From the
HT-29 cells resistant to 10
Mucins are highly O-glycosylated proteins, which
polymerize through the formation of disulfide bonds to form a gel that
is secreted onto the surface of glandular epithelia. In the last 10
years, it has become clear that mucin polypeptide backbones are encoded
by several genes (see Refs. 1 and 2 for reviews). The complete
sequences of MUC1
(3, 4, 5) ,
MUC2
(6, 7, 8) , and MUC7 cDNAs
(9) have
been reported, but only partial sequences are available for
MUC3
(10) , MUC4
(11) , MUC5AC
(12, 13) ,
MUC5B
(14) , and MUC6
(15) . The initial identification of
these genes was allowed by screening of expression libraries with
polyclonal antisera prepared against deglycosylated mucins from a
variety of sources. A notable feature of these clones was that they
contained tandem repeats of sequences coding for Thr- and Ser-rich
regions, which appear to correspond to the highly glycosylated domains
of mucins
(1) . In the case of MUC2, these regions are flanked by
``unique'' sequences, which encode Cys-rich
domains
(7, 8) that are homologous to the pro von
Willebrand factor D domains
(8, 16) . Recently, there
have been a number of other reports of partial mucin cDNAs encoding
Cys-rich regions
(13, 17) .
Although several mucin
genes appear to be expressed in the epithelium of each tissue
examined
(1, 18, 19, 20) , there are
marked differences in the pattern of expression and also in the
relative expression of the genes within different cell types in a
single
tissue
(1, 18, 20, 21, 22) .
Furthermore, changes in expression are observed in epithelial tumors
and in non-neoplastic disease states
(1) . As a tool to
understanding these changes and in order to obtain information about
the regulation of these genes, we have been developing cell culture
models
(23, 24) . We have isolated and characterized two
subpopulations of the colon cancer cell line HT-29 by means of
selection using 5-fluorouracil (FU)
In view of
the gastric immunoreactivity of the mucins produced by these cells, we
have used an antiserum raised against gastric mucosal fraction
containing native mucins to screen a cDNA library prepared from
HT29-MTX cells. We have characterized a cDNA, and here we present
evidence that it may be the 3` end of the MUC5AC gene. We discuss the
relationship ofthis clone to that recently described by Meerzaman
et al.(17) , which was isolated from a nasal polyp cDNA
library.
For ELISA, microtiter plates
(Dynatech, Chantilly, VA) were coated with native or deglycosylated
mucins isolated from the culture medium of HT29-MTX cells (20
µg/ml), KLH, and peptide-KLH conjugates (20 µg/ml in PBS).
Native and deglycosylated MTX mucins were prepared as described
elsewhere
(21) . The peptides tested correspond to MUC1
(VTSAPDTRPAPGSTAPPAHG), MUC2 (PTTTPISTTTVTPTPTPTGTQT), MUC3
(HSTPSFTSSITTTETTS), MUC4 (TSSASTGHATPLPVTD), MUC5AC (TTSTTSAPTTS), and
MUC6 (SFQTTTTYPTPSHPQTTLP and TSLHSHTSSTHHPEVPT) tandem repeat
sequences. MUC5B was not included because it does not contain a
conserved tandem repeat. The peptide synthesis and the coupling to KLH
were performed as described previously
(21) . Reactive sites were
blocked with 1% gelatin for 1 h at 37 °C. Serial dilutions of the
L56/C serum in PBS containing 1% BSA were incubated for 1 h at 37
°C in the antigen-coated wells. After washing with PBS-Tween,
alkaline phosphatase-conjugated swine anti-rabbit IgG (Dako, Glostrup,
Denmark) (0.5 µg/ml) was added for 1 h at 37 °C. Alkaline
phosphatase activity was detected using 4-methylumbelliferyl-phosphate
(Boehringer, Mannheim, Germany) at 1 mg/ml diluted in triethanolamine
buffer, pH 9.5, and fluorescence was measured using a CytoFluor 2300
fluorescence measurement system (Millipore, Bedford, MA).
Poly(A)
All
membranes were prehybridized and hybridized according to previously
used conditions
(25) . MUC2 was detected using SMUC41
(6) ,
MUC5AC with JER58
(12) , and MUC5B with JER 57
(14) . Actin
was detected with cDNA probe pA2
(35) .
