(Received for publication, July 14, 1995)
From the
Terminal Fuc1-3GlcNAc moieties are displayed by
mammalian cell surface glycoconjugates in a tissue-specific manner.
These oligosaccharides participate in selectin-dependent leukocyte
adhesion and have been implicated in adhesive events during murine
embryogenesis. Other functions for these molecules remain to be
defined, as do the tissue-specific expression patterns of the
corresponding
-(1-3)-fucosyltransferase (
1-3FT)
genes. This report characterizes a murine
1-3FT that shares
77% amino acid sequence identity with human ELAM ligand
fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene
maps to mouse chromosome 9 in a region of homology with the Fuc-TIV
locus on human chromosome 11q. In vitro, the murine
1-3FT can efficiently fucosylate the trisaccharide
Gal
1-3Gal
1-4GlcNAc (apparent K
of 0.71 mM) to form an unusual tetrasaccharide
(Gal
1-3Gal
1-4[Fuc
1-3]GlcNAc)
described in periimplantation mouse tissues. The enzyme can also form
the Lewis x determinant from Gal
1-4GlcNAc (K
= 2.05 mM), and the
sialyl Lewis x determinant from
NeuNAc
2-3Gal
1-4GlcNAc (K
= 1.78 mM). However, it does not yield
sialyl Lewis x determinants when expressed in a mammalian cell line
that maintains sialyl Lewis x precursors. Transcripts from this gene
accumulate to low levels in hematopoietic organs, but are unexpectedly
abundant in epithelia that line the stomach, small intestine, colon,
and epididymus. Epithelial cell-specific expression of this gene
suggests function(s) in addition to, and distinct from, its proposed
role in selectin ligand synthesis.
Oligosaccharides represent major components of animal cell
surfaces and are believed to function in cellular interactions during
development and differentiation(1, 2) , oncogenic
transformation(3) , and inflammation(4) .
Identification of specific oligosaccharide ligands for the selectin
family of cell adhesion molecules directly links cell surface
carbohydrates to cell-cell communication in the context of inflammatory
response(5, 6, 7) . The proposed ligands for
E-selectin and P-selectin are fucosylated oligosaccharides (for review,
see (4) and (8) ), whose biosynthesis is catalyzed by
-(1-3)-fucosyltransferases (
1-3FTs). (
)These enzymes are encoded by one or more distinct and
tightly regulated
1-3FT genes.
Much of the interest in
discovering functional roles for oligosaccharides during development is
derived from studies documenting precise temporal-spatial expression
patterns for some oligosaccharides during human and murine
embryogenesis(9, 10, 11, 12) . The
murine stage-specific embryonic antigen-1 (SSEA-1;
(Gal1-4[Fuc
1-3]GlcNAc; Lewis x)), for
example, is expressed coincident with morula compaction at the
8-16 cell stage of the preimplantation mouse
embryo(13, 14) . Since SSEA-1 structural analogs
appear to inhibit compaction, it has been suggested that this antigen
may participate in this process(1, 2) . While it has
been proposed that the SSEA-1 determinant functions to promote
homotypic adhesion(15) , neither the physiological relevance of
this interaction during compaction nor the existence of other
preimplantation-specific receptors for SSEA-1 have been demonstrated.
Furthermore, it has not been possible to identify and directly
demonstrate functional correlates for SSEA-1 expression patterns during
early embryogenesis.
Virtually nothing is known about the molecular
mechanisms that determine the tissue-specific and developmentally
regulated expression patterns of the oligosaccharides implicated in
morphogenic events during early murine embryogenesis. Expression of
surface-localized SSEA-1 molecules may be regulated by differential
expression of 1-3FT(s) required for their synthesis (16) and of sialyltransferases (17) and an
-(1-3)-galactosyltransferase (18) that may mask
SSEA-1 expression(9, 10, 18) . The relative
contributions of these and other regulatory mechanisms, in the context
of the developing embryo, remain undefined.
