(Received for publication, December 14, 1995; and in revised form, January 18, 1996)
From the
Lymphocyte homing to lymph nodes and Peyer's patches is
mediated, in part, by adhesive interactions between L-selectin
expressed by lymphocytes and L-selectin ligands displayed at the
surface of the cuboidal endothelial cells lining the post-capillary
venules within lymphoid aggregates. Candidate terminal oligosaccharide
structures thought to be essential for effective L-selectin ligand
activity include a sulfated derivative of the sialyl Lewis x
tetrasaccharide. Cell type-specific synthesis of this oligosaccharide
is presumed to require one or more (1,3)fucosyltransferases,
operating upon common 3`-sialylated and/or sulfated N-acetyllactosamine-type precursors. The identity of the
(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin
ligands has not been defined. We report here the molecular cloning and
characterization of a murine
(1,3)fucosyltransferase locus whose
expression pattern correlates with expression of high affinity ligands
for L-selectin. In situ hybridization and immunohistochemical
analyses demonstrate that this cDNA and its cognate
(1,3)fucosyltransferase are expressed in endothelial cells lining
the high endothelial venules of peripheral lymph nodes, mesenteric
lymph nodes, and Peyer's patches. These expression patterns
correlate precisely with the expression pattern of L-selectin ligands
identified with a chimeric L-selectin/IgM immunohistochemical probe and
by the high endothelial venule-reactive monoclonal antibody MECA-79.
Transcripts corresponding to this cDNA are also detected in isolated
bone marrow cells, a source rich in the surface-localized ligands for
E- and P-selectins. Sequence and functional analyses indicate that this
murine enzyme corresponds to the human Fuc-TVII locus. These
observations suggest that Fuc-TVII participates in the generation of
(1,3)fucosylated ligands for L-selectin and provide further
evidence for a role for this enzyme in E- and P-selectin ligand
expression in leukocytes.
Cell adhesion events between leukocytes and endothelial cells operate to facilitate the exit of blood leukocytes from the vascular tree. The selectin family of cell adhesion molecules and their counter-receptors function early in this process, mediating transient adhesive contacts between leukocytes and the endothelial cell monolayer. These selectin-dependent adhesive contacts, together with shear forces impinging upon the leukocyte, cause the leukocyte to ``roll'' along the endothelial monolayer. Leukocyte rolling, in turn, facilitates subsequent events that include leukocyte activation, firm leukocyte-endothelial cell attachment, and transendothelial migration(1, 2) .
E- and
P-selectins, expressed by activated vascular endothelial cells,
recognize glycoprotein counter-receptors displayed by leukocytes. Each
of these selectins can operate to mediate leukocyte rolling in the
context of inflammation. L-selectin has also been implicated in
mediating leukocyte adhesion to activated vascular endothelium through
interactions with an as yet poorly understood endothelial cell ligand (3, 4) . By contrast, lymphocyte L-selectin recognizes
glycoprotein counter-receptors displayed by specialized cuboidal
endothelial cells that line high endothelial venules (HEV) ()within lymph nodes and Peyer's patches.
L-selectin-dependent adhesive interactions in this context operate to
facilitate trafficking of lymphocytes (lymphocyte ``homing'')
to such lymphoid aggregates.
The NH-terminal C-type
mammalian lectin domain common to each of the three selectin family
members mediates cell adhesion through calcium-dependent interactions
with specific oligosaccharide ligands, displayed by leukocytes (E- and
P-selectin ligands) (2, 5) or by HEV
(L-selectin)(6) . Physiological ligand activity for E- and
P-selectins is critically dependent on the expression of a nonreducing
terminal tetrasaccharide termed sialyl Lewis x (sLe
)
[NeuNAc
2,3Gal
1,4 (Fuc
1,3)GlcNAc-R] (5) and/or its difucosylated variant(7) . However, E-
and P-selectins recognize this oligosaccharide in different contexts.
P-selectin-dependent cell adhesion is optimal when sLe
is
displayed by serine and threonine-linked oligosaccharides residing on a
specific protein termed P-selectin glycoprotein ligand 1
(PSGL-1)(8, 9) . sLe
-modified P-selectin
glycoprotein ligand 1 also appears to represent a high affinity
counter-receptor for E-selectin(10, 11) . A distinct
leukocyte glycoprotein termed E-selectin ligand 1 (ESL-1) (12) and its
(1,3)fucosylated, asparagine-linked
oligosaccharides may also function as an E-selectin counter-receptor.
Physiological L-selectin counter-receptors on HEV are represented by
the glycoproteins GlyCAM-1(13) , CD34(14) , and
MAdCAM-1(15) . Biochemical studies indicate that L-selectin
ligand activity of these molecules is also critically dependent upon
post-translational modification by glycosylation. Early studies
documented a requirement for sialylation and sulfation(16) ,
implied a requirement for (1,3)fucosylation, and indicated that
these modifications are components of serine- and/or threonine-linked
glycans. More recent oligosaccharide structural analyses extend this
work and imply that high affinity L-selectin ligand activity may depend
upon a sulfated variant of the sLe
determinant,
NeuNAc
2,3(SO
6) Gal
1,4(Fuc
1,3)GlcNAc-R (17, 18, 19) .
