(Received for publication, March 23, 1995; and in revised form, June 26, 1995)
From the
Five different human (1,3)-fucosyltransferase
(
(1,3)-Fuc-T) genes have been cloned. Their corresponding enzymes
catalyze the formation of various
(1,3)- and
(1,4)-fucosylated cell surface oligosaccharides, including several
that mediate leukocyte-endothelial cell adhesion during inflammation.
Inhibitors of such enzymes are predicted to operate as
anti-inflammatory agents; in principle, the isolation or design of such
agents may be facilitated by identifying peptide segment(s) within
these enzymes that interact with their oligosaccharide acceptor
substrates. Little is known, however, about the structural features of
(1,3)-Fuc-Ts that dictate acceptor substrate specificity. To begin
to address this problem, we have created and functionally characterized
a series of 21 recombinant
(1,3)-Fuc-T chimeras derived from three
human
(1,3)-Fuc-Ts (Fuc-TIII, Fuc-TV, and Fuc-TVI) that maintain
shared and distinct polypeptide domains and that exhibit common as well
as idiosyncratic acceptor substrate specificities. The in vivo acceptor substrate specificities of these
(1,3)-Fuc-T
chimeras, and of their wild type progenitors, were determined by
characterizing the cell surface glycosylation phenotype determined by
these enzymes, after expressing them in a mammalian cell line
informative for the synthesis of four distinct
(1,3)- and
(1,4)-fucosylated cell surface oligosaccharides (Lewis x, sialyl
Lewis x, Lewis a, and sialyl Lewis a). Our results indicate that as few
as 11 nonidentical amino acids, found within a
``hypervariable'' peptide segment positioned at the NH
terminus of the enzymes' sequence-constant COOH-terminal
domains, determines whether or not these
(1,3)-Fuc-T can utilize
type I acceptor substrates to form Lewis a and sialyl Lewis a moieties.
Cell surface (1,3)- and
(1,4)-fucosylated
oligosaccharides have received a substantial amount of attention
because some are thought to be essential to the initiation of immune
cell adhesion to vascular endothelium during the inflammatory process
(reviewed in (1, 2, 3, 4, 5) and
6-15). This is perhaps best characterized by the
``rolling'' type of adhesion mediated by interactions between
sialyl Lewis x (sLe
)-bearing glycoconjugates on leukocytes
and P- and E-selectin expressed by activated vascular
endothelium(5) . Animal intervention studies indicate that such
fucosylated oligosaccharide molecules can act as anti-inflammatory
agents by inhibiting leukocyte-endothelial cell
interactions(15, 16, 17, 18) .
Furthermore, absence of such molecules on the leukocytes of individuals
with the rare human leukocyte adhesion II syndrome is associated with
profound defects in leukocyte-endothelial cell adhesion and with
nonpyogenic infections (19, 20) . These observations
suggest that compounds capable of specifically inhibiting leukocyte
sialyl Lewis x expression might represent candidates for
anti-inflammatory pharmacologic agents.
The
(1,3)-fucosyltransferases (
(1,3)-Fuc-Ts) (
)represent a target for such inhibitory agents, since
synthesis and expression of the sLe
molecule and related
(1,3)- and
(1,4)- fucosylated oligosaccharides are
controlled, in large measure, by
(1,3)Fuc-Ts(5) . These
enzymes catalyze the attachment of L-fucose in
anomeric
linkage to one or more distinct oligosaccharide precursors. Biochemical
and molecular cloning studies indicate that the human genome encodes at
least five distinct
(1,3)-Fuc-Ts (9, 21-28; reviewed in
Refs. 5, 29, and 30). Each enzyme can react with one or more
structurally distinct oligosaccharides and can thereby generate a
unique spectra of cell surface
(1,3)-fucosylated oligosaccharide
products. In turn, these molecules may exhibit distinct biologic
functions, including those involving selectin-dependent cell adhesion.
Although the primary sequences are known for several
(1,3)-Fuc-Ts (9, 21, 22, 23, 24, 25, 26, 27, 28) ,
the structural determinants within these enzymes that dictate their
different substrate specificities remain undefined. The purpose of this
work is to identify such protein sequence(s) and to provide a
conceptual background for work to design or identify molecules that
might inhibit
(1,3)-Fuc-Ts by interacting with acceptor substrate
binding sites.