To begin to define, in
detail, the enzymes and mechanisms that determine expression of
Fuc1-3GlcNAc linkages in murine cell surface
oligosaccharides, we have isolated and characterized a murine gene that
corresponds to a human
1-3FT gene termed Fuc-TIV (Refs. 19
and 20; also known as ELFT for ELAM-1 ligand fucosyl transferase, (21) ). The mouse enzyme differs from human Fuc-TIV/ELFT in its
relatively higher apparent affinity for
-(2-3)-sialylated
type acceptor substrates in vitro, and is able to efficiently
synthesize an unusual tetrasaccharide
(Gal
1-3Gal
1-4[Fuc
1-3]GlcNAc)
whose existence in periimplantation mouse tissues has been previously
inferred(10) . Northern blot analyses confirm that transcripts
corresponding to this gene accumulate in leukocytic cell lines and in
leukocyte-rich tissues in the mouse. However, these analyses, and
companion in situ hybridization studies, demonstrate that
Fuc-TIV/ELFT transcripts are unexpectedly abundant in epithelial cells
lining the gastrointestinal and reproductive tracts and suggest that
this sequence, and the cognate Fuc
1-3GlcNAc linkages whose
expression it determines may have unexpected functions in these
tissues.
Figure 1:
Nucleotide and deduced amino acid
sequences of the murine 1-3FT gene and comparison to the
human Fuc-TIV/ELFT gene and its cDNAs. The DNA sequence of the murine
gene, and its predicted protein sequences, correspond to MFT-IV
DNA, and MFT-IV AA, respectively. The DNA sequence of the
human Fuc-TIV/ELFT gene, and predicted protein sequences, correspond to
lines denoted by HFT-IV DNA, and HFT-IV AA,
respectively. The A residues of the putative initiation codon of the
murine
1-3FT gene and of the human ELFT/Fuc-TIV sequence are
assigned as residue 1 of their nucleotide sequence. The methionine
residues corresponding to these codons are assigned as residue 1 of the
corresponding protein sequences. These positions are further denoted by
an arrow pointing to these aligned initiator methionine codons (start of ELFT AA). The initiation codon for the
``long'' form of ELFT (21) is also indicated by arrows (start of ELFT AA; Initiator codon for
ELFT-L protein). The initiator methionine for the long form of the
murine polypeptide is also indicated by an arrow (start of
``long'' mouse polypeptide). This methionine codon is
encompassed within the NcoI site (dottedunderlined) used to construct the vector pcDNAI-mFuc-TIV. Arrows also indicate the 5`-most residues found in the ELFT
and ELFT-L cDNAs (21) (start of ELFT cDNA; start
of ELFT-L cDNA). The sequence alignment was generated using the
BESTFIT and GAP programs of the University of Wisconsin Genetics
Computer Group(39) . The GAP program generates symbols between
aligned amino acids, according to the evolutionary distance between
them, as measured by Dayhoff (50) and normalized by
Gribskov(51) . Amino acid sequence identities are assigned a
score of 1.5, denoted by a verticalbar; related
amino acid residues with scores from 0.5 to 1.4 are denoted by a two dots, less strongly related amino acid residues with
scores between 0.1 to 0.4 are denoted by onedot; no
symbol is placed between dissimilar amino acid pairs with scores less
than 0.1. Gaps in the amino acid sequence alignment are denoted by a dash. Nucleotide sequence identity is indicated by verticallines between aligned corresponding
residues. Gaps in the nucleotide sequence alignment are indicated by a dottedline. The predicted transmembrane domain of
the murine enzyme (amino acids 53-74) is doubleunderlined. Consensus sites for asparagine-linked
glycosylation are underlined.
Figure 3:
pH- and Mn
concentration-
1-3FT activity profiles. A,
pH-activity profiles. The enzymatic activity in COS-7 cells transfected
with pcDNAI-mFuc-TIV or pcDNAI-hFuc-TIV (20) was assayed using
20 mMN-acetyllactosamine and 3 mM GDP-[
C]fucose (see ``Experimental
Procedures''). Reactions contained 50 mM sodium acetate,
; sodium phosphate,
; or Tris-HCl,
. B,
Mn
concentration-activity profiles. The enzymatic
activity in COS-7 cells transfected with pcDNAI-mFuc-TIV or
pcDNAI-hFuc-TIV was assayed using 20 mMN-acetyllactosamine and 3 µM GDP-[
C]fucose (``Experimental
Procedures''). Reactions contained 50 mM Tris-HCl, pH
7.2.
