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(Received for publication, April 14, 1995; and in revised form, May 24, 1995) From the
The human
Most
non-hematopoietic malignancies in adults involve malignant
transformation of epithelial cells of a particular organ. One example
of these highly prevalent diseases is adenocarcinoma of the colon,
where studies have correlated metastasis and invasion with tumor cell
expression of fucosylated lactosaminoglycans(14, 15) .
Interpretation of these results and the underlying mechanisms involved
is difficult due to the complex and changing array of Molecular cloning studies to date have resulted in the
isolation of five human genes encoding
Figure 5:
Schematic and sequence of alternatively
spliced and polyadenylated FUT6 transcripts. A, genomic
structure and position of exons A-G are shown in the top line of the
figure. All transcript structures are confirmed by standard RT-PCR,
extended length RT-PCR, 5`-RACE, and 3`-RACE (``Experimental
Procedures''). Primers used for 5`-RACE and RT-PCR experiments are
shown (U1, U2, L1, and L2 sequences are in Table 2). Major
transcripts are detectable on Northern analyses and in at least three
cDNA clones obtained by hybridization (Major I, six clones; Major II,
five clones; Minor I, two clones; Minor II, two clones; Minor III, one
clone). The 3.5-kb FUT6 transcript visible on Northern blot analyses (e.g. kidney and liver) is mostly comprised of the Major I
species. The 2.5-kb transcript (e.g. colon and liver)
corresponds to Major II. B, sequences of exons A-G. Splice
consensus sequences (intronic) are in lower case. Dotted
underlining in exon G denotes highly repetitive sequences similar
to human Alu motifs (61). Start and stop codons are shown in bold type.
The coding region is as previously published(22) . 3`-RACE
experiments and genomic sequence comparisons confirm use of two
polyadenylation signal sequences (proximal, underlined;
distal, double-underlined) and two downstream cleavage sites
(TA&cjs1219; and CA&cjs1219;,
respectively).
Figure 7:
FUT6 isoform schematic, sequence, and
expression. A, schematic representation of FUT6 isoforms. Numbers in parentheses refer to previously published
codon numbers(22) . Relative to FUT6 wild type sequence,
splicing within the coding region results in the loss of 12 (Isoform I)
and 82 (Isoform II) carboxyl-terminal amino acid residues. Consensus
sequences for both splice events are shown. Asterisks refer to
the stop codon present in the open reading frame of the isoforms.
Restriction enzyme sites used to create isoform expression constructs
are noted (``Experimental Procedures''). B, amino
acid and nucleotide sequence of FUT6 isoforms. Numbers and residues in parentheses refer to previously published
FUT6 sequence (22) . The italicized subsequence in Isoform II
is identical to the sequence of Isoform I. C, RT-PCR was
performed as described under ``Experimental Procedures''
using primers noted in the schematic. cDNAs were generated with L1
(from exon G) and amplified with U/L3. For colon, a second cDNA
synthesis was performed with a lower strand primer from exon F (L2),
followed by the same PCR amplification with U/L3. PCR products were
electrophoresed on a 1% agarose gel and stained with ethidium bromide.
The amplified fragments are 1140 bp (wild type, highest levels), 1045
bp (isoform I), and 865 bp (isoform II). Isoform I is expressed at
higher levels than isoform II (>10-fold) in kidney (K).
Liver (L) shows lower expression of both isoforms than kidney.
For colon (C), amplifiable levels of cDNA are not generated
with the L1 primer from exon G (no wild type or isoform fragments in
first C lane). When colon cDNAs are synthesized with the exon
F lower strand primer (L2), high levels of 1140-bp wild type fragment
are amplified, but no isoform transcripts are detectable (second C
lane).
Figure 1:
Northern blot analyses of human tissues
using coding region probes. 20 µg of poly(A)
Figure 2:
PCR analyses of human tissues and cells
using coding region primers. 0.2 µg of poly(A)
High levels of FUT3
transcript were found in colon, stomach, small intestine, lung, and
kidney (Fig. 1). Lesser amounts were seen in salivary gland,
bladder, uterus, and liver (Fig. 2). The predominant FUT3
transcript present in most tissues is 2.37 kb. FUT5 transcripts were
not detectable by hybridization techniques (data not shown). By RT-PCR,
FUT5 expression was restricted in quantity and distribution to liver,
colon, and testicle (Fig. 2). Trace amounts of FUT5 message were
also detected in unselected T lymphocytes, HL-60 cells, and brain. For
FUT6, a 3.5-kb transcript was seen in kidney, liver, and small
intestine; a 2.5-kb transcript was present in liver, colon, salivary
gland, bladder, and uterus (Fig. 1). Because of the
tissue-specific differences in FUT3 and FUT6 transcripts shown here,
kidney, liver, and colon were chosen for further study.
Figure 3:
Schematic, sequence, and expression of
alternatively spliced FUT3 transcripts. A, genomic structure
and organization of exons A-C are shown in the top line of the figure.