In this study, we chose to explore three human
(1,3)-Fuc-Ts (Fuc-TIII, Fuc-TV, and Fuc-TVI) with informative
structural and catalytic properties. These enzymes share approximately
85% overall amino acid sequence identity(24, 25) . The
COOH termini of these enzymes maintain nearly identical amino acid
sequences, whereas their NH
-terminal regions are punctuated
by foci of nonidentical amino acids; we term these regions
``hypervariable'' segments. These three enzymes maintain
shared and distinct acceptor substrate specificities. In particular,
each of the three can effectively utilize neutral (Gal
1
4GlcNAc-) and
(2,3)-sialylated
(NeuNAc
2
3Gal
1
4GlcNAc-) type II
acceptors to create cell surface Lewis x (Le
) and sLe
determinants, respectively, when tested by transfection in
cultured cells(24, 25) , and when tested with low
molecular weight acceptor substrates in in vitro fucosyltransferase assays. Fuc-TIII can also efficiently utilize
neutral (Gal
1
3GlcNAc) and sialylated
(NeuNAc
2
3Gal
1
3GlcNAc-) type I
acceptor substrates to generate Lewis a (Le
) and sialyl
Lewis a (sLe
) molecules (6, 21) in
transfected cells and in vitro. By contrast, neither Fuc-TV
nor Fuc-TVI directs formation of fucosylated type I antigens (i.e. Le
and sLe
) when tested in transfected
cells, and neither can efficiently utilize low molecular weight
acceptor substrates in vitro(24, 25) . Thus,
the three enzymes' shared, and distinct, catalytic specificities,
along with similarities in, and differences between, their primary
sequences, provide an opportunity to identify amino acid residues
present in Fuc-TIII and/or absent from Fuc-TV and Fuc-TVI that
determine type I oligosaccharide acceptor substrate utilization.
To
identify such residues, we studied the pair of enzymes that differ most
dramatically in their respective abilities to utilize type I acceptors
(Fuc-TIII versus Fuc-TVI; Fig. 1A). We divided
the amino-terminal hypervariable regions of each enzyme into five
subdomains, each consisting of a distinct region of nonidentical
peptide sequence, separated by a region of identity (Fig. 1B). We then created a series of recombinant
(1,3)-Fuc-T chimeras in which these domains, or other domains
derived from the enzymes` COOH-terminal regions, were transposed by
themselves, or in combination, between the three enzymes, at
corresponding positions. Functional analysis of these Fuc-TIII:Fuc-TVI
chimeras (Fuc-TCs), and their wild type progenitors, via expression in
a transfected mammalian cell line, indicates that the acceptor
substrate specificity of these enzymes is determined by peptide
sequences in the hypervariable NH
-terminal segments of
these enzymes. When considered together with results obtained with a
Fuc-TIII:Fuc-TV chimera, these analyses indicate that type I acceptor
substrate utilization is determined by as few as eleven residues within
a 61-amino acid-long segment immediately proximal to the enzymes`
``sequence-constant'' COOH-terminal domains.
Figure 1:
Comparison of
protein and DNA sequences of Fuc-TIII and Fuc-TVI. A, Fuc-TIII
and Fuc-TVI are schematically represented by open rectangles.
The transmembrane domain of each enzyme is denoted by a striped box (). Vertical lines within the rectangles denote the positions of amino acid residues that differ between
the two enzymes. These differences are concentrated in the
hypervariable segment between the AflI I and BglII
restriction sites shown under the rectangles. The
carboxyl-terminal regions of these enzymes, distal to the BglII site, differ at seven positions. The relative positions
of these differences, and their nature (six nonidentical amino acids
and one amino acid/insertion (identified in single letter amino acid
code)) are also indicated in the figure. Sites of potential
asparagine-linked glycosylation are indicated by (
). The disaccharide portions of the
potential oligosaccharide acceptors are also shown in the figure.
``LacNAc'' denotes the type II precursor N-acetyllactosamine. LNB-I denotes the type I
precursor lacto-N-biose I. Arrows near the
disaccharide moieties point to the hydroxyl group that will be modified
by fucosylation. R denotes the underlying glycoconjugate that
displays the disaccharide. R` represents either a hydroxyl
group in the neutral precursors for Lewis x or Lewis a products or
(2,3)-linked sialic acid in the sialylated precursor for the
sialyl Lewis x or sialyl Lewis a products. Fuc-TIII (top) can
utilize GDP-fucose to form products from both type II and type I
precursors, whereas Fuc-TVI (bottom; shaded to match Fig. 2Fig. 3Fig. 4) can utilize GDP-fucose to form
products only from the type II precursors.
B,
subdomains within the hypervariable segments of Fuc-TIII and Fuc-TVI.
The DNA and derived protein sequences of the two enzymes are aligned.
The upper DNA and protein sequences correspond to Fuc-TIII. The lower
DNA and protein sequences correspond to Fuc-TVI. Dotted lines indicate positions in one enzyme that do not have a counterpart in
the other. Residues are numbered beginning with the initiator
methionine codon of Fuc-TIII (residue number 1). Nonidentical amino
acid residues are shaded. Underlined DNA sequences
correspond to the synthetic oligonucleotides used to create or destroy
restriction sites (positions and identity indicated above, for
Fuc-TIII, or below, for Fuc-TVI) that define the boundaries of each
subdomain. The nature of the DNA sequence changes needed to create
these sites are denoted above (Fuc-TIII) or below (Fuc-TVI) the wild type DNA sequence. As noted in the text,
these changes maintain the wild type protein sequence of each
enzyme.