Figure 4:
Apparent Michaelis constants for
GDP-[C]fucose determined for mouse and human
Fuc-TIVs. Apparent K
values were
determined (see ``Experimental Procedures'') in the presence
of 20 mMN-acetyllactosamine, 15 mM Mn
, and 50 mM Tris-HCl, pH 7.2. Cell
extracts prepared from COS-7 cells transfected with pcDNAI-mFuc-TIV
exhibited an apparent K
of 16.6
µM, whereas human Fuc-TIV extracts generated using plasmid
pcDNAI-hFuc-TIV maintained an apparent K
of 27.0 µM.
Figure 5:
Apparent Michaelis constants for N-acetyllactosamine and
-(2-3)-sialyl-N-acetyllactosamine, determined for
murine and human Fuc-TIVs. Apparent K
values were determined (see ``Experimental
Procedures'') in the presence of 3 µM GDP-fucose, 15
mM Mn
, and 50 mM Tris-HCl, pH 7.2. A, N-acetyllactosamine K. Using N-acetyllactosamine as the acceptor, cell extracts prepared
from COS-7 cells transfected with pcDNAI-mFuc-TIV exhibited an apparent K of 2.05 mM, whereas human Fuc-TIV extracts
generated using plasmid pcDNAI-hFuc-TIV exhibited an apparent K of 3.82 mM. B,
2`-sialyl-N-acetyllactosamine K. Apparent K values using
-(2-3)-sialyl-N-acetyllactosamine
as the acceptor were 1.78 mM and 6.74 mM for the
murine and human Fuc-TIVs, respectively. C,
Gal
1-3Gal
1-4GlcNAc K. The apparent K value using Gal
1-3Gal
1-4GlcNAc as the
acceptor was 0.71 mM for murine
Fuc-TIV/ELFT.
Reactions containing neutral acceptors (N-acetyllactosamine, lactose, lacto-N-biose I,
2`-fucosyllactose, Gal1-3Gal
1-4GlcNAc) 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-X2-400, formate form(35, 37) . 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
-(2-3)-sialyl-N-acetyllactosamine 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-X8-200
column (1 ml) prepared in the phosphate form. The reaction product was
collected in the eluate and quantitated as described
previously(43) .
The structure of the product obtained with
-(2-3)-sialyl-N-acetyllactosamine was confirmed by
HPLC fractionation, before and after neuraminidase digestion, as
described previously(20) . The structures of the products
obtained with the neutral acceptor substrates (N-acetyllactosamine, lactose, lacto-N-biose I,
2`-fucosyllactose) were also confirmed by HPLC fractionation, using
methods described
previously(20, 37, 44, 45) . The
structure of the radiolabeled product
(Gal
1-3Gal
1-4([
C]Fuc
1-3)GlcNAc)
obtained with the neutral trisaccharide
Gal
1-3Gal
1-4GlcNAc was confirmed in separate
experiments, using methods described
previously(20, 37) . The product was purified by
chromatography on Dowex 1-X2-400, formate
form(35, 37) , and subsequently fractionated by amine
absorption HPLC (Dynamax 60A column, Rainin Instruments; isocratic
gradient in 70% acetonitrile, 30% water; flow rate of 1
ml/min)(37, 44, 45) . This product was
identified as a tetrasaccharide by virtue of co-elution, at 19 min,
with a radiolabeled trisaccharide standard
(Fuc
(1-2)Gal
1-4([
C]Fuc
1-3)GlcNAc).
The tetrasaccharide product was digested with 0.01 units of jack bean
-galactosidase (Boehringer Mannheim) for 1 h at 37 °C in 100
mM Tris, pH 6.5. The digest was desalted by Dowex
chromatography and fractionated by HPLC exactly as described above for
the product. The product of this digestion was identified as a
trisaccharide by virtue of co-elution, at 13.4 min, with the
trisaccharide standard
(Gal
1-4([
C]Fuc
1-3)GlcNAc.