Splice acceptor sequences for Major transcripts I and II are denoted B" and B`, respectively
(italicized). All transcript structures were confirmed by RT-PCR,
5`-RACE, and 3`-RACE (``Experimental Procedures''). Primers
used for 5`-RACE and RT-PCR experiments are shown (U, L1, L2 sequences
are in Table 2). For this figure, major transcripts are defined
as those present on Northern analyses and in at least two cDNA clones
obtained by cross-hybridization (``Experimental Procedures'';
Major I, two clones; Major II, four clones; Minor I, one clone; Minor
II, one clone). No alternative splicing or polyadenylation events were
found in the 3`-UT region of FUT3. The previously published cDNA for
FUT3 (17) is depicted as Minor II. The 2.37-kb transcript on
Northern analyses is mostly comprised of the Major II species. FUT3
Minor I (with exon B) is found in liver. B, sequences of exons
A-C. Positions of B` and B" are shown (/). Intronic
splice consensus sequences are in lower case. The start codon
is shown in bold type. The mono-exonic coding region is as previously
published(17) . 3`-RACE experiments and genomic sequence
comparison confirm the polyadenylation signal sequence (underlined) followed by the cleavage site (&cjs1219;) 19
nucleotides downstream (CA, differs from previously published GA, (17) ). C, RT-PCR analyses (``Experimental
Procedures'') were performed using lower strand primers from the
FUT3 coding region and an upper strand primer from exon A (Table 2). PCR products were electrophoresed in a 1% agarose gel
and stained with ethidium bromide. Fragments corresponding to Major I
(545 bp), Major II (599 bp), and Minor I (948 bp) are seen in kidney (lane K). In liver, only the Minor I transcript is amplified (lane L). In colon (lane C), the 599-bp fragment
(corresponding to Major II) is found at highest levels. Minor
transcript II is found at lower levels in colon (409 bp). An additional
faint 635-bp fragment, amplified at trace levels in colon, can also be
seen in lane C. This minor species was not detectable in most
specimens and is not depicted in A.
The 5` exons in Fig. 3were mapped by 5`-RACE and genomic sequence analyses (data
not shown). All FUT3 transcripts were shown to start with Exon A.
Preferential selection of the 3` splice acceptor for exon B` (Fig. 3) corresponds to the length of the polypyrimidine tract
preceding it and correlates with high levels of the 2.37-kb message (e.g. colon, Fig. 1). No alternative splicing or
polyadenylation was found by RACE analyses of the 3`-UT region of FUT3
in these tissues. A minor 3`-UT sequence difference with the A431
transcript (17) was noted, with the major transcript
polyadenylation cleavage site CA predominating in normal tissues (Fig. 3). G/T clusters are present in genomic sequence
immediately downstream of this site (data not shown).
Figure 4:
Schematic and sequence of FUT5
transcripts. A, genomic structure and position of exons A and
B are shown in the first schematic. Transcript structures were
confirmed by RT-PCR, 5`-RACE, and 3`-RACE (``Experimental
Procedures''). Primers used for 3`-RACE and RT-PCR experiments are
shown (U1, U2, L1, L2 sequences are in Table 3). FUT5 transcripts
were not detectable by Northern blot analyses but were isolated by
cross-hybridization screenings (``Experimental Procedures'';
Minor I, one clone; Minor II, one clone). Minor transcript I was
present at higher levels than Minor transcript II in all PCR assays
(>10-fold, data not shown). No alternative splicing events were
found in the 5`-UT or 3`-UT regions of FUT5. B, sequences of
exons A and B. Splice consensus sequences (intronic) are in lower
case. The start and stop codons are shown in bold type. The
mono-exonic coding region is as previously published(21) .
3`-RACE experiments and genomic sequence comparisons confirm that the
3`-UT is colinear with genomic sequence until nucleotide 1723. Cleavage
and polyadenylation are directed by two previously unpublished signal
sequences (underlined). Terminal GA and CA sequences
(respectively) are depicted (&cjs1219;). The proximal cleavage and
polyadenylation events are found in most liver and colon
transcripts.
Figure 6:
Northern blot analysis using FUT6 5`-UT
region exonic probes. 15 µg of poly(A)
In the FUT6 3`-UT region, alternative
splicing of two exons containing disparate polyadenylation sites is
shown. The proximal signal, ATTAAA, which is reported to occur in 12%
of mRNAs compiled from many species(32) , reduces 3` maturation
to 4% of control in comparative studies(33) . This low
efficiency signal is responsible for polyadenylation of 3`-UT sequence
that is contiguous with the coding region (Fig. 5).
Cleavage/polyadenylation occurs 12 bp downstream of this signal at a TA
terminus. The distal site contains two overlapping consensus signal
sequences for polyadenylation, AATAAA, but exists on a separate 245-bp
exon (Fig. 5, exon G). Here, cleavage/polyadenylation occurs 21
bp downstream of the poly(A) signal at a CA terminus. Both signals are
flanked by G/T-clusters (data not shown). The proximal, less efficient
polyadenylation event (e.g. colon) and the exon G
splice event (bringing in the distal, more efficient signal, e.g. kidney) appear to be competitive
post-transcriptional
modifications(34, 35, 36) . In liver, these
disparate events occur with equal frequency (Fig. 6).
