(Received for publication, November 4, 1994; and in revised form, January 20, 1995)
From the
Human 1
3fucosyltransferases 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
1
3fucosyltransferases 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.
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,
1
3fucosylated 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 1
3fucosyltransferases (FucTs) (
)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.
Enzyme assays using type 2 H-oligosaccharide 8-methoxycarbonyloctyl glycoside as an acceptor were conducted as described previously(27) .
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.
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.
1
3Fucosyltransferase assays were conducted as described
under ``Experimental
Procedures.''
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 (), and the other was
supplemented with the equivalent volume of water (
). 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.
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.
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 (), GDP (
),
UDP-galactose (
), 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.
Figure 5:
Alignment of sequences of cloned human
1
3fucosyltransferases 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.
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.
, mutant FT-IV;
, 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.
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 (
) and LacNAc
(
). Control activity in the absence of NEM was 358
pmol/h.
Table 2shows a comparison of the K values for GDP-fucose for the native and mutant
chimeric FucT enzymes utilized in this study. The K
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
of 9
± 2 µM for the normal chimeric FucT-III, -IV, and
-V enzymes used in this study(15) . The 3-fold higher K
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.
Multiple, distinct 1
3fucosyltransferases 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
1
3fucosyltransferase 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 1
3fucosyltransferase
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
1
3fucosyltransferases, 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
1
3fucosyltransferases 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
1
3fucosyltransferase 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. (
)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
1
4galactosyltransferase(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
1
4galactosyltransferase 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
1
3fucosyltransferases 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 1
3fucosyltransferases, 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
1
3fucosyltransferases
in particular.