The TTVGPTTVGS tandem repeats described by Meerzaman
et al. (17) and 4 degenerate SSSVA tandem repeats are present
in both sequences (Fig. 5).
In previous studies (see Ref. 1 for review), the use of
antisera raised against deglycosylated mucins to screen expression
libraries has led to the preferential isolation of clones containing
repetitive sequences, possibly due to signal amplification resulting
from the peptide repeats or to their greater immunogenicity. The
isolation of mucin sequences corresponding to the ``unique''
domains has required the secondary screening of libraries with cDNAs
containing repetitive sequences as for MUC5AC clones
(13) , or
use of PCR-amplified products encoding peptides identified through
conventional biochemical techniques as for nasal polyp
mucin
(17) , or using RACE (rapid amplification of cDNA ends)-PCR
and anchor-PCR as for MUC2 NH
The
nucleotide sequence of the largest clone L31 showed a very high level
of identity (98.6%) to the clone NP3a, isolated recently from a nasal
polyp library
(17) . Surprisingly, the deduced amino acid
sequence of the peptides encoded by clones L31 and NP3a showed four
short regions of very low similarity, due to changes in reading frame.
Lower similarity was also observed between the L31 peptide and the
carboxyl-terminal region of MUC2 (36.3%) and was maintained all along
the L31 protein, including the four non-conservative regions mentioned
above. A striking conservation of the number and position of the Cys
residues was also found. These observations provide strong evidence
that clone L31 forms the 3` end of a mucin gene. The relationship to
the NP3a sequence is much harder to evaluate, but it should be noted
that cross-hybridization of the targets would be expected for all the
hybridization procedures used here because of the 98.6% identity of the
two sequences.
Meerzaman and colleagues
(17) proposed that
NP3a corresponds to the 3` end of the MUC5 gene, because, like in one
of partial MUC5 cDNA clones, JER 47, it contained the sequence of
peptides TR-3A and TR-3B, which they had previously determined directly
from tracheobronchial mucin protein fragments
(38) , and also
because NP3a maps to chromosome 11. However, it has more recently been
shown that there are two distinct MUC5 genes, temporarily designated
MUC5AC and MUC5B
(13) . From the partial cDNA sequences, it is
clear that cysteine-rich domains which contain the TR-3A and the TR-3B
peptides occur several times within the MUC5AC gene and that these
regions also show some similarity to regions of MUC2 and MUC5B,
although the similarity with MUC5B is only at the nucleotide
level
(13) . All three genes map to chromosome
11p15
(15, 36, 37) , and we show here that clone
L31 maps to the same region. The pattern of expression of the mRNA
transcripts hybridizing with L31 in the HT-29 subpopulations and in
normal and tumor tissues corresponds to that of MUC5AC and is distinct
from MUC5B, MUC2 and MUC6. Southern blot analysis indicates that the
JER58 (MUC5AC) and L31 sequences are located on the same genomic
fragment (
It is possible that
some of the differences in sequence between L31 and NP3a are due to
genetic polymorphism, but this seems unlikely to be the case for those
differences that lead to changes in reading frame. The possibility that
the two clones correspond to the 3` ends of two adjacent highly similar
MUC5AC genes also seems unlikely because this would mean that the 3`
end of both genes must be located on the single 9.5-kb ScaI
fragment while the tandem repeat regions were located on the same
Two short sequences of the deduced L31 peptide seem to
represent motifs that are conserved in other mucins and mucin-related
proteins. The first of these located between positions 1 and 35 in L31,
is found three times in MUC5AC partial cDNAs, twice in MUC2, and three
times in HGM clone (a potential MUC5AC cDNA that overlaps with JUL32
and the 5` end of NP3a clones). In each case this sequence is located
at junctions with TSP-rich domains (from 28 to 77 in
L31)
(7, 13) . The second sequence, from amino acid 216
to 246, is not present in NP3a and shows a high level of similarity to
part of the four D domains of MUC2 and the pro vWF. We have not found
these two regions in any of the other human mucin cDNAs. This evidence
of conservation suggests functional importance and also the possibility
of intermolecular cross-linking. More sequence and structural
information is required before any evolutionary models can be
constructed.