Figure 2:
Flow
cytometry histograms of COS-7 cells transfected with recombinant Fuc-T
chimeras generated via carboxyl-terminal domain exchanges. Fuc-TIII (open rectangles) and Fuc-TVI (shaded rectangles) are
illustrated as in Fig. 1A. Recombinant chimeras derived
by restriction fragment exchange procedures (see ``Experimental
Procedures'') are composed of segments of Fuc-TVI (shaded
areas) and Fuc-TIII (open areas). Transmembrane segments
are denoted by the darkly shaded regions near the NH termini of each schematically represented enzyme. A symbol (
) denotes the positions of potential
asparagine-linked glycosylation sites. Relative positions of the five
subdomains in the hypervariable region are numbered. Positions of the BglII and BstXI sites used to create the chimeras in
this figure are indicated below each schematically represented enzyme.
COS-7 cells transfected with vectors encoding each enzyme, or a
negative control vector (pcDNAI), were stained with the
monoclonal antibodies indicated in the legend in the figure and
analyzed by flow cytometry. Histograms display the fraction of
antigen-positive cells (normalized for transfection efficiencies, as
described under ``Experimental Procedures'') and represent
the average of either two or three experiments. The mean fluorescence
intensity (M.F.I.) of the antigen-positive cells are indicated
by the numbers at the left of each histogram, above
the arrow (
) at the bottom of each panel. Numbers in parenthesis in this column denote the mean
fluorescence intensity of the antigen-negative cells whenever the
experiment yielded no antigen-positive
cells.
Figure 3:
Flow
cytometry histograms of COS-7 cells transfected with recombinant Fuc-T
chimeras generated via single subdomain exchanges. Symbols, legends,
and nomenclature are identical to that used in Fig. 2. The five
subdomains in the hypervariable region of each enzyme are numbered.
COS-7 cells transfected with vectors encoding each enzyme or a negative
control vector (pcDNAI) were stained with the monoclonal
antibodies indicated in the legend in each panel and were analyzed by
flow cytometry. Histograms display the fraction of antigen-positive
transfected COS-7 cells (normalized for transfection efficiencies, as
described under ``Experimental Procedures'') and represent
the average of either two or three experiments. Mean fluorescence
intensities (M.F.I.) of antigen-positive cells are indicated
by the numbers at the left of each histogram, above
the arrow () at the bottom of each panel. Numbers in parenthesis in this column denote the mean
fluorescence intensity of the antigen-negative cells whenever the
experiment yielded no antigen-positive cells. A, single
subdomains from Fuc-TVI (shaded areas) installed into Fuc-TIII (open rectangles). B, single subdomains from Fuc-TIII (open rectangles) installed into Fuc-TVI (shaded
rectangles).
Figure 4:
Flow cytometry histograms of COS-7 cells
transfected with recombinant Fuc-T chimeras generated via multiple
subdomain exchanges. Symbols, legends, and nomenclature are identical
to that used in Fig. 2and Fig. 3. The five subdomains in
the hypervariable region of each enzyme are numbered. COS-7 cells
transfected with vectors encoding each enzyme or a negative control
vector (pcDNAI) were stained with the monoclonal antibodies
indicated in the legend in each panel and were analyzed by flow
cytometry. Histograms display the fraction of antigen-positive
transfected COS-7 cells (normalized for transfection efficiencies, as
described under ``Experimental Procedures'') and represent
the average of either two or three experiments. Mean fluorescence
intensities (M.F.I.) of antigen-positive cells are indicated
by the numbers at the left of each histogram, above
the arrow () at the bottom of each panel. Numbers in parenthesis in this column denote the mean
fluorescence intensity of the antigen-negative cells whenever the
experiment yielded no antigen-positive cells. A, top set of
histograms, multiple subdomains from Fuc-TVI (shaded areas)
installed into Fuc-TIII (open rectangles) (chimeras Fuc-TC15,
Fuc-TC16, and Fuc-TC19). A, bottom histogram, subdomains 4 and
5 from Fuc-TV (lightly shaded areas) replace subdomains 4 and
5 in Fuc-TIII (chimera Fuc-TC21). B, multiple subdomains from
Fuc-TIII (open rectangles) installed into Fuc-TVI (shaded
rectangles) (chimeras Fuc-TC17, Fuc-TC18, and
Fuc-TC20).
The coding regions of Fuc-TIII and Fuc-TVI were mutated to create unique intersubdomain restriction endonuclease sites. Restriction sites were chosen to correspond to positions within the conserved amino acid sequence between subdomains, to maintain the wild type protein sequence, and to be unique within the plasmid vector. For Fuc-TIII, this involved installation of five new intersubdomain restriction sites and elimination of an MscI site. Creation of six new intersubdomain restriction sites was required for Fuc-VI. Mutagenesis was completed using the p-Alter Mutagenesis kit (Promega), after subcloning the inserts in pcDNAI-Fuc-TIII and pcDNAI-Fuc-TVI between the EcoRI and XbaI sites of the plasmid p-Alter (Promega). Simultaneous multiple oligonucleotide site-directed mutagenesis was performed on single strand templates, using procedures recommended by the manufacturer. Sequences of the mutagenic oligonucleotides are shown in Fig. 1B. Candidate mutant clones were screened for the desired restriction sites by restriction enzyme digestion. Mutated clones containing each of the desired mutations were sequenced (34) in their entirety to identify ones that contained no undesired additional mutations. Single, appropriately mutated, but fully correct clones for p-Alter-Fuc-TIII and p-Alter-Fuc-TVI, were digested with EcoRI and XbaI, and the released mutated inserts were recloned between the EcoRI and XbaI sites of pcDNAI.