Cryosections (10 µm thick) were
prepared with Jung Frigocut 2800N cryostat (Leica) from organs of 129/J
strain mice. Tissue sections were fixed in 4%
paraformaldehyde/phosphate-buffered saline for 30 min at room
temperature. After proteinase K treatment and then acetylation by
acetic anhydride, sections were incubated with antisense or sense
probes (2 10
cpm/µl) for 18 h at 55 °C in
20 mM Tris-HCL, pH 8.0, 300 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 50% formamide, 10%
dextran sulfate, 5
Denhardt's solution, 50 mg/ml heparin,
0.1 M dithiothreitol, and 0.5 mg/ml Escherichia coli tRNA. The slides were washed for 30 min at 65 °C in 50%
formamide, 2
SSC, 10 mM dithiothreitol. The sections
were then subjected to digestion with 10 µg/ml of ribonuclease A at
37 °C to eliminate residual nonbase-paired probe and were then
washed again in the wash solution described above. Slides were exposed
for 2 weeks using Kodak NBT-2 emulsion and processed using D-19
developer and fixer (Eastman Kodak Co.). The sections were subsequently
stained with hematoxylin and eosin.
A description of the probes and RFLPs for the loci linked to Fut4 including murine macrophage metalloelastase (Mmel), low density lipoprotein receptor (Ldlr), and erythropoietin receptor (Epor) has been reported previously(48) . Recombination distances were calculated as described previously (49) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
This
hybridization screen also identified phages containing a 1.4-kb NcoI-SspI fragment that cross-hybridizes with the
human 1-3FT gene encoding
Fuc-TIV(19, 20, 21) . Sequence analysis of
the gene fragment identifies a single long open reading frame (Fig. 1), which begins with a methionine codon located within a
sequence context largely consistent with Kozak's consensus rules
for mammalian translation initiation(52) . Hydropathy analysis (53) of the protein sequence predicted by this open reading
frame identifies a single 22-amino acid hydrophobic segment at the
NH
terminus, implying that the polypeptide has a type II
transmembrane topology typical of mammalian
glycosyltransferases(16, 54) . Sequence comparisons
made between the predicted murine protein and human
1-3FTs
identify significant primary sequence similarities; the murine protein
maintains approximately 33, 33, 39, and 37% amino acid sequence
identity with the human Fuc-TIII(37) , Fuc-TVI(45) ,
Fuc-TVII (55, 56) , and Fuc-TV (57) enzymes,
respectively (data not shown). However, it is most similar to human
Fuc-TIV(19, 20, 21) , which shares 77% amino
acid sequence identity with the murine protein (304 identities at 396
aligned residues; Fig. 1). This sequence similarity includes the
conservation of two consensus sites for asparagine-linked glycosylation (Fig. 1).
Maximal sequence similarity is achieved by aligning
the murine DNA sequence in a colinear manner with the sequence of the
human Fuc-TIV gene (19, 20) and its cDNA(21) .
Like the human Fuc-TIV/ELFT locus, the murine gene apparently maintains
a single coding exon. Primer extension experiments and RNase protection
analyses designed to define the transcriptional initiation site for
this murine gene have failed because of the tendency for its transcript
to form secondary structure that is resistant to denaturation (data not
shown). This is most probably a function, in part, of the
extraordinarily high G+C content within the 5` end of this gene.
We also believe these observations explain our inability to isolate
full-length murine Fuc-TIV cDNAs from multiply screened high quality
cDNA libraries (data not shown). Nonetheless, comparison of the DNA
sequences of the murine and human genes through positions exceeding 400
base pairs proximal to their respective initiation codons discloses
that their respective 5`-flanking regions are identical at 66% of the
aligned positions (Fig. 1). This high level of DNA sequence
identity is maintained throughout the region of the human gene where it
is co-linear with Fuc-TIV/ELFT mRNA transcripts (Fig. 1), as
defined by cDNA cloning experiments(21) , which in turn yield
Fuc-TIV/ELFT or a longer form of this enzyme (ELFT-L; (21) ).
The murine gene also has the potential to yield a longer form of its
polypeptide product. This longer sequence is represented by a
polypeptide initiating at a methionine codon located 33 codons 5` to
the human Fuc-TIV/ELFT initiator methionine codon, extending the
shorter murine polypeptide by 33 amino acid residues at its NH terminus (Fig. 1). This longer murine sequence maintains
52% amino acid sequence identity with the ELFT-L polypeptide in a
region immediately proximal to the Fuc-TIV/ELFT initiator methionine
residue (Fig. 1). These observations further support the
conclusion that the murine gene maintains a structural organization
essentially identical to that of the human Fuc-TIV/ELFT gene.