It is important to emphasize that the L31 clone was
isolated from a cancer cell library and that alterations of mucin
expression have been described in tumor tissues (see Ref. 1 for
review). A notable feature of HT29-MTX cells is their type of
differentiation. Although this cell line is derived from a human colon
tumor, it synthesizes and secretes mucins with gastric
immunoreactivity
(23, 25) . HT29-MTX cells also express
the brush border-associated proteins: dipeptidylpeptidase IV, villin,
and carcinoembryonic antigen
(23) . These features are similar to
those observed for the colon of early gestational fetuses, which also
expresses the same brush border-associated proteins
(39) and
mucins with gastric immunoreactivity
(40) . Few studies have been
reported concerning the developmental expression of mucin genes.
However, as in MTX cells, it seems that several mucin genes are
expressed in colon of 12 weeks of gestation (41).
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Teresa Adell and Estanis Navarro for cDNA
library construction; David Andreu, Carmen Bolós, and Marta
Garrido for the preparation of synthetic peptide conjugates and
deglycosylated MTX mucins; and Amy Brar-Rai and Wendy Pratt for the
synthesis of some of the oligonucleotides and technical assistance. We
are also grateful to James Gum and Young Kim for the gift of MUC2
probe, and Nicole Porchet and Jean-Pierre Aubert for MUC5AC and MUC5B
probes.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
M
methotrexate (HT29-MTX) secrete mucins with gastric immunoreactivity
(Lesuffleur, T., Barbat, A., Dussaulx, E., and Zweibaum, A.(1990)
Cancer Res. 50, 6334-6343). A 3310-base pair mucin cDNA
clone (L31) was isolated from an HT29-MTX expression library using a
polyclonal serum specific for normal gastric mucosa. It shows a high
level of identity (98.6%) to clone NP3a isolated from a nasal polyp
cDNA library (Meerzaman, D., Charles, P., Daskal, E., Polymeropoulos,
M. H., Martin, B. M., and Rose, M. C.(1994) J. Biol. Chem. 269, 12932-12939). However, as a result of changes in
reading frame, the 1042-amino acid deduced peptide contains four
regions of a low similarity to the NP3a peptide. The amino acid
sequence shows 36.3% similarity to part of the carboxyl-terminal
sequence of MUC2 including the so-called D4 domain and 21.3% to the pro
von Willebrand factor. A short amino acid sequence is similar to
cysteine-rich sequences repeated in tracheobronchial, gastric, and
colonic mucin cDNAs. The gene corresponding to L31 is located in the
mucin gene cluster on chromosome 11p15.5. The patterns of mRNA
expression were indistinguishable from those revealed with the JER58
probe (MUC5AC). Southern blot analysis indicates that the L31 and JER
58 sequences are within 20 kilobase pairs of each other. Together,
these results suggest that L31 clone is the 3` end of MUC5AC.
(
)(24) and methotrexate (MTX)
(23) . HT29-FU cells express
colonic-type mucins as judged by antibody analysis, while HT29-MTX
cells express gastric-type mucins, a situation analogous to that of
fetal colon
(23, 24) . HT29-MTX cells form a homogeneous
population of mucus-secreting cells and thus potentially provide a good
model for the study of mucin biosynthesis
(25) . We have shown
that the MUC genes (MUC1-MUC6) are expressed to different extents
in these cells
(25, 26) , but it is not known whether the
major mucin gene expressed is the product of one of them.
Cell Culture
HT29-MTX and HT29-FU cell
populations resulted from adaptation of HT-29 cell line to
10M methotrexate
(23) and
10
M 5-fluorouracil
(24) ,
respectively, and were used after 3 or 4 weekly passages in the absence
of drug. These cells were maintained under the same conditions of
culture as described previously
(23, 24) .
Normal Tissues
A panel of 30 normal adult stomachs
and colons from irreversibly brain-damaged organ donors was obtained
with the help of France Transplant Association, according to protocols
approved by the National Ethical Committee. Samples of mucosa were
snap-frozen in liquid nitrogen and stored in liquid nitrogen until
used.