Expression of sLe is determined by cell lineage-specific expression of one or more
(1,3)fucosyltransferases(20) . These enzymes utilize the
donor substrate GDP-fucose and catalyze a transglycosylation reaction
involving the addition of
1,3-linked fucose to a common 3`-sialyl N-acetyl-lactosamine precursor. It can be presumed that
expression of the sulfated variant of sLe
also depends upon
lineage-specific expression of
(1,3)fucosyltransferase activities
operating on sulfate-modified 3`-sialyl N-acetyl-lactosamine
precursors or that create sLe
moieties modified
subsequently by sulfation.
The identity of the
(1,3)fucosyltransferase(s) responsible for selectin ligand
expression in leukocytes is not well defined, and HEV-specific
(1,3)fucosyltransferases have not been described. To date, five
different human
(1,3)fucosyltransferases have been cloned (21, 22, 23, 24, 25, 26, 27, 28) .
Northern blot and molecular cloning analyses imply that two of these,
termed Fuc-TIV (24, 25, 26) and
Fuc-TVII(27, 28) , are expressed in leukocytic cells
and represent candidates for critical participation in selectin ligand
expression. The role of Fuc-TIV (also known as ELAM-1 ligand fucosyl
transferase) in this process is not clear, however. Although
Fuc-TIV/ELAM-1 ligand fucosyl transferase is able to efficiently
utilize nonsialylated N-acetyl-lactosamine precursors to
direct expression of the Le
moiety(24, 26) , this enzyme cannot determine
sLe
expression in all cellular contexts(29) , and
its ability to do so in leukocytes or in leukocyte progenitors has not
been demonstrated. By contrast, Fuc-TVII is apparently able to
determine sLe
expression in all mammalian cellular contexts
examined, where sLe
synthesis is biochemically
possible(27, 28) . Neither enzyme has been tested for
its ability to participate in the synthesis of L-selectin ligands
represented by sulfated sLe
determinants.
We report here
the isolation and characterization of murine cDNAs and genomic
sequences encoding an (1,3)fucosyltransferase with primary
sequence similarity and catalytic properties analogous to those
assigned to the human Fuc-TVII
(1,3)fucosyltransferase(27, 28) . This murine
locus generates alternatively spliced transcripts that differ in their
respective abilities to encode
(1,3)fucosyltransferase activity.
Expression of this locus is restricted largely to E- and P-selectin
ligand-rich bone marrow cells (30) , where it may participate
in the synthesis of these ligands. Transcripts derived from the
Fuc-TVII locus and the corresponding
(1,3)fucosyltransferase
accumulate to substantial levels in the endothelial cells lining the
HEV of peripheral and mesenteric lymph nodes and of Peyer's
patches. The localized and abundant expression of this
(1,3)fucosyltransferase in HEV, when considered together with the
(1,3)fucosylated oligosaccharides proposed as HEV ligands for
L-selectin, imply a key role for this enzyme in the biosynthesis of
L-selectin ligands.
Figure 2: Nucleotide and deduced amino acid sequence of the isolated mouse Fuc-TVII gene. The DNA sequence was derived from a phage containing the murine Fuc-TVII locus. The DNA sequence present in cDNAs (Fig. 1) is shown in uppercase letters, whereas the DNA sequence corresponding to intronic positions is displayed in lowercase letters. Amino acid sequences predicted by the cDNA sequences are shown in single-letter code. As discussed in detail in the text, alternative splicing events yield different cDNAs that may in turn encode three different polypeptides. One protein is predicted to initiate at the methionine codon localized to nucleotide positions 996-998 (389 residues, 44,492 Da; cDNA 5; Fig. 1). A second protein is predicted to initiate at the methionine codon localized to nucleotide positions 1947-1949 (342 residues, 39,424 Da; cDNAs 6, 10, and 14; Fig. 1). The third protein is predicted to initiate at the methionine codon localized to nucleotide positions 2126-2128 (318 residues, 36,836 Da; cDNA 3, as well as all other cDNAs; Fig. 1).
Figure 1:
Structure and function of the murine
Fuc-TVII gene and cDNAs. A, structures and functional
activities of the murine Fuc-TVII gene and cDNAs. The multi-exon
structure of the murine Fuc-TVII gene is shown at the top.