Figure 2:
Flow cytometry histograms of COS-7 cells
transfected with the murine Fuc-TIV gene. Cells transfected with either
the 1-3FT expression vector pcDNAI-mFuc-TIV or the control
vector pcDNAI were stained with the monoclonal antibodies indicated in
the inset and were subjected to flow cytometry analysis
(``Experimental Procedures''). The data presented are the
mean (linear) fluorescence intensities of the antigen-positive
population of transfected cells (see ``Experimental
Procedures'').
Kinetic analyses of murine and human Fuc-TIV enzymes indicate that
they exhibit typical substrate concentration-dependent Michaelis-Menten
kinetics ( Fig. 4and 5). The calculated apparent Michaelis
constants for the donor substrate GDP-fucose are 16.6 and 27.0
µM for the murine and human enzymes, respectively (Fig. 4). The apparent Michaelis constants for the acceptor
substrates N-acetyllactosamine and
-(2-3)-sialyl-N-acetyllactosamine are 2.05 and 1.78
mM, respectively, for the murine enzyme, and 3.3 mM and 6.74 mM, respectively, for human Fuc-TIV (Fig. 5). Both enzymes utilize the neutral type II acceptor
molecules 2`-fucosyllactose and lactose with efficiencies that are
substantially less than those obtained with either
-(2-3)-sialyl-N-acetyllactosamine or N-acetyllactosamine. For example, a single preparation of the
murine enzyme utilized 2`-fucosyllactose and lactose at rates that were
7% (1.1 nmol/mg
h) and 1% (0.2 nmol/mg
h), respectively, of
the rate obtained with N-acetyllactosamine (16.6
nmol/mg
h). Only trace amounts of fucosylated product were
produced when the neutral type I acceptor lacto-N-biose I was
assayed with the same preparation of murine enzyme (0.05
nmol/mg
h). These results are virtually identical to those
obtained when extracts containing human Fuc-TIV activity are assayed
with the latter three substrates(20, 55) . Thus, when
considered together with the DNA and protein sequence comparisons and
the flow cytometry analyses, these biochemical data indicate that the
open reading frame shown in Fig. 1encodes an
1-3FT
activity that corresponds to a human ``myeloid''-type
1-3FT
(Fuc-TIV/ELFT)(19, 20, 21, 58, 59) .
The Gal1-3Gal
1-4GlcNAc trisaccharide is a cell
surface oligosaccharide epitope expressed by many murine
tissues(60) . There is indirect evidence for a fucosylated form
of this epitope in murine tissues
(Gal
1-3Gal
1-4[Fuc
1-3]GlcNAc; (10) ). In vitro studies using a human
1-3FT suggest that fucosylation can be a terminal step in
the synthesis of this tetrasaccharide(61) . We find that the
murine
1-3FT can efficiently utilize the trisaccharide
Gal
1-3Gal
1-4GlcNAc (with an apparent K
= 0.71 mM; Fig. 5C) to form a fucosylated product corresponding to
the tetrasaccharide
Gal
1-3Gal
1-4[Fuc
1-3]GlcNAc
(see ``Experimental Procedures''). This observation suggests
that murine glycoconjugates containing the
Gal
1-3Gal
1-4GlcNAc trisaccharide represent one
authentic acceptor substrate for this murine
1-3FT. Although
the human Fuc-TIV enzyme can also form this tetrasaccharide product in vitro (data not shown), this reaction has no apparent
physiological relevance since the human genome does not encode a
functional
(1-3)-galactosyltransferase capable of
synthesizing Gal
1-3Gal
1-4GlcNAc precursors(62).
Figure 6:
Fut4 maps in the proximal region of mouse
chromosome 9. Fut4 was placed on mouse chromosome 9 by
interspecific backcross analysis. The segregation patterns of Fut4 and flanking genes in 138 backcross animals that were typed for
all loci are shown at the top of the figure. For individual
pairs of loci, more than 138 animals were typed (see text for details).
Each column represents the chromosome identified in the backcross
progeny that was inherited from the (C57BL/6J M.
spretus)F
parent. The shadedboxes represent the presence of a C57BL/6J allele, and whiteboxes represent the presence of M. spretus allele. The number of offspring inheriting each type of chromosome
is listed at the bottom of each column. A partial chromosome 9
linkage map showing the location of Fut4 in relation to linked
genes is shown at the bottom of the figure. Recombination
distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes
are shown to the right. References for the human map positions
of loci cited in this study can be obtained from the Genome Data Base,
a computerized database of human linkage information maintained by The
William H. Welch Medical Library of The Johns Hopkins University
(Baltimore, MD).