Preparation of a Polyclonal Antiserum Raised against
Normal Gastric Mucosa
The antiserum used was prepared 20 years
ago by the same protocol used for the characterization of the so-called
WZ polymorphic colon antigens
(27, 28) . Normal stomach
and colon from the same blood group O donor were used. The stomach
mucosa was scraped, homogenized in distilled water (Ultra-turrax,
Jankee and Kunkel, Stauffer, Germany), and centrifuged (1 h, 48,000
g). The supernatant was heated for 1 h in a boiling
water bath and further centrifuged (1 h, 48,000
g).
The resultant supernatant was dialyzed against distilled water for 2
days at 4 °C and lyophilized (crude gastric extract). Scraped
colonic mucosa was homogenized in acetone, dried at room temperature,
and then passed through a sieve (200-mesh) in order to obtain a thin
dry powder. One rabbit (L56) was immunized with the crude gastric
extract in Freund's complete adjuvant according to the protocol
previously reported
(27) . The immunized rabbit was of the
A
phenotype
(29) in order to avoid the presence
of anti-blood group A antibodies. The immune serum was further absorbed
with the colonic mucosal dry acetone powder to remove
non-gastric-specific antibodies and stored in aliquots at -20
°C. The absorbed immune serum is referred to as L56/C.
Immunological Assays
Indirect immunofluorescence
was performed on frozen cryostat sections of tissues and HT29-MTX cells
as described earlier
(23) .
Preparation and Screening of a HT29-MTX cDNA
Library
Poly(A) RNA was extracted from
postconfluent HT29-MTX cells (day 21 after seeding) as described
previously
(23) . A
ZAP-cDNA library was constructed using 5
µg of mRNA as template and amplified once according to instructions
in the ZAP-cDNA® synthesis kit (Stratagene, La Jolla, CA) and was
kindly provided by E. Navarro and T. Adell (Institut Municipal
d'Investigació Mèdica, Barcelona, Spain). The host
strain was Escherichia coli XL1 blue. The library was plated
in soft agar at a density of 30,000 plaque-forming units/150-mm plate.
Plates were incubated at 37 °C until plaques began to appear, then
overlaid with isopropyl
-D-thiogalactopyranoside-saturated
nitrocellulose membranes and incubated for an additional 3 h. The
filters were removed and blocked with 3% BSA. For the immunoscreening,
L56/C antibodies were used at a 1:50 dilution and preabsorbed for 1 h
with 1% BSA and XL1 blue cells, lysed or killed by UV light (each XL1
blue preparation corresponding to a pellet of 50 ml overnight culture
for 50 ml of diluted serum). After primary incubation with absorbed
L56/C serum for 3 h at room temperature and washing, filters were
incubated with alkaline phosphatase-conjugated swine anti-rabbit IgG
diluted 1:1000 (Dako). After washing, positive plaques were identified
by detection of alkaline phosphatase activity with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase
buffer (Promega, Madison, WI) and purified by successive rounds of
screening. Using helper phage R408, the pBluescript® plasmids
containing positive cDNA were released from
ZAP phage, and
transferred into XL1 blue for storage and sequencing.
cDNA Sequencing
The clone L31 was sequenced in its
entirety on both strands using the dideoxynucleotide chain termination
method while the other clones were partially sequenced. Sequencing was
performed with double-stranded plasmid and T7 DNA polymerase kit,
according to the recommended procedure (Pharmacia, Saint Quentin en
Yvelines, France). The primers used were the T3 sequence and a 20-mer
oligonucleotide (5`-CCCAAAAGGGTCAGTGCTGC-3`) in pBluescript for the 5`
and 3` end sequences, respectively, and 11 pairs of oligonucleotides
(both sense and antisense) corresponding to positions 237-255,
464-484, 821-840, 1155-1173, 1413-1431,
1674-1693, 1906-1925, 2184-2203, 2459-2478,
2698-2717, and 2954-2973 in the L31 sequence. The
oligonucleotides were synthesized on an Applied Biosystems Inc.