Finally, chimera Fuc-TC21 was constructed by replacing subdomains 4 and 5 of Fuc-TIII with a region corresponding to subdomains 4 and 5 of Fuc-TV. The PCR and oligonucleotide primers containing a 5` Bst11071 site and a 3` BglII site (sense primer: CCGACTCCAGTGTATACCCACAGGCAGAC; antisense primer: GTCGGAGTCAGATCTGTAGGACAT) were used to generate this segment. PCR amplification conditions were chosen to minimize Taq polymerase-induced errors (annealing at 94 °C for 3 min; 20 cycles consisting of 1 min at 94 °C, 45 s at 65 °C, and 1 min at 72 °C; a final 7-min extension at 72 °C). The PCR product was digested with Bst1107I and BglII, and the resulting fragment was used to replace the corresponding Bst1107I and BglII fragment (subdomains 4 and 5) of the Fuc-TIII coding region. An error-free clone identified by sequence analysis was termed Fuc-TC21 and was used for transfection analyses.
In some instances, a fucosyltransferase was found to maintain a very low inherent specific activity toward one or both low molecular weight acceptor substrates, such that the rate of transfer was found to be directly proportional to acceptor substrate concentrations between 0.25 mM and at least 167 mM. In these instances, data in Table 1indicates that the apparent Michaelis-Menten constant exceeds 50 mM. As a measure of the relative efficiencies with which the enzymes utilize N-acetyllactosamine and lacto-N-biose I, the specific activity of each enzyme was determined for each of these two acceptor substrates (20 mM concentration). The ratio of the specific activity was calculated and displayed in Table 1.
This hypothesis was tested by first
characterizing the substrate utilization patterns of recombinant Fuc-T
chimeras in which the nearly identical COOH termini of Fuc-TIII and
Fuc-TVI were exchanged with each other (Fig. 1A and 2).
Substrate utilization was determined by expressing recombinant chimeras
in COS-7 cells, which maintain acceptor substrate precursors for
surface-localized Le, sLe
, Le
, and
sLe
epitopes (6, 21) and characterizing
the chimera-determined cell surface glycosylation patterns of the
transfected cells using flow cytometry.
Four chimeras informative
for the COOH termini of these enzymes were evaluated. In two of these
chimeras (Fuc-TC1 and Fuc-TC3), the exchanged segments were between
amino acid residue 301 of Fuc-TIII or 300 of Fuc-TVI (at positions
corresponding to a BstXI site in both genes; Fig. 1A and 2) and the COOH terminus of each enzyme.
This represents a 60-amino acid-long peptide segment in Fuc-TIII and a
59-residue-long peptide in Fuc-TVI. These segments differ from each
other at three amino acid positions and by the presence (Fuc-TIII) or
absence (Fuc-TVI) of a valine residue at position 353 (in Fuc-TIII) (Fig. 1A). In the other two chimeras (Fuc-TC2 and
Fuc-TC4; Fig. 2), the entire COOH-terminal regions were
exchanged. These transposed segments encompass all seven amino acid
sequence differences distal to the BglII site corresponding to
amino acid 160 in Fuc-TIII and 159 in Fuc-TVI ( Fig. 1and 2).
COS-7 cells transfected with chimeras Fuc-TC3 and Fuc-TC4 (which
maintain the NH-terminal segment of Fuc-TVI and portions of
the COOH terminus of Fuc-TIII) express the Le
and sLe
antigens, but not the type I-based Le
or sLe
epitopes (Fig. 2). By contrast, COS-7 cells transfected
with chimeras Fuc-TC1 and Fuc-TC2 (which maintain the
NH
-terminal segment of Fuc-TIII and portions of the COOH
terminus of Fuc-TVI) express the Le
, Le
, and
sLe
epitopes; however, neither chimera determines
expression of the sLe
antigen (Fig. 2). The reason
that these latter two chimeras do not yield sLe
expression
is not known. Nonetheless, these results clearly demonstrate that the
NH
-terminal hypervariable segments of Fuc-TIII and Fuc-TVI
determine their respective abilities to utilize or disregard the type I
acceptor substrates expressed by COS-7 cells.