Immunohistologic and biochemical surveys of non-diseased
human organs containing a large proportion of epithelial cells suggest
that at least two Transcript surveys of leukemia and epithelial
carcinoma cell lines (28) have shown the presence of multiple
fucosyltransferase transcripts in a given line, with highest expression
levels of FUT3 and FUT6 transcripts in transformed epithelia, and much
lower levels of these messages in leukemia cells. Conversely, the
We studied expression of the 19p13.3
In kidney, high levels of Lewis x
and sialyl Lewis x are found on glomerular podocytes and proximal
tubular cells(27, 37) . Transitional epithelia of the
calyx, ureter, and bladder appear to have lower levels of fucosylated
glycan expression. The functional significance of this distribution is
not known. Our results are consistent with high renal parenchymal
expression of sialyl Lewis x and confirm that FUT3 and FUT6 are
responsible for this biosynthesis. Our results also correlate with
enzymatic data showing fully differentiated kidney to possess high
levels of Lewis (calyceal) and plasma (tubular) fucosyltransferase
activities(13, 27) , with developing kidney having a
myeloid-like pattern. In situ hybridization with FUT3 and FUT6
riboprobes composed of flanking exons will be required to verify this
distribution. Normal human liver tissue contains both plasma and
Lewis fucosyltransferase
activities(9, 12, 38) . Previous studies have
correlated increased In contrast, normal colon contains high levels of the
FUT6 2.5-kb transcript and virtually no 3.5-kb transcript. Other
gastrointestinal mucosa do not contain high amounts of FUT6 message,
but instead show strong expression of the FUT3 2.37-kb transcript (e.g. stomach, Fig. 1). Our results correlate with
recent enzymatic analyses of plasma and Lewis fucosyltransferase
activities in benign and malignant gastrointestinal
mucosa(41) . Normal stomach mucosa shows low
Another human tissue expressing high levels of FUT6
2.5-kb message was salivary gland. The high level of expression of FUT6 versus FUT3 transcripts was unexpected, given the
concentration of Lewis enzyme activity in
saliva(42, 43) , but was also highly reproducible (n = 8 specimens). Enzymatic assays of salivary gland
and other epithelial tissue extracts do not necessarily correlate with
FUT3 and FUT6 undergo extensive post-transcriptional
processing of the 5`-UT region. While their coding regions are highly
homologous(22) , the 5`-UT regions share no homology, are
expressed in a tissue-specific manner, and have different
organizations. Similar splicing complexity restricted to the 5`-UT
region has been described in other human gene families. Diverse mRNAs
encoding tryptophan hydroxylase, for example, are generated from a
single promoter followed by extensive splicing upstream of the
translation start site(49) . FUT3 5`-UT exonic organization (Fig. 3) and upstream genomic sequence are also consistent with
alternative splicing following transcription initiation from a single
site. In the more complex case of FUT6, we cannot presently
rule out transcription initiation from alternative promoters followed
by differential splicing of intervening sequences in the 5`-UT region.
Particularly suggestive of this organization is the presence of exon A
only in the 3.5-kb transcript, exon C in both the 3.5- and 2.5-kb
transcripts, and the apparent absence of a common upstream exon in
full-length cDNAs(53) . Exon B, which does not have strong
splice consensus sequences(51) , is only rarely
skipped(50) , and contains putative initiation site(s) for the
2.5-kb transcript. Despite its highly homologous open reading frame (which encodes a
catalytically active enzyme in vitro, (21) and (55) ), low expression levels of FUT5 were seen in
differentiated tissues. Colon and liver were found to contain modest
but reproducible amounts of FUT5 message, thus allowing isolation of
flanking sequences by RACE, PCR cloning, and cross-hybridization
methods. Possible explanations for low FUT5 expression include
order-dependent transcriptional switching during tissue
differentiation(56) , differential splice events at temporally
specific developmental stages(54) , lack of message transport
from nucleus to cytoplasm(32) , and/or lack of stable message
due to degradation(32) . Given the lack of consensus
polyadenylation signal sequences in FUT5 mRNAs, the 10-fold excess of
the proximally cleaved species, and the low levels of FUT5 message in
transformed cells, post-transcriptional transport, and/or degradation
mechanisms seem most likely. Site-directed mutagenesis of FUT5
polyadenylation signal sequences (33) and determining the
actual rate of FUT5 mRNA synthesis at various stages of development
will be required to address this hypothesis. FUT6 also uses
alternative polyadenylation signals, but unlike FUT5, undergoes a
pivotal 3`-UT splice event. The exclusive use of the proximal,
``low efficiency'' signal ATTAAA (33) in colon, which
has high levels of 2.5-kb FUT6 message, is in sharp contrast to the
predominant use of two overlapping AATAAA sites in kidney 3.5-kb
transcripts, which contain exon G. 3`-RACE experiments show that liver
uses both sequences equally. These data are consistent with a
competition model of 3` alternative splicing and polyadenylation (34, 35, 36) , which suggests that in the
case of FUT6, the fate of the RNA precursor is mainly determined by
competition between the exon G splice event and
cleavage/polyadenylation at the proximal signal sequence (36) .
Tissue-specific factors influencing post-transcriptional
``choice'' of these two species have not been identified, and
it is not known what the actual FUT6 transcription rates are preceding
these events in each tissue. Functional analyses will be required to
compare the effects of these 3` end modifications(34) . One
notable effect of the exon G splice event is that additional
tissue-specific splice events can occur, resulting in isoforms of FUT6
which are catalytically inactive in assays with oligosaccharide
acceptors. Sequence comparisons of these full-length transcripts
(particularly in the putative stem regions(22) , and upstream
exons (this work)) are not consistent with the presence of another
highly homologous fucosyltransferase gene or pseudogene. Based on
previously defined mutations in FUT6(57) , the particular
carboxyl-terminal regions deleted would indeed be expected to result in
loss of function (i.e. no detectable
fucosyltransferase activity). Single amino acid changes in
carboxyl-terminal residues have been shown to have profound effects on
fucosyltransferase synthetic capability, while upstream coding region
changes appear to have less effect(43, 57) . It is not
yet known, however, whether complex carbohydrate acceptors can be
fucosylated by FUT6 isoforms. It is also not clear whether use of
exon G is part of a concerted regulatory process or merely an event
associated with prolonged half-life of FUT6 precursor transcripts. The
tissue specificity of this splice event suggests down-regulation.