PCR-mate or purchased from Bioprobe (Bioprobe, Montreuil sous Bois,
France). The sequencing difficulties due to the high (G+C)
concentration of the insert were solved by the use of 7-deaza-GTP
(Pharmacia). Enzymatic pauses were suppressed using a lower quantity of
DNA template (1 µg/reaction). Analysis of nucleotide and amino acid
sequence data was performed using Bisance
(30) and the Wisconsin
GCG package on the HGMP-RC computer
(31) .
Chromosomal Localization
Fluorescent in situ hybridization to metaphase chromosomes was conducted as described
previously
(32, 33) , using 200 ng of plasmid DNA from
clone L31 in pBluescript biotinylated by nick translation.
Northern Blot Analysis
Total RNA was extracted
from frozen samples of normal human tissues as described
previously
(34) . RNA (15 µg) was denatured in loading buffer
containing formamide and formaldehyde, fractionated by 1% agarose gel
electrophoresis, and transferred onto nitrocellulose (Schleicher &
Schuell, Dassel, Germany).
RNA isolation
from HT29-MTX and HT29-FU cells, electrophoretic separation, and
Northern blotting were as reported previously
(25) .
Southern Blot Analysis
Southern blot analysis of
human genomic DNAs using Hybond N filters (Amersham)
was carried out following standard procedures, as recommended by the
manufacturer. The final stringent wash was done with 0.1
SSC at
65 °C. The relative sizes of the fragments were determined by
comparison with Raoul markers (Appligene, Durham, United Kingdom),
which were probed with
P-labeled PUC18 for their
detection.
Reactivity of Antiserum L56/C
The antiserum
L56/C was tested by indirect immunofluorescence on cryostat sections of
the panel of normal gastric and colonic mucosa samples. The antiserum
showed strong reactivity with mucus droplets of mucus-secreting cells
in all normal gastric samples tested (Fig. 1a). In
contrast no reactivity was observed with colonic goblet cells
(Fig. 1b). Mucus droplets in HT29-MTX cells showed
strong reactivity (Fig. 1c).
Figure 1:
Immunofluorescence detection
of gastric mucins with the polyclonal antiserum L56/C. Indirect
immunofluorescence staining, with L56/C antibodies, of ethanol-fixed
cryostat sections of normal gastric mucosa (a), normal colon
mucosa (b), and postconfluent HT29-MTX cells (day 21 after
seeding) (c). Note that gastric and MTX mucus secretions are
immunoreactive, whereas no mucus positivity is observed in colon.
Bar = 100 µm.
Since these antibodies
were not prepared against purified gastric mucins and the epitopes that
they recognize had not been identified, reactivity of the antiserum
against native and deglycosylated mucins isolated from HT29-MTX cells,
and against the tandem repeat peptide-KLH conjugates (see
``Materials and Methods''), was analyzed by ELISA. As shown
in Fig. 2, the antibodies recognize both native and
deglycosylated mucins purified from HT29-MTX cells. In contrast, they
do not react with the synthetic peptides corresponding to the mucin
tandem repeats. These results suggested that the antiserum L56/C
contains antibodies that are reactive either with epitopes outside the
tandem repeat domains of the MUC1-MUC6 apomucins or reactive with a new
apomucin.
Figure 2:
Reactivity of the polyclonal antiserum
L56/C with MTX mucins and peptide-KLH conjugates in ELISA assays. 20
µg/ml native () or deglycosylated (
) MTX mucins, KLH
or peptide-KLH conjugates (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6) were
coated onto plates and incubated with serial dilutions of antiserum.
The basal reactivity level of L56/C with KLH was first subtracted from
MUC-KLH conjugate data. The assays were conducted in duplicate and on
two occasions. The levels of reactivity of L56/C with the different MUC
peptides were very similar, and only MUC5AC result is represented
(
) in order to simplify the figure. No reactivity was detected
with the synthetic peptides, whereas strong reactivity with native and
deglycosylated mucins is noted.