This conclusion is
reinforced by in vitro determinations of the relative
efficiencies with which the fucosyltransferases utilize a pair of
representative, neutral low molecular weight type I and type II
acceptor substrates, N-acetyllactosamine and
lacto-N-biose I (Table 1). Wild type Fuc-TIII exhibits
similar Michaelis-Menten constants for these type II and type I
substrates (8.1 and 12.7 mM, respectively), although the
enzyme exhibits a substantially higher relative specific activity with
the type I substrate. By contrast, Fuc-TVI maintains a relatively low
apparent K for the type II acceptor (4.9
mM), a relatively high apparent Michealis-Menten constant for
the type I acceptor (greater than 50 mM), and exhibits a
substantially higher relative specific activity toward the type II
substrate (Table 1). The similarity between Fuc-TC4 and Fuc-TVI
observed by flow cytometry analysis (Fig. 2) is also evident
from the enzymatic analyses; Fuc-TC4 exhibits apparent Michealis-Menten
constants for the two acceptors, and a specific activity ratio, that do
not differ substantially from those obtained with wild type Fuc-TVI (Table 1). Fuc-TC2, the chimeric counterpart to Fuc-TC4 exhibits
an inherently low specific activity toward both substrates, as
reflected by the high apparent K
's
for the two substrates (Table 1). Nonetheless, this enzyme, like
wild type Fuc-TIII, utilizes the type I acceptor substrate much more
efficiently than it does the type II acceptor (Table 1). This
result is also consistent with the observation that Fuc-TC2 directs
type I product expression in COS-7 cells with an efficiency that equals
or exceeds its ability to determine type II product expression (Fig. 2).
We used this approach to construct a series of 17
additional recombinant (1,3)-Fuc-T chimeras derived from Fuc-TIII
and Fuc-TVI. Each chimera was evaluated for its ability to determine
expression of cell surface Le
, sLe
,
Le
, and sLe
epitopes in transfected COS-7
cells, using the flow cytometry procedures described above for chimeras
Fuc-TC1, Fuc-TC2, Fuc-TC3, and Fuc-TC4.
A series of single subdomain
transpositions were used to identify either a Fuc-TVI subdomain that
inactivates Fuc-TIII type I substrate utilization or a Fuc-TIII
subdomain that confers type I acceptor utilization when transposed into
Fuc-TVI (Fig. 3, A and B). Each of the five
Fuc-TIII chimeras containing a single Fuc-TVI subdomain expresses each
of the four cell surface antigens determined by wild type Fuc-TIII (Fig. 3A). Nonetheless, two of these chimeras (Fuc-TC8
and Fuc-TC9), representing substitutions in subdomain 4 or 5,
respectively, yields a cell surface antigen profile that differs
substantially from the Fuc-TIII profile. These two chimeras yield
populations of transfectants that exhibit a relative reduction in the
number of cells expressing the type I-based Le and
sLe
antigens and a relative increase in the fraction of
cells expressing the type II-based epitopes Le
and
sLe
. Specifically, the ratio of the fraction of Le
positive cells (29.8%) to the fraction of Le
positive
cells (37.2%) for wild type Fuc-TIII in this experiment is 0.80. By
contrast, for Fuc-TC8, this ratio is 0.19 (9.1% Le
positive versus 47.9% Le
positive), and the ratio is 0.29
for Fuc-TC9 (10.2% Le
positive versus 35.4%
Le
positive). Similar ratios are observed using results
obtained with antibodies against the sialylated forms of these antigens (Fig. 3A). These two chimeras thus yield a
glycosylation phenotype that is roughly intermediate between the
phenotypes observed for Fuc-TIII and Fuc-TVI. In contrast to the
results shown in Fig. 3A, substitution of single
Fuc-TIII subdomains at corresponding positions in Fuc-TVI yields a set
of chimeras that each determine a glycosylation phenotype virtually
identical to that determined by wild type Fuc-TVI (Fig. 3B). Taken together, these results indicate that
sequences within subdomain four and five of Fuc-TIII, by themselves,
can strongly, if not completely, influence the type I acceptor
substrate utilization efficiency of this enzyme.
By contrast, results obtained with an
additional set of chimeras indicate that subdomains 4 and 5, when
transposed together (with or without subdomain 3), strongly influence
type I substrate usage. Replacement of subdomains 3, 4, and 5 in
Fuc-TIII with the corresponding subdomains from Fuc-TVI yields a
fucosyltransferase chimera (Fuc-TC16; Fig. 4A) that
utilizes type II substrates exclusively to generate cell surface
Le or sLe
determinants. This phenotype is
virtually identical to that generated by wild type Fuc-TVI (Fig. 4A).
In a similar manner, replacement of
subdomains 3, 4, and 5 in Fuc-TVI with the corresponding subdomains
from Fuc-TIII creates a fucosyltransferase chimera (Fuc-TC18; Fig. 4B) with a phenotype qualitatively similar to that
of wild type Fuc-TIII. Specifically, COS-7 cells expressing this
chimera express all four epitopes analyzed (Fig. 4B),
although at reduced absolute levels (Le, 8.1% positive;
sLe
, 3.0% positive; Le
, 4.8% positive; and
sLe
, 4.0% positive) relative to levels generated by wild
type Fuc-TIII. The diminished amount of cell surface antigen expression
yielded by Fuc-TC18 is apparently not a consequence of decreased
expression of the chimeric enzyme, since Western blot analysis of
Fuc-TC18 transfectants identifies amounts of immunoreactive Fuc-TC18 in
these cells that are approximately equivalent to the amounts of wild
type Fuc-TVI and nearly equivalent to wild type Fuc-TIII accumulations
(data not shown). Enzyme activity assays suggest that the decreased
cell surface fucosylated antigen expression we observe with this
chimeric fucosyltransferase is due to the enzyme's inherently low
specific activity and not because it is mislocalized or otherwise not
able to effectively interact with its intracellular target
oligosaccharides.