On/off gene regulation at the level of splicing has been estimated to
occur in at least 5% of regulatory ``decisions'' in Drosophila(58) , but its frequency in other species is
unknown. Detection of such non-functional mRNA species is frequently
hampered by size and stability of the alternatively spliced
species(58) , but that does not seem to be the case for FUT6,
since the catalytic domain splice events occur only when the consensus
polyadenylation signal in exon G is used. While some examples of this
form of regulation involve a single promoter directing constitutive
expression of a primary transcript, many other isoform families are
generated in the context of tissue specificity(59) ,
alternative promoter use(53) , and diverse splicing
options(60) . Our data for FUT6 show that isoform species are
another post-transcriptional modification associated with
tissue-specific
DNA
sequences have been deposited in GenBank: FUT3 Major I,
U27326[GenBank]; FUT3 Major II,
U27327[GenBank]; FUT3 Minor I,
U27328[GenBank]; FUT5 Minor I,
U27329[GenBank]; FUT5 Minor II,
U27330[GenBank]; FUT6 Isoform I,
U27331[GenBank]; FUT6 Isoform II,
U27332[GenBank]; FUT6 Major I,
U27333[GenBank]; FUT6 Major II,
U27334[GenBank]; FUT6 Minor I,
U27335[GenBank]; FUT6 Minor II,
U27336[GenBank]; FUT6 Minor III,
U27337[GenBank].
Volume 270,
Number 34,
Issue of August 25, pp. 20112-20122, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(1,3)-Fucosyltransferase Genes in Normal
Tissues
ALTERNATIVE SPLICING, POLYADENYLATION, AND ISOFORMS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(1,3)-fucosyltransferase genes FUT3, FUT5, and
FUT6 form a cluster on chromosome 19p13.3. Expression was studied using
reverse transcriptase-polymerase chain reaction, rapid amplification of
cDNA ends, and Northern analyses. FUT3 and FUT6 were expressed at high
levels, while FUT5 expression was lower and restricted to fewer cell
types. Alternatively spliced transcripts were identified for FUT3 and
FUT6 in kidney, liver, and colon. A 2.37-kilobase pair (kb) FUT3
transcript, detected at high levels in kidney and colon, was absent in
liver. FUT6 expression was characterized by a 3.5-kb transcript present
in kidney and liver, and a 2.5-kb transcript in colon and liver. Two
polyadenylation sites were shown for FUT5, but absence of consensus
sequences suggests reduced efficiency for cleavage and polyadenylation.
Two polyadenylation sites were also shown for FUT6, with the
alternatively spliced downstream signal in tissues expressing high
levels of FUT6. In these tissues, additional splicing results in
isoforms with catalytic domain deletions. No detectable (1,3)- or
(1,4)-fucosyltransferase activity was found in assays of cells
transfected with FUT6 isoform cDNAs. Thus, tissue-specific
post-transcriptional modifications are associated with expression
patterns of FUT3, FUT5, and FUT6.
(1,3)- and (1,4)-fucosylated (2,3)-sialylated
lactosaminoglycans are components of ligands for the cell adhesion
receptors E-selectin and P-selectin(1) . Cell surface
expression of sialyl Lewis x, sialyl Lewis a, and related fucosylated
antigens varies during development and malignant
transformation(2, 3, 4, 5, 6, 7) .
Enzymatic data imply that corresponding (1,3)- and
(1,4)-fucosyltransferase activities also change during these
dynamic processes(8, 9, 10) . Recent studies,
for example, have examined expression of sialyl Lewis x and
(1,3)-fucosyltransferase activities during hepatic
inflammation(11, 12) . Transition from an
``embryonic'' (1,3)-fucosyltransferase activity to
``adult'' forms (Lewis and plasma type) during renal
development has also been recently described(13) .(1,3)- and
(1,4)-fucosyltransferase activities found in normal epithelial
cells of gastrointestinal, genitourinary, and gonadal
organs(16) . In general, Lewis and plasma fucosyltransferase
activities have been highest in normally differentiated epithelium, but
it is not known how expression of (1,3)-fucosyltransferases is
controlled.(1,3)-fucosyltransferases(17, 18, 19, 20, 21, 22, 23, 24, 25) .
The (1,3)-fucosyltransferase genes FUT3 (encoding the Lewis
(1,3/1,4)-fucosyltransferase), FUT5 (an unspecified type of
(1,3)-fucosyltransferase), and FUT6 (the plasma
(1,3)-fucosyltransferase) form a cluster on human chromosome
19p13.3 (26) . It has been postulated that these tandemly
arranged homologues are regulated in a manner analogous to
developmental switching of globin genes(27) . Surveys of human
leukemia and epithelial carcinoma cell lines suggest that FUT3 and FUT6
transcripts are expressed at high levels in transformed
epithelia(24, 28) . Given these data, we sought to
study expression of FUT3, FUT5, and FUT6 at the transcript level in
normally differentiated human tissues. Our analyses show that
(1,3)-fucosyltransferase expression patterns in kidney, liver, and
colon depend, at least in part, on differential processing of FUT
transcripts.
Human Tissue Collection, Cell Culture, and RNA
Isolation
Human tissues were obtained through the Cooperative
Human Tissue Network (CHTN), which provides pathologic review and
accompanying tissue block specimens by protocol. Tissues were
snap-frozen less than 30 min from harvest. Specimens with documented
fibrosis, malignancy, or inflammation were excluded. Approximately 70
samples meeting these criteria were processed for the studies reported.
To facilitate comparison with other published
results(24, 28) , human HL-60 cells and unselected
peripheral blood T lymphocytes were also prepared. HL-60 cells were
grown in RPMI 1640 medium with 10% fetal calf serum. For T lymphocyte
preparations, peripheral blood buffy coat was washed with 1% fetal calf
serum. Lymphocytes were stimulated for 2 days in 20% fetal calf serum
and 1% phytohemagglutinin. The T cell population was then expanded by
placing the cells in 20% fetal calf serum, 10% T-cell conditioned
medium(29) , 50 units/ml interleukin-2, and 1% sodium pyruvate
in RPMI 1640 medium. Total cellular RNA was extracted from homogenized
cells and tissues with guanidine isothiocyanate and purified by cesium
chloride gradient centrifugation(30) . Poly(A) RNA was prepared using oligo(dT)-cellulose
chromatography(30) .