Numbering below the schematic corresponds to the nucleotide
positions of intron-exon boundaries, and the first (1) and
last(3594) nucleotides of the known sequence of the locus. Intron-exon
boundaries are defined by comparison of the cDNA sequences to the
corresponding genomic DNA sequence (see Fig. 2). The numbering above the schematic, immediately above the ATGs, corresponds to the nucleotide position of the first
nucleotide in each of the three potential initiation codons as
discussed in detail in the text. The numbering above the schematic, immediately above the STOPs,
corresponds to the nucleotide position of the translational termination
codon (TGA; base pairs 1900-1902) localized to exon 3b that
truncates the potential open reading frame initiated by the start codon
at nucleotide 996-998 in cDNA classes represented by cDNAs 6 and
10 (see text for details). Representative members of the five
structurally different classes of Fuc-TVII cDNAs isolated from the
murine cytotoxic T-lymphocyte cell line 14-7fd are schematically
represented below the gene structure schematic. The
cDNAs shown are the representative member of each class with the
longest 5` extension. The number of cDNAs isolated in each class is
indicated in the column labeled number of cDNA's. Each
cDNA was transiently expressed in COS-7 cells (see ``Experimental
Procedures''). The transfected COS-7 cells were then subjected to
flow cytometry analysis to characterize the cell surface glycosylation
phenotype determined by each cDNA. The fraction of sialyl Lewis
x-positive cells in the transfected population (positive staining with
the monoclonal antibody CSLEX-1, normalized to transfection efficiency
as determined by chloramphenicol acetyltransferase activity encoded by
a co-transfected plasmid vector encoding this enzyme
(``Experimental Procedures'') is indicated in the column
labeled % CSLEX-1 positive. These results represent the
fraction of antigen-positive cells observed above a background of 2%
staining obtained with the negative control vector pCDM8. Extracts were
also prepared from the transfected cells and were subjected to in
vitro (1,3)fucosyltransferase assays using 5 mM sialyl N-acetyllactosamine as an acceptor (see
``Experimental Procedures''). The specific activity of the
(1,3)fucosyltransferase activity encoded by each cDNA (normalized
for transfection efficiency) is indicated in the column labeled Sp.
Act. (cpms/µg/hr). B, Western blot analysis of the
polypeptides expressed in COS-7 cells by cDNAs 3, 5, 6, 10, and 14. The
extracts used in the
(1,3)fucosyltransferase assays discussed in A were also subjected to Western blot analysis using an
antigen affinity purified anti-FucTVII antibody. The amounts of protein
analyzed from each type of transfected cell extract were varied to
achieve normalization to the transfection efficiencies, exactly as
indicated in A for the flow cytometry and
(1,3)fucosyltransferase activity analyses. Cell extracts were
fractionated by SDS-polyacrylamide gel electrophoresis and
electroblotted to a polyvinylidene difluoride membrane, and the
Fuc-TVII expression vector-encoded polypeptides were identified by
probing with an antigen affinity purified rabbit anti-Fuc-TVII
antibody, goat anti-rabbit IgG-peroxidase conjugate, and a commercially
available enhanced chemiluminescence reagent (ECL, Amersham Corp.) as
described under ``Experimental
Procedures.''
Transiently transfected cells were harvested 72 h after transfection and were stained with monoclonal antibodies diluted in staining medium as described previously(22) . Anti-Lewis a, anti-H, and anti-sialyl Lewis x antibodies were used at 10 µg/ml. Anti-Lewis x was used at a dilution of 1:1000. Anti-sialyl Lewis a was used at a dilution of 1:500. Anti-VIM-2 antibody was used at a dilution of 1:200. Cells were then stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM or anti-mouse IgG and subjected to analysis on a FACScan (Becton-Dickinson) as described previously(22) . Cells were also co-transfected with the plasmid pCDM8-CAT(32) , and extracts prepared from these cells were subjected to chloramphenicol acetyltransferase activity assays (32) to allow for normalization of flow cytometry and Western blot data to transfection efficiency.
Reactions containing neutral acceptors (N-acetyllactosamine, lactose, lacto-N-biose I,
2`-fucosyllactose, all from Sigma) were terminated by the addition of
20 µl of ethanol and 560 µl of water. Samples were centrifuged
at 15,000 g for 5 min and a 50-µl aliquot was
subjected to scintillation counting to determine the total amount of
radioactivity in the reaction. An aliquot of 200 µl was applied to
a column containing 400 µl of Dowex 1
2-400, formate
form(21, 23) . The column was washed with 2 ml of
water, and the radioactive reaction product not retained by the column
was quantitated by scintillation counting. Reactions with the acceptor
NeuNAc
2
3Gal
1
4GlcNAc (Oxford
Glycosystems, Inc.) were terminated by adding 980 µl of 5.0 mM sodium phosphate buffer, pH 6.8. Samples were then centrifuged at
15,000
g for 5 min, and a 500-µl aliquot was
applied onto a Dowex 1
8-200 column (1 ml) prepared in
the phosphate form. The reaction product was collected and quantitated
as described previously(22) .
Recombinant E. coli-derived Fuc-TVII was fractionated by SDS-polyacrylamide gel electrophoresis, and segments of the gel containing Fuc-TVII were excised and used subsequently as antigen for rabbit immunizations. Rabbit immunization services were purchased (Pel-Freeze Biologicals, Rogers, AR). Each of three rabbits were initially immunized subcutaneously with a total of approximately 200 µg of Fuc-TVII in pulverized polyacrylamide gel slices mixed with complete Freund's adjuvant. Subsequent immunizations were completed in an essentially identical manner at 14-day intervals, except that antigen was administered in incomplete Freund's adjuvant. Antisera were harvested 10 days following the last of a total of approximately six secondary immunizations.