(1,3)-Fuc-T activity assays indicate that the
specific activity of this chimera is substantially lower than either
Fuc-TIII or Fuc-TVI when assayed with the low molecular weight type I
and type II acceptor substrates lacto-N-biose I and N-acetyllactosamine (apparent K
values exceeding 50 mM for these substrates; Table 1). Nonetheless, this chimeric enzyme exhibits the same in vitro ``preference'' for the type I acceptor
substrate observe for wild type Fuc-TIII (Table 1). Thus, even
though the inherent efficiency of this chimeric fucosyltransferase is
apparently substantially less than that of the wild type enzyme, the
presence in this chimera of subdomains 3, 4, and 5 from Fuc-TIII is
associated with retention of a phenotype that is qualitatively similar
to wild type Fuc-TIII, in vitro (Table 1) and in
vivo (Fig. 4B).
Subdomain 3 in Fuc-TIII and
Fuc-TVI varies only by the presence (Fuc-TVI) or absence (Fuc-TIII) of
a consensus sequence for asparagine-linked glycosylation (Fig. 1B). This sequence, by itself, is without
apparent effect on acceptor substrate utilization as judged from
transfection analyses, since exchange of this subdomain alone does not
alter the glycosylation phenotype determined by the corresponding
mutant Fuc-TIII and Fuc-TVI chimeras (Fig. 3A, Fuc-TC7, and Fig. 3B, Fuc-TC12).
Thus, when this observation is considered together with other data in Fig. 2and 3, and with the results obtained with chimeras
Fuc-TC15, Fuc-TC16, Fuc-TC17, and Fuc-TC18, it can be supposed that
type I acceptor substrate discrimination (i.e. (1,4)-Fuc-T activity) can be conferred upon a
``generic''
(1,3)-Fuc-T scaffold, derived from either
Fuc-TIII or Fuc-TVI, by a set of sequences between the
NH
-terminal boundary of subdomain 4 and the COOH-terminal
boundary of subdomain 5.
This hypothesis is confirmed by analysis of
chimeras in which these two subdomains have been transposed together.
These chimeric (1,3)-Fuc-Ts (Fuc-T19 and Fuc-T20) generate
glycosylation phenotypes (Fig. 4) that correspond nearly exactly
to the phenotypes generated by chimeras constructed by exchange of
subdomains 3, 4, and 5 (Fig. 4). Specifically, Fuc-TC19
(subdomains 4 and 5 from Fuc-TVI installed into Fuc-TIII) utilizes type
II acceptor substrates with an efficiency approximately equivalent to
wild type Fuc-TVI, but does not determine expression of type I
molecules (Fig. 4A). The in vitro properties
of this chimera are consistent with these flow cytometry results.
Chimera Fuc-TC19 maintains a relatively low apparent K
for the type II acceptor (1.1
mM), a relatively high apparent Michealis-Menten constant for
the type I acceptor (greater than 50 mM) and displays a high
relative specific activity toward the type II substrate (Table 1). The chimeric enzyme Fuc-TC19 is therefore similar to
wild type Fuc-TVI and to the other ``Fuc-TVI-like'' chimera
Fuc-TC4 analyzed in vitro.
The complementary chimera
Fuc-TC20 (subdomains 4 and 5 from Fuc-TIII transposed into the
corresponding position in Fuc-TVI) utilizes neutral and sialylated type
I molecules, as well as neutral type II molecules, in vivo, to
generate Le (6.3% positive cells), sLe
(3.3%
positive cells), and Le
(5.4% positive cells) antigens.
Western blot analyses indicate that Fuc-TC20 accumulates in COS-7 cells
to levels nearly equivalent to those observed when wild type Fuc-TVI is
expressed in this cell line (data not shown). In vitro,
fucosyltransferase assays demonstrate that this chimeric enzyme
maintains an inherently low specific activity toward the type I and
type II acceptor substrates examined (apparent K
values exceeding 50 mM; Table 1). However,
Fuc-TC20 utilizes the type I acceptor substrate much more efficiently
than it does the type II acceptor (Table 1) and thus maintains
the in vitro type I acceptor substrate preference exhibited by
wild type Fuc-TIII. These results are analogous to those obtained with
Fuc-TC18, above. They indicate that the low level of surface antigen
expression determined by Fuc-TC20 is due at least in part to an
inherently diminished general specific activity. However, they also
indicate that this enzyme, like Fuc-TC18 above, has acquired a
Fuc-TIII-like phenotype by virtue of subdomains 4 and 5 derived from
Fuc-TIII.