Isolation and Sequencing of Mucin cDNAs
The
antiserum L56/C was therefore used to screen approximately 300,000
recombinants of the HT29-MTX cDNA library, and 37 positive clones were
isolated. Of these, 18 clones were related, as determined by
hybridization analysis (data not shown). The size of the inserts varied
from 3,310 to 300 bp. The sequence of the largest clone, named L31, was
determined in its entirety on both strands and is given in
Fig. 3
. Other clones used in this study (L18, L10, L21, L34, and
L17) were partially sequenced, manually or automatically, and
correspond to L31 sequence from position 337, 790, 1216, 1279, and 1507
to 3` end, respectively. The L31 cDNA sequence is 3,310 bp long with a
70% (G+C) content and ends with a 22-bp poly(A) tail. Comparison
with other cDNA sequences from mucins showed that the L31 sequence is
very similar (98.6% of identity) to clone NP3a (nucleotide 273 to the
3` end) isolated from a nasal polyp cDNA library by Meerzaman et
al.(17) . The comparison revealed 14 insertions, 3
deletions, 7 inversions, and 9 substitutions (presented in
Fig. 3
) all along the L31 sequence. Each deletion involved a
single nucleotide, while insertions involved one to three nucleotides.
These differences were confirmed on both strands by at least three
sequencing reactions with dGTP or deaza-GTP. Some of the differences
detected were assessed by digestion with restriction enzymes
(Fig. 4). In each case the fragment number and sizes were as
predicted from the L31 sequence.
Figure 3:
cDNA sequence of the clone L31. Sequence
differences from the NP3a clone (GenBank accession number U06711, 1993)
(17) are shown: nucleotide substitutions or inversions (underlined
twice), nucleotide insertions (), or nucleotide deletions
(▾). The stop codon (TGA) and the potential polyadenylation
signal at the 3` end of the clone are underlined
once.
Figure 4:
Analysis of L31 restriction fragments.
A, restriction maps showing sites for five enzymes,
AccIII, BamHI, BglI, MslI, and
NaeI, within the EcoRI-KpnI L31 insert (3342
bp), were predicted from sequence analysis. The sites that are absent
in the NP3a sequence are indicated with , and sites that are
present in the NP3a but not in L31 are represented by
. The
vertical bar corresponds to sites present in both sequences.
B, L31 insert (lane2) was digested by
BamHI (lane3), BglI (lane4), NaeI (lane5),
MslI (lane7), and AccIII (lane8). Fragments were separated on a 1.5% agarose gel and
visualized by ethidium bromide staining. Their sizes were determined by
comparison with
BstEII (Biolabs, Beverley, MA) (lane1) and the 100-base pair ladder (Pharmacia) (lane6) molecular weight markers. Note that all the fragments
have the expected size; partial digestion with NaeI is due to
site preference (Biolabs).
The open reading frame of L31 cDNA
encodes a 1042-amino acid polypeptide rich in cysteine (9.2%)
(Fig. 5). Comparative analysis of L31 and NP3a deduced amino acid
sequences shows 83.5% identity (Fig. 5). Large identical regions
(>98%) alternate with four smaller regions with a low level of
identity (<12.6%). The regions that diverge result from nucleotide
insertions and deletions that introduce several shifts in the reading
frame and are shown in Fig. 5. There is one additional amino acid
in each of the first two domains, and a 74-amino acid extension of the
carboxyl-terminal region; the first in-frame stop codon (TGA) is
located at position 3129-3131 because of the nucleotide
insertions and deletions. The untranslated sequence of clone L31 is
therefore shorter. Immunoblot analysis of bacterial lysates revealed
that the electrophoretic mobilities of the fusion proteins expressed in
XL1 blue by clones L31, L18, L10, L21, or L17 were in good agreement
with that expected if the first stop codon is at position
3129-3131 (data not shown).
Figure 5:
Comparison of the deduced amino acid
sequences of clones L31 and NP3a. The sequences are aligned with gaps
inserted to give maximum identity. Four regions (underlined
once) show low similarity with NP3a. Amino acids that are not
identical in L31 and NP3a conserved regions are underlined
twice. Two repetitive sequences, SSSVA () and TTVGPTTVGS (-
), are conserved in the two apomucins. Cysteine residues are
marked with asterisks. The majority of potential
N-glycosylation sites (▾) are present in both sequence.
Stop codons are indicated by
.