The proximal region of mouse chromosome 9 shares a region of homology with human chromosomes 11q and 19p (summarized in Fig. 6). The human Fuc-TIV gene (locus designation FUT4) was initially assigned to human 11q12-qter(63) . More recently, the human map position has been refined to 11q21(64) . These studies provide additional support for concluding that this murine gene is the human homologue of Fuc-TIV gene, and confirm and extend the region of homology between mouse chromosome 9 and the long arm of human chromosome 11.
Figure 7: Tissue-specific expression patterns of the murine Fuc-TIV gene. Polyadenylated RNA samples (3 µg) prepared from various murine tissues (panelA) and cell lines (panelB) were subjected to Northern blot analysis as described under ``Experimental Procedures.'' The blot was probed with the NcoI-SspI fragment using hybridization and wash conditions detailed under ``Experimental Procedures.'' 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). RNA molecular size standards, in kb, are indicated at the left.
Northern blot analysis of cultured blood cell-type cell lines indicates that the Fuc-TIV transcript is relatively abundant in the murine erythroleukemia cell line MEL(26, 27) , substantially less abundant in the RAW macrophage-derived line(28, 29) , and not detectable in the P388 macrophage-derived cell line (30) or the three lymphoid cell lines we examined (T-cell line EL4, (23) ; mature B-cell lines (hybridomas) S107, (22) ; TH2.54.63, (24) ; and 180.1, (25) ). These results suggest that the murine Fuc-TIV gene, like its human counterpart, may be expressed in cells derived from the myeloid lineage but not in abundance in cells of the lymphoid lineage.
In situ hybridization analysis was used to delineate the cell types within some of the organs where relatively abundant levels of this transcript are present (Fig. 8). These experiments demonstrate that the Fuc-TIV transcript accumulates to substantial levels within the epithelial cells lining the stomach and colon (Fig. 8). The Fuc-TIV transcript accumulates to a lesser degree within the epithelial cells (both absorptive cells and goblet cells) lining the small intestinal villi, and the mucus glands within the small intestine. Fuc-TIV transcripts are also evident in the epithelial cells lining the epididymus (Fig. 8) but are not visible within the testis proper (data not shown). These observations correspond well with Fuc-TIV transcript abundance seen on the Northern blot analyses (Fig. 7). No Fuc-TIV transcripts are detected in the kidney or the lung by in situ hybridization (data not shown), even though low to moderate levels of the Fuc-TIV transcript are evident on Northern blot analysis. It remains to be determined if this apparent discrepancy may be accounted for by the relatively insensitive nature of the in situ hybridization method and/or by Fuc-TIV transcripts in blood cells within the larger vessels of these organs. These cells may contribute to the Northern blot signals but may be eliminated from the tissues prior to or during preparation for in situ hybridization. Fuc-TIV transcripts are not detected in murine neutrophils (data not shown); we have yet to define which blood cell lineages, and which maturation stage(s) of those lineages, are responsible for the Fuc-TIV transcripts observed in bone marrow mRNA.
Figure 8:
In situ hybridization analysis of Fuc-TIV
transcripts in murine tissues. Mouse stomach (A-C),
small intestine (D-F), colon (G-I), and
epididymus (J-L) were hybridized with antisense (A, B, D, E, G, H, J, K) or sense (C, F, I, L) mouse Fuc-TIV probes. PanelsA, D, G, and J are
bright-field photomicrographs, and panels B, C, E, F, H, and I are dark-field
photomicrographs, each taken at 40 magnification. MS,
muscle layer; GL, glands; VL, villi. Arrows in panel J point to the tubular lumenae within the
epididymus.
Biochemical studies indicate that selectin ligand expression
in myeloid and lymphoid lineages is controlled in part by cell
type-specific expression of one or more 1-3FTs and that
these enzymes consequently play a pivotal role in the human
inflammatory response(5, 6, 7, 8) .
