(Received for publication, June 28, 1995; and in revised form, January 12, 1996)
From the
A series of molecular biology experiments were carried out to
identify the catalytic domain of two human
1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V),
and to identify amino acids that function in acceptor substrate
binding. Sixty-one and 75 amino acids could be eliminated from the N
terminus of FucTs III and V, respectively, without a significant loss
of enzyme activity. In contrast, the truncation of one or more amino
acids from the C terminus of FucT V resulted in a dramatic or total
loss of enzyme activity. Results from the truncation experiments
demonstrate that FucT III
(containing amino
acids 62-361) and FucT V
(containing amino
acids 76-374) are active, whereas shorter forms of the enzymes
were inactive. The shortest, active forms of the enzymes are more than
93% identical at the predicted amino acid level, but have distinct
acceptor substrate specificities. Thus, FucT III is an
1,4-fucosyltransferase, whereas FucT V is an
1,3-fucosyltransferase with disaccharide substrates. All but one
of the amino acid sequence differences between the two proteins occur
near their N terminus. Results obtained from domain swapping
experiments demonstrated that the single amino acid sequence difference
near the C terminus of these enzymes did not alter the enzyme's
substrate specificity. However, swapping a region near the N terminus
of the truncated form of FucT III into an homologous region in FucT V
produced a protein with both
1,3- and
1,4-fucosyltransferase
activity. This region contains 8 of the amino acid sequence differences
that occur between the two proteins.
1,3/4-Fucosyltransferases (FucTs) (
)catalyze the
final step in the synthesis of a wide spectrum of fucosylated
glycoconjugates including those that function in leukocyte
adhesion(1, 2) . Cell type-specific and developmental
antigens are also products of these enzymes, as are tumor-associated
antigens. These proteins are members of a large group of proteins (i.e. glycosyltransferases) that are responsible for the
synthesis of glycolipids, glycoproteins, and proteoglycans (for a
recent review, see (3) ). Glycosyltransferases are type II
membrane proteins with four recognized protein domains: cytoplasmic,
transmembrane, stem, and catalytic(4, 5) . The
presence of a stem region in these proteins is based on the observation
that soluble, catalytically active forms occur naturally and are formed
by proteolytic cleavage of the full-length enzyme in what is thought to
be an extended protein domain (i.e. the stem region). The
catalytic region is proposed to be a globular domain that contains the
active site of the enzyme. Recently, we (6) have utilized a PCR
based approach to determine which portions of the N- and C-terminal
domains of murine and marmoset
1,3-galactosyltransferases can be
eliminated without loss of activity (i.e. catalytic domain).
The location of the catalytic domain of other glycosyltransferases is
unknown. In this report, the location of the catalytic domain of two
members of the human FucTs, FucT III and V, is presented. These two
proteins were chosen for analysis because they share a high level of
amino acid sequence similarity and yet have distinct acceptor substrate
specificity(7, 8) . As illustrated in Fig. 1,
there are only 30 amino acid differences between the full-length
sequences of these two proteins. In addition to these sequence
variations, FucT V contains two regions not found in FucT III (shown as
dashes in the FucT III sequence). A majority of the amino acid sequence
differences between FucT III and V occur at the N terminus of these
enzymes, with only one amino acid difference occurring over the last
200 amino acids. Therefore, precisely locating the catalytic domain
should also identify the number of amino acid residues that account for
the distinct acceptor substrate specificities of FucT III and V. The
results presented in this study provide a precise location of the
catalytic domains of FucT III and V and demonstrate there are less than
25 differences in the amino acid sequences of the catalytic domain of
the two enzymes. Portions of the N-terminal domain from the truncated
enzymes have been swapped producing chimeric proteins that were
analyzed for acceptor specificity. These analyses demonstrated that the
distinct acceptor specificity of the FucTs results from as few as 8
amino acid sequence differences.
Figure 1: A comparison of the predicted amino acid sequences of FucTs III and V. Sequences are those previously reported(7, 8) .
The results presented in Table 2demonstrate that the
truncated forms of FucT V with 299 amino acids (aa 76-374)
retained activity; shorter forms were inactive. The studies shown in Table 2were done with a disaccharide (type 2) acceptor which is
known to be a poorer acceptor than the corresponding H-type structure.
To rule out the possibility that the inactive proteins had simply lost
the ability to utilize a single acceptor, each chimeric protein
identified as inactive with the disaccharide acceptor was tested with
several acceptors (results not shown). Only one protein (i.e. FucT V with a 76-amino acid deletion at the N terminus aa
77-374) had activity with any of the acceptors. This enzyme was
minimally active (
5% as active as the longer enzyme forms) with an
H-type 2 acceptor. Finally, the other active forms of FucT V were
analyzed with a range of acceptor substrates and found to have an
acceptor substrate specificity similar to the full-length enzyme as
previously reported (9) (not shown).