Northern Blot Analysis
Poly(A) RNA (20 µg, divided into 5 µg per lane) was denatured and
fractionated with 1.2% formaldehyde agarose gel electrophoresis (30) and transferred to Hybond-N membranes (Amersham). The
membranes were cut into four separate strips, which were then
prehybridized (19) for 2-4 h at 42 °C. Gene-specific
probes were amplified using previously described PCR (
)conditions (22) and the following primer sets:
FUT3 lower strand (L) base pairs (bp) 287-316 and upper strand
(U) bp 157-186(17) ; FUT5 L bp 253-282 and U bp
139-167(21) ; and FUT6 L bp 207-236 and U bp
86-115(22) , corresponding to fragment sizes of 160, 144,
and 151 bp, respectively. Hybridization to 
-actin message was used
as control(28) . Probes were labeled with
[P]dCTP by random priming and purified from
unincorporated isotope at a specific activity of 1
10
cpm/µg or higher(22) . Hybridization (19) was
carried out at 42 °C for 16-20 h. The final wash was carried
out at 65 °C with 0.5 SSC and 0.2% SDS for 30 min.
Autoradiography was performed with an intensifying screen at -80
°C for 10-14 days. Autoradiograms were scanned on a
Pixelcraft Pro Imager scanner (Oakland, CA). The images were sized,
grouped, and labeled using Adobe Photoshop 3.0 (Mountain View, CA).
RT-PCR Analysis of Coding Regions
First strand
cDNA was prepared using 0.2 µg of poly(A) RNA.
Synthesis of cDNA was carried out in a 30-µl reaction volume with
13 units of Moloney murine leukemia virus reverse transcriptase and 125
ng of the common lower strand primer (denoted L in Table 1). This
primer was chosen to ensure that FUT catalytic domain sequence was
present in all amplified fragments(22) . PCR consisted of 30
cycles for FUT3 and FUT6, while FUT5 required 35 cycles. Amplifications
were performed using the cDNA primer and gene-specific upper strand
primers (denoted U in Table 1) according to the following
temperature profile: denaturation 94 °C, 1.5 min; annealing 70
°C, 1.5 min; extension 72 °C, 2 min; and final extension 72
°C for 8 min. To control for genomic contamination, parallel
amplifications of all samples with no reverse transcriptase were
performed (data not shown). RT-PCR of human cytoplasmic
-actin was
performed with previously reported primers (28) to verify the
quality/quantity of RNA and further exclude the possibility of genomic
contamination (25 cycles). PCR products were cloned into the pCRII
vector (Invitrogen) and sequenced by the dideoxy chain termination
method. Sequence analysis and alignments were performed with the
MacVector (International Biotechnologies, Inc.) and Entrez (NLM)
sequence analysis programs.
Human cDNA Library Screening
Approximately 1
10
recombinant phages from normal human kidney and
liver cDNA libraries (insert sizes ranging from 0.6 to 4.5 kb in
gt10, Clontech) were screened by plaque hybridization as described
previously (19) using probes derived from the FUT6 sequence.
These probes included a 1.2-kb HindIII fragment (22) spanning the FUT6 coding region (recognizes all three FUT
genes) and a 330-bp fragment derived from the FUT6 5`-UT sequence
(recognizes only FUT6, see Fig. 5, exon C, bp 1-330).
Filters were processed as described above. Twenty-six cDNA clones were
isolated (sixteen FUT6, eight FUT3, and two FUT5), characterized by
Southern blot analysis(22) , cloned into the pcDNA-1 vector
(Invitrogen), and sequenced as described above.
Rapid Amplification of 5` and 3` cDNA Ends
Rapid
amplification of cDNA ends (RACE) was performed with the 5`-AmpliFINDER
RACE system (Clontech) on kidney, liver, and colon poly(A) RNA (the same specimens used in Northern and RT-PCR analyses).
5`-RACE first strand cDNA was generated with gene-specific lower strand
primers (L1, Table 2) followed by PCR amplification with nested
gene-specific lower strand primers (L2, Table 2). 3`-RACE was
performed as two separate PCR amplifications with gene-specific upper
strand primer sets (U1 and U2, Table 3). Following 30 cycles, 2
µl of a one-tenth diluted primary PCR product was applied to the
secondary nested amplification of 40 cycles. Where genomic and cDNA
sequences were colinear, parallel amplifications of samples with no
reverse transcriptase were performed (data not shown). PCR products
were cloned and sequenced as above. Flanking exons in 5`- and 3`-RACE
clones were verified by RT-PCR assays and Northern blot analyses.
Extended Length PCR of cDNA Contigs
Extended
length PCR (XL-PCR, Perkin Elmer) was performed according to the
manufacturer's protocol in a 100-µl reaction volume in the
GeneAmp PCR System 9600 (Perkin Elmer). The reaction mixture consisted
of 2 µl of kidney, liver, and colon cDNAs (oligo-dT primed
poly(A) RNA), 125 ng of each primer, 200 µM dNTPs, 10
reaction buffer, and 4 units of rTth DNA
polymerase XL. Parallel amplifications of samples with no reverse
transcriptase added were performed as above. Hot start PCR (30) and 30 cycles of amplification were employed with the
following temperature profile: denaturation 94 °C, 1 min;
annealing/extension 72 °C, 4 min; and final extension 72 °C, 10
min. cDNA contigs were amplified with the following primer sets: FUT3 L (Table 3) and U (Table 2); FUT5 L1 (distal poly(A), Table 3) and U (Table 2); FUT6 L1 (distal poly(A), Table 3) and U1 (3.5 kb, Table 2).