Evidence also suggests that the aberrant expression of these enzymes in
malignancy may facilitate the spread of transformed cells via
selectin-dependent metastatic
processes(5, 6, 7, 63, 64, 65, 66, 67) .
The developmentally regulated expression patterns of
-(1-3)-fucosylated oligosaccharides in mammalian
embryos(2, 9, 10, 11, 12) further imply that these molecules have additional,
undefined functions. Given logistical and ethical considerations, the
laboratory mouse is a useful system to analyze embryonic and adult
1-3FT gene expression patterns and to perturb these patterns
through transgenic and embryonic stem cell approaches.
The
polypeptide product of the murine gene described here shares 77% amino
acid sequence identity with human
Fuc-TIV(19, 20, 21) . Inter-species sequence
comparisons have not previously been made for any 1-3FT
sequences; the degree of sequence identity observed is comparable with
levels reported for other cross-species comparisons of
glycosyltransferase sequences (68, 69, 70) and suggest that this murine
gene is the orthologous homologue (71) of human Fuc-TIV. This
assignment is further supported by their corresponding chromosomal
localizations and by the genomic organization of the murine gene and
the human Fuc-TIV gene, both of which apparently maintain intronless
coding sequences.
Comparison of the catalytic properties of the
murine enzyme with those of the human Fuc-TIV enzyme provides
additional evidence for their homologous nature. Transfection studies
indicate that both enzymes can determine expression of
surface-localized Lewis x molecules, but not sialyl Lewis x moieties or
products based on type I precursors. The mouse and human enzymes
maintain similar affinities for the type II acceptor N-acetyllactosamine, in vitro (2.05 and 3.3
mM, respectively), but differ somewhat in their apparent
affinities for -(2-3)-sialyl-N-acetyllactosamine.
Specifically, the human enzyme exhibits a rather higher apparent K
(6.7 mM) for the sialylated substrate
than does the mouse enzyme (1.8 mM). Nevertheless, the murine
enzyme does not utilize oligosaccharides terminating with
-(2-3)-sialylated type II chains when it is expressed in the
cultured cell lines used here. The apparent discrepancy presented by
the ability of Fuc-TIV to utilize
-(2-3)-sialylatedtype II
oligosaccharides in vitro, but not in vivo, may be
resolved by considering recent results indicating that Fuc-TIV resides
in a Golgi compartment proximal to
-(2-3)-sialyltransferase
and thus may not have an opportunity to operate upon such substrates
within a cell(72) . These observations further emphasize that
it is difficult to reliably predict the spectrum of oligosaccharide
products that will be constructed in a specific cell lineage by a given
glycosyltransferase solely by considering the results obtained from in vitro assays using low molecular weight oligosaccharide
acceptors. This notion is strongly reinforced by results indicating
that the human Fuc-TIV gene can, under some circumstances, determine
cell surface sialyl Lewis x expression in transfected cells, and that
this outcome is critically dependent upon the glycosylation phenotype
of the host cell in which Fuc-TIV is expressed(73) . Thus,
while it seems likely that the murine Fuc-TIV enzyme creates
Fuc
1-3GlcNAc linkages in murine tissues, it is not yet
possible to predict which murine glycoconjugate acceptors will be
utilized by this
1-3FT, and, consequently, we cannot yet
predict the cell surface products created by this enzyme.
However,
one such molecule may correspond to the terminal oligosaccharide
structure Gal1-3Gal
1-4GlcNAc. This determinant is
widely expressed in murine tissues(60) , and, as we have shown
here, is efficiently utilized, in vitro, by the murine
Fuc-TIV/ELFT enzyme. Prior work has provided indirect evidence for the
expression of the
-(1-3)-fucosylated form of this molecule
(Gal
1-3Gal
1-4[Fuc
1-3]GlcNAc)
by murine cell surface glycoconjugates during the periimplantation
period(10) . This tetrasaccharide has also been synthesized in vitro by a sequential enzyme-assisted synthetic scheme
involving
-(1-3)-galactosylation of N-acetyllactosamine to form
Gal
1-3Gal
1-4GlcNAc, followed by
-(1-3)-fucosylation with an
1-3FT isolated from
human milk(61) . However, since in some cell types the murine
1-3 galactosyltransferase responsible for synthesis of the
Gal
1-3Gal
1-4GlcNAc precursor trisaccharide is
most probably localized to a Golgi compartment coincident with
-(2-3)-sialyltransferase, and distal to the compartment
where Fuc-TIV is located(74) , it seems probable that
simultaneous expression of the Fuc-TIV and
-(1-3)-galactosyltransferase genes will not necessarily
yield expression of this tetrasaccharide. Additional information
concerning the expression pattern of this tetrasaccharide will await
the development of antibodies directed against this molecule.