Based on the FucT V
results, a more limited analysis of FucT III constructs was done. Thus,
a FucT III containing amino acids 62-361 had an activity
equivalent to that obtained with forms of the enzyme previously
characterized(9) , whereas a shorter form (aa 67-361) was
inactive. Therefore, a form of FucT III with 300 amino acids (aa
62-361) retained activity. Assays with various acceptors
demonstrated that these forms of FucT III had a substrate specificity
identical to that previously reported (7, 9) (not
shown).
The current model of the protein domain structure of glycosyltransferases indicates that the C-terminal portion of a glycosyltransferase constitutes the catalytic domain, but little is known about the importance of amino acids at the C terminus for catalytic activity. To investigate this issue, FucT V proteins were prepared which lack one or two of the C-terminal amino acids of the full-length enzyme. The results presented in Table 2demonstrate that removal of one amino acid from the C terminus of FucT V drastically alters catalytic activity. A protein missing two of the C-terminal amino acids was inactive, even when tested with an H-type 2 acceptor.
It is possible that the lack of detectable enzyme activity for the shorter FucT III and V constructs is due to the fact that the COS cells do not secrete these forms of the proteins, or that these forms are rapidly degraded. To rule out these possibilities the medium from COS cells, transfected with plasmids containing inserts encoding various FucT constructs, was mixed with IgG-agarose beads and the bound proteins were analyzed on Western blots. Fig. 2shows that the inactive constructs (Table 2) were produced and secreted into the medium. Furthermore, the relative amounts of inactive chimeric proteins were similar to that of the active proteins. Finally, these proteins appear to have the expected molecular weight and thus, inactivity does not appear to be due to proteolytic degradation.
Figure 2: Western blot analysis of protein A-FucT chimeric proteins separated by SDS-PAGE. Proteins were purified by affinity chromatography on IgG-agarose, separated by SDS-PAGE, transferred to nitrocellulose, and detected with an alkaline phosphatase detection system as described under ``Experimental Procedures.'' Lane 1, FT III(62-361); Lane 2, FT III(67-361); Lane 3, FT V(76-374); Lane 4, FT V(77-374); Lane 5, FT V(78-374); Lane 6, FT V(76-372); Lane 7, FT V(76-373).
To further evaluate the two forms of FucTs, the acceptor specificity of the non-protein A forms of FucT III and V were compared with similar protein A constructs (Table 3). Both forms of FucT III were most active with acceptors based on a type 1 core structure, whereas the FucT V constructs produced significantly more product with type 2 acceptor substrates. Although some quantitative differences were apparent, the overall acceptor specificity pattern was similar for each enzyme pair.
Figure 3:
A comparison of the predicted amino acid
sequences of the N-truncated forms of FucTs III (62-361) and V
(76-374). Sequences are based on those previously
reported(7, 8) . Underlined amino acid
residues in FucT III are those found to differ (i.e. L to F
and T to A) in the constructs described in this report compared to
those previously reported. The position of Cys and
Cys
of FucT III and V, respectively, is shown in outline
type.
Domain swapping experiments were designed to obtain a more precise location of amino acids that contribute to the acceptor specificity of FucT III and V. Fig. 4shows a diagrammatic representation of the four proteins that were constructed by the domain swaps. The first domain swap (FucT III-62-227/FucT V-241-374) was designed to produce a protein that contained all of the amino acids found in FucT III except residue 336 (Asp) which was exchanged for the residue found in FucT V (Ala). This protein was active and had an acceptor substrate specificity similar to FucT III (Table 4). The second domain swap (FucT V-76-240/FucT III-228-361) produced a protein that contained all of the amino acids found in FucT V except residue 349 (Ala) which was exchanged for the residue found in FucT III (Asp). This protein was active and had a substrate specificity similar to FucT V. Thus, the single amino acid difference that occurs at the C terminus of FucT III and V does not contribute to the acceptor specificity of these enzymes.
Figure 4: Diagrammatic representation of the four proteins produced by domain swaps. The portions of the diagram that are underlined by a single line represent FucT III sequences, whereas those underlined by the double line represent FucT V sequences. The top two constructs were designed to produce proteins which differ in only a single amino acid residue compared to their parent protein. The bottom two constructs where designed to produce proteins which contain approximately half (i.e. 8 and 12 amino acid sequence differences in regions designated 1 and 2, respectively) of the N-terminal amino acid differences occurring between FucT III and V. The lower case letters in the FucT III sequence represent the amino acid sequence differences between those originally published and those found in the FucT III sequence of the proteins analyzed in this study.