Genomic Mapping of FUT Flanking Exons
XL-PCR
(above) and Southern blot analyses (22) were performed on total
human genomic DNA using exonic probes and primers listed in Tables
I-III. In addition, overlapping genomic clones encompassing the 19p13.3
FUT locus (26) and selected subclones (data not shown) were
used to verify splice sites, map exons, and provide intronic sequence.Subcloning of FUT6 Isoform Constructs
FUT6 isoform
transcripts were amplified using the primers listed in Table 3and Fig. 8. The resulting fragments were cloned into the
pCRII vector (Invitrogen) and sequenced to exclude PCR errors. To
construct expression vectors consisting largely of the coding sequences
of the isoforms, the catalytic domain from the FUT6 ``wild
type'' clone, pcDNA1-Fuc-TVI(22) , was replaced with the
truncated catalytic domain from each of the isoform clones. The first
isoform expression plasmid was constructed by BstXI digestion
of pcDNA1-Fuc-TVI and replacement of the released fragment with the BstXI fragment from the pCRII Isoform I clone. (Fig. 7A; second BstXI site in pCRII
polylinker). The second isoform construct was made by PstI
digestion of pcDNA1-Fuc-TVI and replacement of the released fragment
with the PstI fragment from the pCRII Isoform II clone (Fig. 7A; second PstI site in pCRII
polylinker). Representative plasmids containing the inserts in the
sense orientation with respect to the pcDNA-1 cytomegalovirus promoter
were verified by sequence analysis and designated pcDNA1-Fuc-TVI-I
(encoding Isoform I) and pcDNA1-Fuc-TVI-II (encoding Isoform II).
Transfection of COS-7 Cells with FUT6 Isoform Constructs
and Preparation of Cell Extracts
COS-7 cells, cultured as
described (17) , were transfected with plasmid DNAs (pcDNA1,
Invitrogen; pcDNA1-FucTVI, (22) ; pcDNA1-Fuc-TIII, (17) ; pcDNA1-Fuc-TVI-I, this work; or pcDNA1-Fuc-TVI-II, this
work), using a previously described DEAE-dextran
procedure(17) . Cell extracts containing 1% Triton X-100 and
25% glycerol were prepared from sonicated transfected COS-7 cells after
a 72-h incubation period(17) .Fucosyltransferase Assays
Fucosyltransferase
assays with low molecular weight acceptor substrates were performed as
described previously(21, 22) . Acceptor substrates
were added to a final concentration of 20 mM (LacNAc, N-acetyllactosamine, Gal14GlcNAc and LNB-I,
lacto-N-biose I, Gal
13GlcNAc, and sialyl LacNAc,
(2,3)sialyl-N-acetyllactosamine,
NeuNAc23Gal
14GlcNAc). Control assays with no
added acceptor were performed using the same conditions. Reactions were
incubated at 37 °C for 1 h(22) . In addition, longer
incubations were performed to detect possible trace activities
(typically 4 h). Product separations for assays with neutral acceptors
(LacNAc and LNB-I) were performed using columns with Dowex
1X2-400, formate form(17) . For assays with sialyl
LacNAc, separations were performed with Dowex 1-X8
(PO4
) columns, equilibrated as
described(31) . Flow-through fractions and elutions were
collected, pooled, and counted to measure product formation. Product
identifications were performed as described(22) .
FUT3 and FUT6 Transcripts Are Expressed at High Levels
in Several Normal Tissues, while FUT5 Expression Is Lower and
Restricted in Distribution
Biochemical surveys of human tissues
and cells have suggested the presence of at least three
(1,3)-fucosyltransferase activities in epithelia(9) .
Lewis (1,3/1,4)-fucosyltransferase activity is co-expressed with
two other (1,3)-fucosyltransferase activities in some
instances(8) . Given these data, we performed semiquantitative
studies of fucosyltransferase transcripts in normally differentiated
tissues ( Fig. 1and Fig. 2). For each tissue, Northern
blot analyses were performed in parallel with ``stem'' region
probes containing divergent coding region sequence (``Experimental
Procedures''). For PCR analyses, reverse transcription was carried
out with a lower strand primer common to the catalytic domain of all
three FUT genes, followed by amplification with gene-specific upper
strand primers derived from stem regions ( Table 1and (22) ). These approaches allow comparison of message levels by
tissue (Fig. 1) and gene (Fig. 2).
RNA
was prepared from a single pathologically reviewed specimen for each
tissue type, divided into 5 µg/lane, electrophoresed, and
transferred to a single membrane. Each resulting blot was then
subdivided into strips, and hybridized with a
P-labeled
coding region probe specific for FUT3, FUT5, FUT6, or
-actin
(``Experimental Procedures''). Autoradiographic films were
exposed for 10-14 days (except for -actin lanes, A,
which were exposed for 12-15 h). Numbers refer to RNA size in kb.
The major transcript size noted for FUT3 (lane 3) was 2.37 kb.
For FUT6 (lane 6), 2.5- and 3.5-kb transcripts are noted. No
hybridizing signal was detected for FUT5 in the tissues studied (data
not shown).
RNA
was reverse-transcribed with a common lower strand primer (except
-actin; see Table 1) and used for PCR analyses as described
under ``Experimental Procedures.'' FUT3 and FUT6
amplifications were performed for 30 cycles, FUT5 for 35 cycles, and
-actin for 25 cycles. Control amplifications with no reverse
transcriptase are not shown. PCR products were electrophoresed in 1.2%
agarose gels and stained with ethidium bromide. Numbers refer to sizes
of amplified fragments (bp). Lane 1, kidney; lane 2,
liver; lane 3, lung; lane 4, colon; lane 5,
small intestine; lane 6, stomach; lane 7, T
lymphocytes; lane 8, bladder; lane 9, uterus; lane 10, ovary; lane 11, testicle; lane 12,
salivary gland; lane 13, brain; lane 14, HL-60
cells.