Sections to be stained with anti-Fuc-TVII were fixed in 2%
paraformaldehyde in phosphate-buffered saline for 20 min on ice. The
sections were rinsed with phosphate-buffered saline at room
temperature, were quenched with 50 mM NHCl in
phosphate-buffered saline at room temperature, and then were rinsed
briefly with water. The tissues were then permeabilized with 100%
methanol for 20 min on ice, rehydrated in phosphate-buffered saline,
and then incubated for 30 min at room temperature with blocking
solution A (phosphate-buffered saline containing 2% goat serum, 0.05%
Triton X-100, 0.05% Tween 20). The blocking solution was aspirated, and
the sections were incubated overnight at 7 °C with antigen affinity
purified anti-Fuc-TVII used at a final concentration of 5 µg/ml in
blocking solution A. After the overnight incubation, the
anti-Fuc-TVII/blocking solution was removed, and the slides were washed
with phosphate-buffered saline and were incubated for 1 h at room
temperature with a FITC-conjugated goat anti-rabbit IgG reagent (Sigma)
diluted 1:200 in blocking solution A. The slides were then washed at
room temperature in phosphate-buffered saline, mounted with citifluor
(Citifluor Products, Chemical Laboratory, The University, Canterbury,
Kent, UK), and examined by immunofluorescence microscopy (Leitz DM RB
microscope).
Sections to be stained with the monoclonal antibody
MECA-79 (48) were fixed on ice for 20 min in 2%
paraformaldehyde in phosphate-buffered saline, washed at room
temperature with phosphate-buffered saline, and quenched for 20 min at
room temperature with 50 mM NHCl in
phosphate-buffered saline. The slides were then rinsed briefly in
water, permeabilized with 100% methanol for 20 min on ice, rehydrated
in phosphate-buffered saline, and then incubated overnight at room
temperature with blocking solution A (phosphate-buffered saline
containing 2% goat serum, 0.05% Triton X-100, 0.05% Tween 20). The
blocking solution was then aspirated, and the sections were incubated
for 1 h at 7 °C with MECA-79 at a concentration of 5 µg/ml in
blocking solution A. Sections were then washed extensively with
phosphate-buffered saline at room temperature. The washed sections were
then incubated for 1 h at room temperature with a tetramethyl rhodamine
isothiocyanate-conjugated goat anti-rat IgM reagent (Jackson
ImmunoResearch) used at a dilution 1:200 in blocking solution A. The
slides were then washed three times at room temperature with
phosphate-buffered saline, mounted with citifluor, and examined.
Sections to be stained with the L-selectin/IgM chimera were fixed in
1% paraformaldehyde and 0.1 M cacodylate, pH 7.1, for 20 min
on ice, were then washed with Tris-buffered saline, pH 7.4. The
L-selectin/IgM chimera was applied to the sections at a concentration
60 µg/ml in blocking solution B (Tris-buffered saline, pH 7.4,
containing 2% goat serum) supplemented with either 3 mM CaCl or with 5 mM EDTA, and were allowed to
incubate overnight at 7 °C. Sections were then washed extensively
with ice-cold Tris-buffered saline supplemented with 3 mM CaCl
. Sections were then incubated for 1 h at 7 °C
with a biotinylated goat anti-human IgM reagent (Sigma), diluted 1:200
in blocking solution B, and supplemented either with 3 mM CaCl
or with 5 mM EDTA. The sections were
then washed with ice-cold Tris-buffered saline supplemented with 3
mM CaCl
and were incubated for 1 h at 7 °C
with a FITC-conjugated streptavidin reagent (Vector Labs, Burlingame,
CA) diluted 1:200 in blocking solution B supplemented with 3 mM CaCl
. The slides were washed with ice-cold
Tris-buffered saline supplemented with 3 mM CaCl
,
mounted with citifluor, and examined by immunofluorescence microscopy
(Leitz DM RB microscope).
To identify transcripts corresponding to this genomic sequence, a
segment of the phage insert representative of the open reading frame
was used to probe Northern blots prepared from mouse cell lines and
tissues. Transcripts corresponding to this probe were identified in the
murine cytotoxic T-cell line 14-7fd(42, 43) . A cDNA
library constructed from this cell line (32) was screened by
hybridization with a segment of the phage insert, yielding 16
hybridization positive colonies. The sequences of all 16 cDNA clones
were determined, as was the sequence of the corresponding genomic DNA (Fig. 1A). Analysis of this sequence data indicates
that this locus yields multiple structurally distinct transcripts
derived from alternative splicing events and possibly also from
alternative transcription initiation events. Five classes of cDNAs were
identified (Fig. 1). Analysis of these cDNA sequences identifies
three methionine codons that may function to initiate translation of an
open reading frame with amino acid sequence similarity to human
Fuc-TIII, Fuc-TIV, Fuc-TV, and Fuc-TVI (Fig. 2). The positions
of these methionine codons predicts the synthesis of
(1,3)fucosyltransferases with different cytosolic domains (encoded
by exons 2 and/or 3) but with identical Golgi-localized catalytic
domains (encoded by exon 4). One relatively abundant class of cDNAs
(represented by cDNA 14) maintains an open reading frame initiating at
the methionine codon at nucleotide 1947. This reading frame predicts a
342-residue, 39,424-Da type II transmembrane protein with a hydrophobic
transmembrane segment derived from amino acids 9-31 (Fig. 2). An in-frame methionine codon at nucleotide 2126
predicts a 318-residue, 36,836-Da polypeptide that initiates within the
hydrophobic transmembrane segment of the polypeptide predicted by the
longer reading frame initiated at nucleotide 1947. A similar structural
arrangement is found in two other cDNA classes, represented by cDNAs 6
and 10. However, these two cDNAs differ from cDNA 14 in that they
contain an additional upstream exon with a methionine codon
corresponding to nucleotide 996. The translational reading frame
initiated by this methionine codon is truncated by a termination codon
in exon 2 at a position proximal to the methionine codon at nucleotide
1947 and thus cannot generate a polypeptide that shares similarity to
the human
(1,3)fucosyltransferases. However, in cDNA 5, the
absence of exon 2 allows the translational reading frame generated by
the methionine codon at nucleotide 996 to continue in frame with
sequence in exon 4. This arrangement predicts the synthesis of a
389-residue, 44,492-Da type II transmembrane protein with the same
putative transmembrane segment defined for the protein predicted by
cDNA 14 (Fig. 2). Finally, cDNA 3 is representative of a
relatively abundant class of cDNAs that each initiate between the
splice acceptor site of exon 4 and the methionine codon at nucleotide
2126. This class of cDNAs predicts a 318-residue, 36,836-Da polypeptide
that initiates within the transmembrane segment predicted for the
proteins corresponding to the other cDNA classes.