The
Gal1-3Gal
1-4[Fuc
1-3]GlcNAc
tetrasaccharide represents a noncharged analogue of the sialyl-Lex
determinant, prompting a suggestion that it may represent a possible
ligand for the selectins (61) . It is not yet known if this
tetrasaccharide can participate in selectin-dependent cell adhesion
processes or if it is expressed by murine neutrophils, although the
precursor trisaccharide determinant is displayed by these cells. (
)It is interesting to note in this context that the
oligosaccharide portion of the murine E- and P-selectin
counter-receptors are not yet defined, although the sialyl Lewis x
determinant is not expressed on murine leukocytes(75) , at
least as defined by the use of the monoclonal antibody CSLEX. The
tissue-specific expression patterns, and functions, if any, of this
tetrasaccharide molecule thus remain an open and interesting question.
Northern blot analyses identify a single mouse Fuc-TIV transcript in all tissues where the gene is expressed, whereas the human Fuc-TIV gene generates multiple transcripts(19, 20, 21) . By analogy to other mammalian glycosyltransferase mRNAs(16, 54) , the relatively large size of the mouse Fuc-TIV transcript suggests that it contains substantial amounts of 3`- and/or 5`-untranslated segments. The structure of this transcript remains to be determined by cloning and sequencing of the cDNA(s) derived from this gene. While the murine transcript is detectable in bone marrow, the specific marrow cell types that express this gene remain to be precisely defined. It is likely, however, that the marrow transcripts are derived in part from myeloid-lineage cells since the human Fuc-TIV gene is expressed in myeloid cells(19, 20, 21) . The murine erythroid lineage may also contribute to expression in the bone marrow since transcripts are detected in the mouse erythroleukemia line MEL. The murine transcript is also relatively abundant in the epithelial cells lining the colon and stomach, with somewhat lesser amounts in small intestinal epithelial cells. The function of this enzyme in these locations remains unknown, although it is interesting to speculate that it serves to participate in the synthesis of fucosylated mucins that may operate to protect the gastrointestinal lining from destructive effects of digestive enzymes or from ingested pathogens. The low levels of the Fuc-TIV transcript in the testes is accounted for by transcripts that accumulate in the epididymus. The low levels of Fuc-TIV message detected in the kidney, and the trace amounts identified in brain and heart, may represent transcripts derived from specific cell types within the parenchyma of these organs. Further in situ hybridization experiments will be required to confirm this possibility.
These results indicate that while the murine and human
Fuc-TIV genes are similarly organized, there may be substantial
interspecies differences in the structural, functional, and regulatory
properties of the orthologous 1-3FTs. Evidence suggests that
the functional roles of their oligosaccharide products may also differ
in significant ways. The expression patterns exhibited by the mouse
1-3FT gene are different from those shown by a rat
-(2-6)-sialyltransferase gene (76, 77) , a
constitutively expressed mouse
-(1-4)-galactosyltransferase
gene(78, 79) , a mouse
-(1-3)-galactosyltransferase gene(60) , and a series
of human sialyltransferase genes. When considered together with these
data, our results provide additional support for the notion that
transcriptional control of glycosyltransferase gene expression
regulates cell surface glycosylation(54) .
Functional
correlates for the regulation of this 1-3FT remain to be
explored. In this context, we have compared our interspecific map of
chromosome 9 with a composite mouse linkage map that reports the map
location of many uncloned mouse mutations. (
)Fut4 maps in a region of the composite map that lacks mouse mutations
with a phenotype that might be expected for an alteration in this locus
(data not shown). The task of assigning function to this locus may be
facilitated by a more complete understanding of the types, structures,
and expression patterns of this and other murine glycosyltransferase
genes and their cognate oligosaccharide products, in concert with
approaches that utilize transgenic and gene ablation technologies in
the mouse.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33457 [GenBank]and U33458[GenBank].