Alternatively Spliced FUT3 Transcripts Show
Tissue-specific Expression of 5`-UT Region Exons
To better
understand the expression of FUT3, RACE and hybridization screenings of
normal human colon, kidney, and liver libraries were performed
(``Experimental Procedures''). Fig. 3summarizes the
results of these analyses. The predominant FUT3 2.37-kb transcript was
designated ``Major II'' because it was isolated in 4 of 8
cDNA clones and showed high levels in colon and kidney (599-bp
fragment). This transcript was absent in liver, which contains a
2.72-kb message (948-bp fragment). A previously reported FUT3 cDNA
species, originally isolated from A-431 epithelial carcinoma
cells(17) , is shown as Minor II in Fig. 3; it is
present in colon at low levels (409-bp fragment).
FUT5 Transcripts Use Two Polyadenylation
Sites
FUT5, which encodes an enzyme with substrate specificity
that closely resembles FUT6(21) , was found to be expressed at
low but consistent levels in liver and colon (Fig. 2).
Considering the above data showing alternative splicing for FUT3, we
postulated that post-transcriptional modifications of FUT5 might affect
its expression. Using the same RACE and hybridization screenings
described above, no alternative splicing was found in 5`-UT, coding, or
3`-UT regions (Fig. 4). RACE and genomic analyses showed that
two polyadenylation signal sequences were used in liver and colon.
These putative signals differ from the typical
AA
U
A
A
A
and other published consensus motifs(32) . Minor
transcript I, which uses the proximal polyadenylation signal CACAAA, is
present at approximately 10-fold higher levels than minor transcript
II, which uses GATAAT. Both transcripts contain terminal elements
indicative of 3`-UT processing: GA and CA terminal sequences,
respectively, and G/T clusters (data not shown).
Alternatively Spliced and Polyadenylated FUT6 Transcripts
Are Differentially Expressed in Kidney, Liver, and Colon
As
shown in Fig. 1, FUT6 transcripts include a 3.5-kb message
present in kidney and liver, and a 2.5-kb species in colon and liver.
To determine the structure of these transcripts, RACE and hybridization
screenings of colon, kidney, and liver libraries were performed as
described above. In addition, extended length RT-PCR was performed to
verify the exonic composition of full-length alternative transcripts
(``Experimental Procedures''). Fig. 5and Fig. 6summarize these results. The kidney 3.5-kb major
transcript begins with exon A and contains an additional 5`-UT region,
exon B, compared to the major 2.5-kb transcript from colon. Liver
contains equivalent amounts of both major species. Rare transcripts
were also found in liver that lacked exon B (Minor III) but contained
exons A and C. The low abundance of this spliced species (one cDNA
clone isolated by hybridization, confirmed by RACE and extended length
RT-PCR) correlates with the absence of splice consensus sequences for
exon B (Fig. 5).
RNA was
prepared from a single pathologically reviewed specimen for each tissue
type, divided into 5 µg/lane, electrophoresed, and transferred to a
single membrane. Each resulting blot was then divided into three
strips, hybridized with
P-labeled 5`-UT probes, and washed
at high stringency (``Experimental Procedures'').
Autoradiographic films were exposed for 10 days, except for
-actin
lanes, which were for 12 h. Probe 2, containing sequence from exon C
(bp 1-330), recognizes both major transcripts (3.5 and 2.5 kb),
while probe 1, which contains exon A sequence (bp 1-285),
hybridizes only with the 3.5-kb transcript. Kidney (lane K)
contains only the 3.5-kb transcript, liver (lane L) contains
both, and colon (lane C), the 2.5-kb
species.
Tissues That Express High Levels of FUT6 Transcripts
Containing Exon G Undergo Additional Splicing within the Coding Region
to Form Isoform Transcripts
Further PCR characterization of
clones obtained by RACE and hybridization techniques revealed that a
subset of kidney and liver transcripts existed with aberrant sizes.
These truncated transcripts were only observed when exon G was present
and comprised 10-25% of clones containing the distal
polyadenylation signal (data not shown). Representative transcripts
from kidney and liver were cloned and sequenced, revealing two
additional splice events occurring within the FUT6 coding region (Fig. 7A). Sequence analysis of these clones confirmed
identity with the FUT6 stem region(22) . Extended length PCR
was used to verify the relation of these isoform splice variants to
upstream exonic sequence (data not shown). The removal of wild type
carboxyl-terminal nucleotides and replacement with isoform residues
results in a new putative open reading frame (Fig. 7B).
RT-PCR analysis confirms the tissue-specific distribution of isoform
transcripts, which correlates with expression level of exon G (Fig. 7C).Transcripts Encoding FUT6 Isoforms Do Not Direct
To
determine if the isoform coding regions could direct (1,3)-Fucosyltransferase Activity in Transfected Cells(1,3)- and/or
(1,4)-fucosyltransferase activity, the putative open reading
frames were used to construct expression plasmids and transfected into
mammalian cells as described(22) . Extracts prepared from
transfected cells were tested for (1,3)- and
(1,4)-fucosyltransferase activity in assays containing low
molecular weight oligosaccharides(17) . Control extracts
prepared from pcDNA1-transfected COS-7 cells transferred no detectable
fucose to any acceptor. Extracts prepared from cells transfected with
pcDNA1-Fuc-TVI and pcDNA1-Fuc-TIII were found to contain
fucosyltransferase activities similar to previously published values (17, 22) . Under the same assay conditions, no
(1,3)- or (1,4)-fucosyltransferase activity was found in any
of the isoform extracts (specific activity less than 1.0 pmol/mg/h).
These results suggest that the alternatively spliced isoform
transcripts do not encode functional (1,3)- or
(1,4)-fucosyltransferases.