Because the
polypeptides predicted by these murine cDNAs share primary sequence
similarity to the four human (1,3)fucosyltransferases known at the
time (Fuc-TIII, IV, V, and VI), we anticipated that one or more of them
would function as an
(1,3)fucosyltransferase. However, because the
murine peptide sequence shares approximately equivalent sequence
similarity to each of these human enzymes, we expected that it did not
represent the murine homologue of any of them and consequently named it
Fuc-TVII. This appellation has been justified by subsequent work in
which this murine gene has been used to isolate cDNAs encoding the
human Fuc-TVII (27) .
None of the three putative initiation
codons are embedded in a sequence context consistent with Kozak's
rules for translation initiation (Fig. 2)(60) . To
determine which, if any, of these initiation codons and cognate cDNAs
function to encode the predicted polypeptide(s) and to confirm that
this locus encodes an (1,3)fucosyltransferase, COS-7 cells were
transfected with a cDNA representative of each class, and the
transfectants were subjected to assays to (i) identify cDNA-determined
cell surface-localized fucosylated oligosaccharide antigens, (ii)
identify and quantitate the polypeptides encoded by cDNAs, and (iii)
identify and partially characterize cDNA-determined
(1,3)fucosyltransferase activity in transfectant cell extracts
using in vitro
(1,3)fucosyltransferase activity assays.
cDNAs representative of three of the five classes (cDNAs 6, 10, and
14) (Fig. 1) each determine relatively high levels of cell
surface-localized sLe expression (35.1, 21.8, and 16.5%,
respectively, above a 2% background) when introduced into COS-7 cells
by transfection. cDNA 5 also directs cell surface sLe
expression in COS-7 cells but at a level (9% positive cells) that
is lower than the sLe
expression levels determined by cDNAs
6, 10, and 14. By contrast, none of these four cDNAs directs expression
of Lewis x, Lewis a, or sialyl Lewis a determinants (data not shown).
These results indicate that one or both of the two potential methionine
initiator codons in each cDNA can efficiently direct translation to
yield
(1,3)fucosyltransferase activity. These observations further
indicate that this
(1,3)fucosyltransferase activity can utilize
(2,3)sialylated lactosamine-based glycan structures to form
sLe
determinants but indicate that the activity does not
efficiently utilize neutral type II oligosaccharide Lewis x precursors
nor neutral or
(2, 3)sialylated type I precursors to the Lewis a
isomers. Because all four of these cDNAs direct qualitatively identical
cell surface antigen profiles in COS-7 cells, it seems likely that
individually or together, each directs the expression of polypeptides
that individually or together maintain essentially identical acceptor
substrate specificities (at least for the four antigens examined).
In contrast to the results obtained with cDNAs 5, 6, 10, and 14,
cDNA 3 does not direct detectable sLe expression. This
result suggests that the methionine codon at nucleotide 2126 in this
cDNA does not efficiently promote initiation of translation of the
cognate mRNA and thus does not encode functionally significant levels
of enzyme activity. Alternatively, this cDNA may encode a polypeptide
without
(1,3)fucosyltransferase activity.
Qualitatively
identical results were obtained when these five cDNAs were expressed in
another cell line (CHO-Tag cells) (32) informative for
expression of the Lewis x and sLe determinants (data not
shown). Unlike COS-7 cells, this cell line is also capable of forming
the internally fucosylated VIM-2 determinant
(NeuAc
2,3Gal
1,4GlcNAc
1,3Gal
1,4(Fuc
1,3)
GlcNAc-R)(22) . We found that none of the cDNAs directs
expression of the VIM-2 epitope when expressed in the CHO-Tag cells
(data not shown). Considered together, these results indicate that
some, though not all, of the cDNAs can encode an
(1,3)fucosyltransferase activity that can catalyze
(1,3)fucosylation of the N-acetylgalactosamine moiety on
a terminal
(2,3)sialylated lactosamine unit but not to internal N-acetylgalactosamine moieties on
(2,3)sialylated
polylactosamine precursors nor to neutral type II precursors.