Fuc-TC20 is unusual in that it does not yield cell surface sialyl Lewis x expression, but does direct expression of the other three fucosylated cell surface antigens (Fig. 4B). Given that this chimera differs from Fuc-TC18 only by the absence of a potential asparagine-linked glycosylation site within subdomain 3, the observations derived from these two chimeras suggested the possibility that display of an asparagine-linked oligosaccharide by subdomain 3, at least in the context of an otherwise intact Fuc-TVI scaffold, enables catalysis of sialyl Lewis x formation, whereas its absence disables sialyl Lewis x formation. This possibility is supported by the results obtained with chimeras Fuc-TC1 and Fuc-TC2 (Fig. 2), which determine a glycosylation phenotype analogous to that of Fuc-TC20 (Fig. 4B). In chimeras Fuc-TC1 and Fuc-TC2, absence of an asparagine-linked oligosaccharide modification site in subdomain 3, in the context of a full (Fuc-TC2) or partial (Fuc-TC1) COOH-terminal Fuc-TVI scaffold, also correlates with an inability to determine cell surface sialyl Lewis x formation. However, we note that Fuc-TC12 (Fig. 3B), representing a fully wild type Fuc-TVI sequence, except for absence of the asparagine-linked glycosylation site in subdomain 3, determines robust expression of the cell surface sialyl Lewis x antigen (Fig. 3B). Clearly then, the unusual phenotype exhibited by chimeras Fuc-TC20, Fuc-TC1, and Fuc-TC2 cannot be explained by the simple absence of a single asparagine-linked glycosylation site. Naturally, additional experiments will be required to determine if this site is actually modified by glycosylation in the transfected cells and how it may participate in this unusual phenotype.
In aggregate, the results obtained with the recombinant fucosyltransferase chimeras derived from Fuc-TIII and Fuc-TVI indicate that some complement of the 21 nonidentical amino acid residues encompassed within subdomains 4 and 5 of Fuc-TIII and Fuc-TVI (Fig. 1B) can determine whether or not these enzymes efficiently utilize type I oligosaccharide acceptor substrates in COS-7 cells and strongly influence their ability to discriminate between type I and type II acceptors in vitro.
Figure 5: Protein sequence alignments of Fuc-TIII, Fuc-TV, and Fuc-TVI across subdomains 4 and 5. The protein sequences of subdomains 4 and 5 of Fuc-TIII, Fuc-TV, and Fuc-TVI are shown in the single letter code. Numbers at the left indicate the residue number of the leftmost amino acid of each peptide sequence. Dashed lines represent positions in Fuc-TV and Fuc-TVI that are identical to corresponding residues in Fuc-TIII. Nonidentical residues are explicitly indicated by the single letter amino acid designation. Residues present in Fuc-TVI, but not found in either Fuc-TV or in Fuc-TIII (``unique'' to Fuc-TVI) are displayed below, among residues (indicated by an asterisk) that are common to at least two of the three enzymes. The asterisks indicate residues in Fuc-TVI that are also in either Fuc-TV or Fuc-TIII or both.
These experiments demonstrate that utilization of type I
oligosaccharide substrates by the human (1,3)-fucosyltransferases
Fuc-TIII, Fuc-TV, and Fuc-TVI is determined in large measure by a
relatively short peptide segment, encompassed by subdomains 4 and 5,
and localized to the NH
terminus of each enzyme's
Golgi lumenal domain. Examination of the residues within this segment,
in each enzyme, suggests the simple possibility that, in the context of
a ``generic''
(1,3)-fucosyltransferase background,
efficient type I substrate utilization is allowed by residues shared by
Fuc-TIII and Fuc-TV and found within subdomains 4 and 5, whereas type I
substrate utilization is ``disabled'' by residues in this
segment that are unique to Fuc-TVI.
In the context of this simple model, the more obvious ``Fuc-TVI-unique'' amino acid residues (Fig. 5) that might operate singly, or in concert with other neighboring residues, to disable type I acceptor substrate utilization, include the nonconservative differences represented by (i) the arginine residue at position 110 in Fuc-TVI that corresponds to a tryptophan residue that occupies the analogous position in both Fuc-TIII and Fuc-TV, (ii) a pair of arginine residues in Fuc-TVI at positions (122 and 126) that correspond to a pair of proline residues in Fuc-TIII and Fuc-TV, (iii) a tryptophan residue (143) in Fuc-TVI that corresponds to glutamine or arginine in Fuc-TIII or Fuc-TV, respectively, and (iv) a lysine residue at position 146 in Fuc-TVI that corresponds to a glutamic acid residue in Fuc-TIII and Fuc-TV. It is also possible that type I acceptor substrate utilization may be influenced by the more conservative amino acid sequence differences identified in Fig. 5.
