Free Full Text]
  43. Yadav, S. P., and Brew, K. (1991) J. Biol. Chem. 266, 698-703 [Abstract/Free Full Text]
  44. Nishihara, S., Narimatsu, H., Iwasaki, H., Yazawa, S., Akamatsu, S., Ando, T., Seno, T., and Narimatsu, I. (1994) J. Biol. Chem. 269, 29271-29278 [Abstract/Free Full Text]

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