To
confirm that the sLe expression efficiency characteristic
of each cDNA correlates with the level of expression of the
corresponding protein, cell extracts of the transfected COS-7 cells
were subjected to Western blot analysis using an affinity purified
rabbit polyclonal antibody generated against a recombinant form of the
predicted polypeptide (Fig. 1B). Cells transfected with
cDNAs 6, 10, and 14 express two major forms of the protein, with
molecular masses of 35 and 37 kDa. Smaller amounts of several other
proteins are also evident in these cells. The amount of immunoreactive
protein generated by these three cDNAs correlates with the level of
sLe
expression directed by each. This observation indicates
that the relative sLe
expression level directed by each is
a function of the efficiency with which each corresponding mRNA is
translated and thus the relative intracellular accumulation of the
cognate polypeptide.
Cells transfected with cDNA 5 cells also
contain multiple immunoreactive polypeptides (Fig. 1B).
The most abundant pair of these proteins migrate more rapidly than do
the proteins detected in cells transfected with cDNAs 6, 10, and 14 yet
are approximately similar in quantity to the immunoreactive protein
directed by cDNAs 6 and 10. Because cDNA 5 directs lower levels of cell
surface sLe expression than these two cDNAs, it is
therefore possible that the lower M
immunoreactive
polypeptides found in cDNA 5-transfected cells maintain substantially
lower specific enzyme activity than do the proteins encoded by cDNAs 6,
10, and 14 or are otherwise less able to direct sLe
expression in COS-7 cells. Finally, cells transfected with cDNA 3
do not contain any detectable immunoreactive proteins. This implies
that the putative initiator codon at base pair 2126 in this cDNA does
not initiate translation of an immunoreactive product and is consistent
with the observation that this cDNA does not yield sLe
expression following transfection into COS-7 cells.
Conclusions derived from the flow cytometry and Western blot
analyses summarized above are supported by the results of in vitro (1,3)fucosyltransferase assays completed on the same cell
extracts. These assays demonstrate that cells transfected with cDNAs 5,
6, 10, and 14 contain enzyme activity that can transfer
C-labeled fucose from the nucleotide donor substrate
GDP-fucose to the low molecular weight acceptor 3`-sialyl N-acetyllactosamine (NeuNAc
2, 3Gal
1, 4GlcNAc) (Fig. 1A). For each cDNA, the product of this reaction
co-elutes with a radiolabeled sLe
tetrasaccharide standard
when fractionated by ion suppression amine adsorption high pressure
liquid chromatography (data not shown). The
(1,3)fucosyltransferase activity directed by each of these four
cDNAs does not utilize the neutral acceptor substrates N-acetyllactosamine or lacto-N-biose I. Extracts
prepared from cells transfected with cDNA 3 do not contain detectable
(1,3)fucosyltransferase activity when tested with 3`-sialyl N-acetyllactosamine nor when tested with the neutral acceptor
substrates N-acetyllactosamine or lacto-N-biose I.
These results are entirely consistent with the flow cytometry data
summarized above and indicate that this locus encodes an
(1,3)fucosyltransferase activity that apparently requires type II
acceptor substrates that are terminally substituted with an
(2,3)-linked sialic acid residue. Considered together, these
results suggest that differential splicing and/or transcriptional
initiation events can control the level of
(1,3)fucosyltransferase
activity and thus cell surface sLe
expression level through
mechanisms that depend on the efficiency with which each transcript is
translated.
Figure 3: Tissue-specific expression patterns of the murine Fuc-TVII gene. Oligo(dT) purified mRNA (5 µg) purified from murine tissues and cell lines was fractionated by agarose gel electrophoresis, blotted to a nylon hybridization membrane, and probed with a 974-base pair DNA segment derived from the coding region of the mouse Fuc-TVII locus (nucleotides 2228-3207; see Fig. 2and ``Experimental Procedures''). RNA molecular size standards, in kilobase pairs, are indicated at the left in each panel. Each blot was subsequently stripped and reprobed with a radiolabeled chicken glyceraldehyde 3-phosphate dehydrogenase probe to confirm that RNA samples were intact and loaded in equivalent amounts (see ``Experimental Procedures,'' data not shown). A, polyadenylated RNA isolated from mouse tissues and from the murine T-lymphocyte cell line 14-7fd (14FD). B, polyadenylated RNA isolated from mouse bone marrow and spleen and from cultured murine leukocyte cell lines. Cell lines represent the following lineages: MEL, murine erythroleukemia cell line; P388 and RAW (RAW 264.7), macrophage; EL4, T cell; S107, 63 (TH2.54.63) and 180.1, B cell lines (hybridomas).
Both the marrow and lung maintain several differently sized transcripts, including two abundant transcripts of approximately 1.6 and 2.2 kilobase pairs in size and a fainter transcript at approximately 3.0 kilobase pairs. These three transcripts are similar in size to the three most abundant transcripts observed in the murine 14-7fd cytotoxic T cell line. These observations suggest that cells in the bone marrow and lung yield alternatively spliced transcripts similar in structure to those characterized by cDNA cloning studies in the 14-7fd cells. These data also suggest that in the marrow, the Fuc-TVII locus is transcribed in cells assigned to the myeloid and T-lymphoid lineages but not in B-lymphoid lineage cell types and suggest that expression of this fucosyltransferase correlates with selectin ligand expression on myeloid and T-lymphocyte lineage cell types.