The third and forth domain swaps were designed to produce proteins which contain approximately half (i.e. 8 and 12 amino acid sequence differences in regions designated 1 and 2, respectively) of the N-terminal amino acid differences occurring between FucT III and V. The third domain swap (FucT III-62-110/FucT V-124-374) produced a protein containing 8 amino acids representative of FucT III, attached to the remaining sequence of FucT V. This protein catalyzed fucose transfer to both type 1 and type 2 acceptors and therefore, had a new and broader acceptor specificity than FucT III and FucT V. The final domain swap (FucT V-76-123/FucT III-111-361) produced a protein containing 8 amino acids representative of FucT V, attached to the remaining sequence of FucT III. This protein catalyzed the transfer of fucose to the type 1, but not the type 2 acceptor and therefore, had an acceptor specificity similar to FucT III.
Very little information is currently available about the
substrate binding and catalytic sites of glycosyltransferases. The
studies that are available in the literature have provided information
on amino acid residues that may form part of the nucleotide sugar
binding site. For example, Yadav and Brew (14) have used a
chemical modification approach to obtain evidence that regions near
Lys and Lys
of bovine
1-4
galactosyltransferase (
1-4GalT) are involved in UDP-Gal
binding. Aoki et al.(15) have obtained evidence that
Tyr
of
1-4GalT is also involved in UDP-Gal
binding. Recently, Wang et al.(16) used a
site-directed mutagenesis approach to obtain evidence that Cys
is involved in UDP-Gal binding by
1,4-galactosyltransferase.
In a study of blood group A and B transferases, Yamamoto and Hakomori (17) have demonstrated the importance of a limited number of
amino acid residues for determining the nucleotide sugar specificity (i.e. UDP-Gal versus UDP-GalNAc) of these enzymes. A
recent study by Datta and Paulson (18) has provided evidence
that some residues within the so called ``sialyl motif'' of
sialyltransferases can influence the affinity of the
2,6-sialyltransferase for CMP-sialic acid(18) . Finally,
we (19) have recently completed a study that demonstrates that
Cys
and Cys
of FucT III and V,
respectively, are in or near the binding site for GDP-Fuc. Our current
efforts are directed at more precisely defining the amino acids that
affect acceptor specificity.
The protein domain structure proposed
several years ago for glycosyltransferases indicates that the
C-terminal portion of these enzymes contains their catalytic domain.
The results we have obtained in this study demonstrate that about 20%
of the N-terminal amino acids of the FucT III and V sequences are not
required for enzyme activity and therefore, constitute the other three
recognized protein domains (i.e. cytoplasmic, transmembrane,
and stem). In contrast, truncation of the C terminus of these enzymes
results in their inactivation. We have reported similar results for two
forms of 1,3-galactosyltransferase(6) . In all of these
cases approximately 300 amino acids have been found to be required for
enzyme activity. However, this does not appear to be the minimum length
required by glycosyltransferases since forms of
1-4
galactosyltransferase missing 127 out of 400 amino acids are
active(15, 16) .
Our truncation studies also
demonstrate that the two amino acid segments that occur in FucT V which
make it 13 amino acids longer than FucT III are located between the
transmembrane domain and the catalytic domain (i.e. in the
stem region). Thus, FucT V has a stem region that is 42 amino acids
long, whereas FucT III's stem region is approximately 13 amino
acids shorter. This result is reminiscent of the observation made by
Joziasse et al.(20) that mice produce three forms of
1,3-galactosyltransferase which differ only in the length of their
stem regions.
The most important result of the truncation study was that active forms of FucT III and V only differ at 23 out of about 300 amino acid residues. Since these enzymes have distinct acceptor specificities, it allowed us to conclude that some or all of the amino acid differences occurring between the two enzymes must account for their distinct acceptor specificities. This led us to carry out a series of domain swapping experiments. The results of the domain swapping experiments allow us to conclude that: (i) the single amino acid differences at the C terminus of FucT III and V do not affect acceptor substrate specificity, (ii) a protein with either region 1 or 2 of FucT III has a type 1 acceptor specificity, (iii) the combination of region 1 of FucT III with region 2 of FucT V produces an enzyme with both type 1 and type 2 acceptor specificity, and (iv) a protein containing amino acids in region 1 of FucT V does not have a type 2 acceptor specificity, whereas one containing the 12 amino acids of region 2 of FucT V does. Taken together these results demonstrate that the amino acids in regions 1 and 2 of FucT III and V are critically involved in defining the acceptor substrate specificity of these two enzymes.