Although these studies have identified
candidate residues for acceptor substrate discrimination, they do not
allow us to discern the mechanism(s) through which these residues
operate in this process. However, previous work by others in this area
suggests a role for hydrogen bonding interactions in acceptor substrate
binding and utilization. For example, in an evaluation of deoxygenated
oligosaccharide acceptor substrates for two different human
fucosyltransferases (corresponding to Fuc-TIII and Fuc-TVI), Hindsgaul et al. (43) report that acceptor substrates
deoxygenated at the reactive hydroxyl position do not act as inhibitors
of these enzymes. Hindsgaul et al.(43) interpret
these results to mean that these fucosyltransferases, as well as other
glycosyltransferases, bind to their acceptor substrates, and
subsequently effect catalysis, through hydrogen bonding interactions
between the reactive hydroxyl group on the acceptor and one or more
basic groups in the enzyme. In the context of this hypothesis, we note
that, in aggregate, the ``Fuc-TVI-specific'' residues shown
in Fig. 5are largely basic in character, relative to the
corresponding residues in Fuc-TIII and Fuc-TV. The nonconserved
tryptophan residues in subdomains 4 and 5, and other such polar
residues, may also mediate fucosyltransferase acceptor substrate
recognition, again presumably through hydrogen bonding interactions, as
suggested from studies of
(1,4)-galactosyltransferase(44) . Whether or not these
residues operate to determine binding to, and fucosylation of, type II
acceptor substrates exclusively (and perhaps at the expense of type I
acceptor substrate binding) remains to be experimentally determined.
On a more global scale, it seems probable that fucosyltransferase-specific substrate recognition and modification properties will also be found to be more than simply a function of the ability of the relevant subdomains to ``bind'' to the oligosaccharide acceptor. For instance, we can anticipate that some of the residues in these subdomains may interact in space with amino acids far away along the linear sequence of these enzyme, to alter acceptor substrate utilization. Alternatively, residues in these subdomains may influence acceptor substrate recognition by residues elsewhere in the molecule, via transmitted conformational change dictated by the specific residues within these subdomains. Finally, it is possible that subdomain-dependent proteolytic events indirectly determine acceptor substrate specificity by controlling the liberation of distinct soluble fucosyltransferase fragments with different acceptor substrate specificities. The relevance of these possibilities to our observations will await an exploration of the biosynthesis and in vitro catalytic properties of the recombinant wild type and chimeric enzymes and the availability of the tertiary structures of these enzymes.
We emphasize that our studies have focused on detecting the products of these enzymes at a relatively low level of resolution (i.e. at the level of cell surface antigen expression). This approach is advantageous in that it measures product(s) formed by an enzyme within its correct cellular environment. Furthermore, since the fraction of antigen-positive cells we observe correlates very well with catalytic activities measured in vitro, this parameter seems to accurately reflect the biochemical nature of the enzyme under study. By contrast, the mean fluorescent intensities of the antigen-positive cells fall within a similar range, regardless of the enzyme analyzed, and do not correlate with in vitro activity. We do not understand this phenomenon, although we speculate that this can be accounted for by the possibility that acceptor substrate concentration and/or accessibility within COS cells is limiting to the point that utilization of the small amounts of the available acceptor substrate molecules yields a virtually maximal, though easily detectable, signal. The broad spectrum of enzyme protein expression level obtained within a population of transiently transfected cells may play upon such limiting substrate levels to yield an off-on or digital signal response that is a function of the efficiency of the enzyme under study. For example, in a population of cells expressing an enzyme with robust inherent activity, many cells, including those that express a relatively small amount of enzyme protein, will contain sufficient intracellular enzyme activity to convert them to antigen positivity (hence the relatively larger fraction of antigen-positive cells seen with efficient enzymes), even though the limiting amounts of substrate operate to place a strict upper boundary on the ``brightness'' of such cells. By contrast, in a population of cells expressing an enzyme with a low inherent activity, only the relatively small fraction of cells that have accumulated relatively large amounts of enzyme protein will contain sufficient enzyme activity to exceed the threshold for substrate conversion (hence the relatively small fraction of antigen positive cells with inefficient enzymes), and will become antigen-positive, with a mean fluorescence intensity that can be roughly maximal. Additional work will be required to confirm or refute this notion.
Our approach
cannot tell us much about the nature of the glycoconjugate(s) that
displays the fucosylated antigens or about the structures of the
glycoconjugate precursors that serve as substrates for these enzymes
within these cells (aside from the fact that some of these moieties
terminate with neutral and 2
3-sialylated type I and type II
structures). It will be important to eventually know this information,
however, since it is possible that some of the apparent
subdomain-dependent acceptor substrate specificity we observe is also a
function of the type of glycoconjugate that displays the acceptor di-
or trisaccharide moieties with which we have concerned ourselves here.
We note that there are well known precedents for this type of
cis-modulation of glycosyltransferase substrate specificity by the
underlying glycoprotein sequence(45, 46) . Whether or
not this is actually the case for the fucosyltransferases studied here
will first require a biochemical characterization of the cellular
products formed by the transfected enzymes.