Figure 4:
In situ hybridization analysis of
Fuc-TVII transcripts in murine lymph nodes and Peyer's patches.
Sequential 10-micron-thick frozen sections of an axillary lymph node (A, B, and C), a mesenteric lymph node (D, E, and F), and Peyer's patches (G, H, and I) were stained with hematoxylin
and eosin (row labeled H & E, panels A, D, and G, photograph at 5 magnification using
bright field illumination) or were processed for in situ hybridization as described under ``Experimental
Procedures.'' Adjacent sections subjected to in situ hybridization were probed with an
S-labeled antisense
RNA probe derived from base pairs 2196-2497 of the murine
Fuc-TVII locus (row labeled antisense, panels B, E, and H) or were probed with an
S-labeled negative control sense RNA probe derived from
base pairs 2196-2497 of the murine Fuc-TVII locus (row labeled sense, panels C, F, and I).
Sections processed for in situ hybridization were photographed
at 5
magnification using dark field illumination. The white
areas in panels B, E, and H correspond
to sites (i.e. high endothelial venular endothelial cells in panels B and E; high endothelial venular endothelial
cells and lumenally positioned cells (the white-stained
``caps'' on the lymphoid aggregates) in panel H)
where the Fuc-TVII antisense probe identifies Fuc-TVII transcripts (see
text for details).
As noted above, not all
Fuc-TVII-derived transcripts yield a protein product.
Immunohistochemical analyses were therefore used to confirm that the
Fuc-TVII transcripts detected in HEV are accompanied by Fuc-TVII
polypeptide expression and to confirm that such expression co-localizes
with L-selectin ligand expression. A rabbit polyclonal antibody raised
against the Fuc-TVII peptide yields an intracellular staining pattern
in the endothelial cells within HEV in all three lymphoid aggregates (Fig. 5). The perinuclear intracellular staining pattern seen
with the anti-Fuc-TVII antibody is consistent with the notion that this
enzyme is localized to the Golgi apparatus, where it may participate in
the synthesis of fucosylated oligosaccharides with L-selectin ligand
activity. In each of the three types of lymphoid aggregate, expression
of immunoreactive Fuc-TVII co-localizes with expression of epitopes
recognized by the MECA-79 antibody shown previously to stain HEV and to
interfere with L-selectin binding to HEV(48) . Fuc-TVII
expression also co-localizes with expression of L-selectin ligands on
HEV, as detected with a recombinant mouse L-selectin/human IgM chimeric
protein. These observations imply that Fuc-TVII may participate in the
synthesis of the sialylated, sulfated, and (1,3)fucosylated
candidate oligosaccharide components of HEV-derived L-selectin ligands.
Figure 5:
Immunohistochemical co-localization of
expression of Fuc-TVII, MECA-79, and L-selectin ligands in lymphoid
aggregate high endothelial venular endothelial cells. a,
co-localized expression of Fuc-TVII, MECA-79, and L-selectin ligands in
HEV. Sequential 10-micron-thick frozen sections of axillary lymph
nodes, mesenteric lymph nodes, and Peyer's patches were stained
with an antigen affinity purified rabbit polyclonal anti-murine
Fuc-TVII, with the monoclonal antibody MECA-79, with a murine
L-selectin/human IgM chimera, or with the L-selectin/IgM chimera
stained in the presence of EDTA. Detection of section-bound primary
immunohistochemical reagents was subsequently accomplished using
secondary immunochemical reagents labeled with flurochromes
(FITC-conjugated (green) anti-rabbit IgG for Fuc-TVII;
FITC-conjugated (green) anti-human IgM for L-selectin/IgM;
rhodamine-conjugated (red) anti-rat IgM for MECA-79), as
described under ``Experimental Procedures.'' Photomicrographs
were taken at 40 magnification using fluorescent microscopic
procedures as described under ``Experimental Procedures.'' b, high power magnification of peripheral lymph node HEV
staining with anti-Fuc-TVII. A 10-micron-thick frozen section of an
axillary lymph node was stained simultaneously with the antigen
affinity purified anti-Fuc-TVII antibody and with anti-MECA-79 antibody (A). Section-bound antibodies were detected with
fluorochrome-conjugated secondary antibody reagents exactly as
described in the legend for a and under ``Experimental
Procedures'' (Fuc-TVII, green; MECA-79, red).
The immediately adjacent 10-micron-thick frozen section of the same
axillary lymph node was instead stained simultaneously with a pair of
negative control antibodies (normal rabbit IgG, and normal rat IgM; B) and developed in a manner identical to that used in A. Photomicrographs were taken at 400
magnification
using fluorescent microscopic procedures described under
``Experimental Procedures.''