The recognition of complex oligosaccharides by proteins,
including lectins, antibodies, and enzymes is accomplished primarily
through interactions with particular hydroxyl groups on the
carbohydrate, but van der Waals interactions also occur in most
instances primarily through stacking of the underface of pyranose
residues with aromatic amino acids(21) . Many of the
interactions occur through hydrogen bonds between the sugar hydroxyls
and side chains of amino acids. In a recent study we evaluated the
ability of FucTs III and V to utilize several modified forms
(deoxygenated and containing modified amino groups on the GlcNAc
residue) of type 1 and type 2 disaccharides as acceptors. An important
result from this analysis was that both enzymes had an absolute
requirement for a hydroxyl group at carbon C-6 of galactose. The
minimum energy conformations of type 1 and 2 disaccharides have been
recognized to have different molecular topographies. Since a correctly
oriented Gal residue appears to be essential for enzyme activity (based
on the absolute requirement of its C6-hydroxyl group), the minimum
energy conformations of type 1 and 2 structures, in effect, invert the
relative orientation of the Gal and GlcNAc residues of the
disaccharides by approximately 180°. Therefore, the positions of
the NHAc and CHOH groups of the GlcNAc residue are
effectively interchanged. The present study suggests that the active
site of FucT III and V forms a pocket capable of discriminating between
OH-6 of the Gal residue and either the NHAc or CH
OH group
of the GlcNAc residue, and this differentiation is realized by
interaction with amino acids in the N-terminal domain of these enzymes.
Another interesting result from the domain swapping experiments was
that the amino acids (Asp and Ala
of FucT
III and V, respectively) that differ near the C terminus of FucTs III
and V did not alter their activity or acceptor specificity compared to
the parent enzymes. Based on a report by Nishihara et al.(22) we had anticipated that the first domain swap shown
in Fig. 4would produce an inactive protein. These workers (22) had reported that the coding region of FucT III of some
Lewis negative individuals contained a single nucleotide base change
that resulted in an Asp
Ala mutation. This would
produce a FucT III that contained a catalytic region similar to the
first domain swap protein shown in Fig. 4. Thus, we had
predicted that this domain swapped protein would be inactive. Recently,
the same research group reported that their original sequencing results
were incorrect and that the actual mutation in these Lewis negative
individuals is Ile
Lys(23) .
DNA sequencing of the FucT III and V revealed that all of the FucT V constructs had a sequence that corresponded to that previously reported for the full-length enzyme, whereas all of the FucT III constructs contained two nucleotide base differences compared to the sequence previously reported for the full-length enzyme. These differences many represent natural variations in the DNA sequence obtained from different sources of DNA. Our original template for cloning FucT III was human placental DNA, whereas the source of template for the original report on FucT III was the human tumor cell line A431(7) . Regardless of the origin, the resulting changes in the amino acid sequence for the FucT III proteins prepared for our studies did not have a major affect on enzyme activity or acceptor substrate specificity. This is in contrast to other single amino acid substitutions detected in Lewis negative individuals(22, 23, 24, 25, 26, 27) . Furthermore, the domain swap construct that had an altered acceptor substrate specificity did not contain these amino acids and thus, these amino acids do not seem to be involved in acceptor substrate recognition.
During the review of our manuscript, Legault et
al.(28) published a study which also demonstrated that a
discrete peptide fragment within 1,3/1,4-fucosyltransferases is
responsible for discriminating among different oligosaccharide acceptor
substrates. They identified a so called ``hypervariable
region'' in the fucosyltransferases that contains as few as 11
amino acids and participates in the binding of type I acceptor
substrates. This area is very near the region we have found to affect
acceptor substrate specificity. In contrast to the work presented here,
Legault et al.(28) utilized full-length constructs of
the enzymes and relied largely on cell surface staining with antibodies
to various type I and II carbohydrate epitopes to analyze the effect of
swapping different domains between fucosyltransferases. In spite of the
differences between our approach and those of Legault et
al.(28) , the conclusions drawn are similar. This adds
strength to the concept that a small peptide region at the N terminus
of the enzymes' sequence-constant C terminus is critical for
determining acceptor substrate specificity. Future studies will
determine which amino acids in this region are critical for substrate
recognition.
The results presented here offer some useful insights into the active site of glycosyltransferases. Identification of the amino acids that control acceptor substrate recognition will refine the domain structure that has defined glycosyltransferases for several years. Since several glycosyltransferases recognize either a type 1 or, more often, a type 2 acceptor it will be interesting to determine if a common set of amino acid residues can be defined among a group of glycosyltransferases that have similar acceptor substrate specificities.