(1,3)-fucosyltransferase genes are expressed at
high levels (FUT3, corresponding to Lewis
(1,3/1,4)-fucosyltransferase activity and FUT6, corresponding to
plasma (1,3)-fucosyltransferase activity). Coexistence of other
fucosyltransferase activities is frequently noted, however(9) .
Molecular analyses have become useful in defining the relative roles of
these enzymes.(1,3)-fucosyltransferases FUT4 and Fuc-TVII are expressed at high
levels in normal and transformed leukocytes(24) . FUT5, which
is highly homologous to FUT3 and FUT6 (21, 22) and
part of the 19p13.3 locus(26) , appears to have lower levels of
expression in both epithelial and hematopoietic cell lines. FUT4, which
appears to be expressed earlier in development(27) , is less
homologous and non-syntenic(26) . When considered together,
this genomic organization and expression pattern suggest that control
at transcriptional and/or post-transcriptional levels may underlie
developmental and tissue-specific fucosyltransferase expression
patterns.(1,3)-fucosyltransferase genes in normal human tissues to find
underlying molecular determinants for this specificity. Our results
correlate transcript levels with the high FUT3 and FUT6 enzyme
activities observed in epithelial tissues of specific types, confirm
lower overall expression levels of FUT5, and suggest that RNA
processing serves as a point of control for
(1,3)-fucosyltransferase expression, particularly for FUT6. Large
differences in mature transcript structure were found in full-length
kidney, liver, and colon cDNAs.(1,3/1,4)-fucosyltransferase activity with
bile duct proliferation, while (1,3)-fucosyltransferase activity
was markedly increased in hepatic parenchymal
injury(12, 39) . Enzymatic assays performed on
cellular fractions suggest that hepatocytes are the source of
approximately 75% of (1,3)-fucosyltransferase activity and 25% of
(1,3/1,4)-fucosyltransferase activity detected in whole tissue
extracts(38) . Normal hepatocytes, however, do not express high
levels of sialyl Lewis x on their surface(11, 40) .
Increased levels of surface-expressed sialyl Lewis x have been shown in
hepatic necro-inflammatory lesions and hepatocellular
carcinoma(11, 40) . High level expression of both the
2.5- and 3.5-kb FUT6 transcripts are found in biopsy specimens with
hepatocellular inflammation (
)and in transformed
hepatocytes(28) . It is not yet clear, however, whether changes
in FUT6 message levels underlie these enzymatic and immunohistochemical
observations.(1,3)-fucosyltransferase and high (1,4)-fucosyltransferase
activity, while normal colon mucosa has high
(1,3)-fucosyltransferase and moderate
(1,4)-fucosyltransferase activity. FUT3 and FUT6 co-expression is
not shown by these enzymatic data nor by the analysis of transcript
levels presented here. Of note, FUT6 2.5-kb transcripts may appear as a
composite signal with 2.37-kb FUT3 transcripts when probes containing
catalytic domain sequence are used(17, 28) . Upstream
probes described in this report allow separation of these messages (Fig. 6).(1,3/1,4)-fucosyltransferase activity purified from saliva nor
with Lewis blood
type(43, 44, 45, 46, 47) .
Possible explanations for these findings include differential splicing
of FUT3 and/or FUT6 transcripts resulting in species not characterized
or the presence of additional, as yet undescribed enzyme(s) capable of
(1,4)-fucosylation(48) . None of the alternatively spliced
FUT6 transcripts isolated to date were found to direct
(1,4)-fucosyltransferase activity when tested in transfection
assays (Table 4). In addition, our RT-PCR screening assays (Fig. 2) used primers spanning most of the coding region for all
three 19p genes, making additional splice events an unlikely
explanation.
All FUT3 transcripts start with exon A, i.e. they are alternatively spliced forms originating
from one pre-mRNA species. Exon A ends in a canonical splice donor
sequence, while relative use of 3` splice acceptors within exon B
likely depends on competition(50) . Frequency of 3` splice
acceptor selection approximately corresponds to the length of
polypyrimidine tract and presence of AG in the intronic sequence
preceding the exon(51, 52) . As seen in Fig. 3,
potential splice acceptor competition exists between sites B, B`, and
B" (which are all preceded by AG), but sequence preceding B` has the
longest uninterrupted polypyrimidine tract. This model is consistent
with the predominance of Major II FUT3 transcripts reported here. The
2.72-kb Minor I transcript in liver suggests that tissue-specific
trans-acting factors also influence site selection and/or message
stability. In contrast, exon C has a very strong
splice donor site and can be followed by the differentially spliced
exons D or E, likely on the basis of the 3` splice acceptor competition
model(51) . In general, mRNAs that maintain such 5`-UT
diversity and length are transcribed from alternative promoters whose
activation may be associated with tissue and developmental stage
specificity (e.g. human insulin-like growth factor
II, (54) ). For FUT6, functional testing of putative promoter
regions will be required to definitively map transcriptional sites. (1,3)-fucosyltransferase gene expression.
)23Gal
14[Fuc
13]GlcNAc;
sialyl Lewis a,
NeuNAc
23Gal
13[Fuc
14]GlcNAc;
(1,3)-fucosyltransferase,
GDP-fucose:-D-N-acetylglucosaminide-3--L-fucosyltransferase;
kb, kilobase; bp, base pair; 5`-UT, 5`-untranslated; 3`-UT,
3`-untranslated; RACE, rapid amplification of cDNA ends; RT-PCR,
reverse transcriptase-mediated polymerase chain reaction; XL-PCR,
extended length polymerase chain reaction; U and L, upper and lower,
respectively.)
We thank Susan Gidwitz and Kate Senger for technical
assistance, Doug Mokaren for expert illustration work, and Bill
Marzluff, Gilbert White, and Beverly Mitchell for advice and support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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