In an effort to understand the functions of cell surface
fucosylated oligosaccharides in animals, we have established a program
to isolate murine (1,3/4)fucosyltransferase genes to be used
initially as reagents to characterize tissue-specific expression
patterns of the loci that control expression of cell surface
fucosylated oligosaccharides. These reagents and the information
gathered from their application will be used eventually with transgenic
approaches to uncover functions of their cognate cell surface
fucosylated oligosaccharides by perturbing their expression patterns.
A cross-hybridization approach outlined here yielded a novel genomic
sequence that cross-hybridizes with segments derived from the conserved
portions of the human Fuc-T's III, V, and VI genes, in a position
corresponding to their catalytic domains. Following the isolation of
this murine genomic locus, functional analyses indicated that it
encoded an (1,3)fucosyltransferase, termed Fuc-TVII, with
structural features and catalytic activities that were, at the time of
its isolation, unique to the
(1,3)fucosyltransferase family. In
particular, this locus was the only
(1,3)fucosyltransferase known
to maintain a coding region distributed over more than one exon and the
first fucosyltransferase with multiple distinct initiation codons with
the potential to yield structurally distinct polypeptides characterized
by different cytoplasmic domains but with essentially identical
catalytic activities. The catalytic activity of each Fuc-TVII isoenzyme
is characterized by an ability to utilize an
(2,3)sialylated type
II N-acetyllactosamine precursors without the ability to
utilize neutral type II, neutral type I, or sialylated type I N-acetyllactosamine substrates. Similar observations have been
made for the human homologue of Fuc-TVII isolated subsequently by our
group (27) and by others(28) . This catalytic
specificity and the leukocyte-specific expression pattern of this gene
strongly suggest that it plays a pivotal role in the biosynthetic
scheme that yields the
(2,3)sialylated and
(1,3)fucosylated
lactosaminoglycans essential to E- and P-selectin ligand activity.
Other observations made in the work described here suggest a role
for Fuc-TVII in directing synthesis of the oligosaccharide components
of the ligands for L-selectin. As Rosen and colleagues have shown,
L-selectin ligands on HEV correspond to O-linked carbohydrate
determinants displayed by the mucin-type glycoproteins GlyCAM-1, CD34,
and MAdCAM-1(13, 14, 15) . Their earlier
biochemical analyses indicate that the oligosaccharides relevant to
L-selectin ligand activity are sialylated, sulfated, and possibly
fucosylated (16) . More recent structural analyses from the
Rosen group are consistent with the hypothesis that the capping groups
on such oligosaccharides correspond to sulfated versions of the sialyl
Le moiety, with sulfate attached via the 6-hydroxyl of the
terminal galactose moiety
[NeuNAc
2,3(SO
6)Gal
1,4(Fuc
1,3)GlcNAc-R],
the 6-hydroxyl of the subterminal N-acetyl-glucosamine moiety
[NeuNAc
2,3Gal
1,4(SO
6)(Fuc
1,3)GlcNAc-R],
or both(17, 18, 19) . Nonfucosylated forms of
these structures were also identified, however, and the evidence that
fucose is required for activity of physiological L-selectin ligands
remains circumstantial.
The identification of such nonfucosylated
structures suggests the possibility that these sialylated and sulfated
molecules represent acceptor substrates for
(1,3)fucosyltransferases expressed in HEV endothelial cells. Our
observation that expression of the Fuc-TVII locus co-localizes with
L-selectin ligand expression in such cells suggests that Fuc-TVII may
operate in this context. The notion that sulfated and sialylated
lactosamine moieties represent acceptor substrates for enzymes like
Fuc-TVII is supported by studies suggesting that Fuc-TIII (62) and Fuc-TV (63) can utilize sialylated, sulfated
lactosamine-type acceptors. Indirect evidence derived from studies on
the biosynthesis of GlyCAM-1 are also consistent with this hypothesis (17, 64) . However, our results indicate that the
nonsulfated entity NeuNAc
2,3Gal
1,4GlcNAc is used in vitro and in vivo by Fuc-TVII. It therefore remains to be
determined if the two sulfated forms of this substrate are also
utilized by Fuc-TVII or if alternatively in HEV endothelial cells,
sialylation and then fucosylation in turn precede sulfation. Thus, the
biosynthetic scheme for such molecules remains to be defined,
especially in the context of the possible role for Fuc-TVII in this
pathway, and the relative order(s) of addition of sialic acid, sulfate,
and fucose, on such molecules. This work is currently in progress in
this laboratory.
There is evidence to suggest that L-selectin expressed by granulocytes and other leukocytes mediates adhesion of these cells to activated vascular endothelium through as yet undefined extracellular endothelial cell counter-receptors(3, 4) . Because the chemical nature of this counter-receptor(s) is not known, a role for Fuc-TVII in the synthesis of such ligands remains speculative and is a subject of current exploration in this laboratory. In any event, the demonstration here that Fuc-TVII is co-expressed with L-selectin ligands on HEV, when considered together with previous observations demonstrating that Fuc-TVII is expressed in leukocytes and can direct synthesis of ligands for E- and P-selectin, suggests that Fuc-TVII may represent a master control locus for the synthesis of ligands for all three selectins and thus for controlling selectin-dependent leukocyte trafficking. Experiments are in progress to address these possibilities through the generation and analysis of mice that are deficient in the Fuc-TVII locus.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U45980[GenBank].