The changes in reading frame led
to notable differences in the deduced amino acid composition of
non-conserved regions. In particular, there is an increased number of
Cys residues all along the L31 sequence, 15 out of 16 being located in
the non-conserved regions (Fig. 5). A number of other nucleotide
changes were detected and in 6/10 cases led to amino acid substitutions
in Arg codons. The deduced amino acid sequence of L31 contains 12
potential N-glycosylation sites, 10 of which are present in
clone NP3a.
Sequence Similarities between L31 Peptide and Other
Proteins
The deduced amino acid sequence of clone L31 also shows
36.3% identity to part of the carboxyl-terminal region of MUC2
apomucin. This region, which extends from amino acid 4277 to 5173,
includes the Cys-rich D4 domain of MUC2. Lower level of identity
(21.3%) is found with the pro vWF, from amino acid 1857 to 2654.
Comparison of this domain in L31 and the four related D domains in MUC2
and in the pro vWF
(8) reveals that a subregion at the start of
each domain shows a particularly high degree of similarity across all
the sequences. Fig. 6a shows the comparison and the
derivation of a consensus sequence. This sequence is not present in
NP3a. The L31 sequence has no significant similarity with the published
sequences of MUC1, MUC3, MUC4, MUC5AC, MUC5B, MUC6, or MUC7 proteins.
However the first 35 amino acids of clone L31 show a very high degree
of similarity to short amino acid sequences in the Cys-rich domains
described recently in MUC5AC
(13) and are also present three
times in a HGM clone coding for a partial gastric
mucin.The peptide encoded by the clone NP3a
also contained this short sequence. A similar sequence was also found
twice in the amino-terminal part of MUC2 (Fig. 6b). From
this sequence comparison, a consensus sequence was determined
(Fig. 6b).
Figure 6:
Comparison of L31 peptide sequences with
other protein sequences. a, alignment of the
NH-terminal part of the D domains D
,
D
, D
, and D
of MUC2, and the pro
vWF. Note that D
, D
, and D
occur in
the amino-terminal half of MUC2 and the pro vWF, and D
is
in the carboxyl-terminal half. b, alignment of a Cys-rich
sequence present in L31 with similar sequences present in HGM (gastric
mucin), JER47, JER62, and JUL32 (MUC5AC), and the amino-terminal region
of MUC2. Two consensus sequences are deduced from these comparisons and
correspond to a part of the consensus sequences previously described by
Gum et al. (8) and Guyonnet et al. (13),
respectively. Residues identical in more than 7 sequences or in 5 and 6
sequences are indicated with capital letters or lowercase
letters, respectively.
Chromosomal Localization
In situ hybridization using clone L 31 was conducted on two different sets
of experiments and a total of at least 50 dividing cells were examined.
Despite the fact that the signal was weak and not detectable on some
chromatids, the hybridization was localized specifically and
unambiguously in the most distal short arm band of chromosome 11,
11p15.5 (Fig. 7), where a cluster of mucin genes (MUC2, MUC5AC,
MUC5B, and MUC6) is located
(15, 36, 37) .
Figure 7:
Fluorescent in situ hybridization
with clone L31 probe. This partial metaphase shows one signal per
chromosome 11.
Expression Studies of L31 using Northern
Blotting
Since the expression of mucin mRNAs differs markedly in
HT29-MTX and HT29-FU cells and varies according to the stage of the
culture, we used mRNA from these cells to compare the expression of L31
mRNA with that of MUC2, MUC5AC, and MUC5B mRNAs
(Fig. 8A). Large transcripts (12 kb) were detected
in both HT29-MTX and HT29-FU cells. A high level of L31 mRNA was found
in postconfluent as compared with preconfluent HT29-MTX cells. No such
increase in expression occurred in HT29-FU cells where the levels
remained low in post-confluent cells. This differential and
growth-related expression of L31 mRNA is similar to that of MUC5AC but
greatly differs from that observed with MUC2 and MUC5B probes
(Fig. 8) or with MUC6, which is poorly expressed in MTX
cells
(26) .
Figure 8:
Northern blot analysis of HT-29
mucus-secreting cells. Poly(A) RNAs from HT29-MTX
(MTX) and HT29-FU (FU) cells in relation to cell growth (7, 14, and 21
days after seeding) were hybridized with L31, MUC5AC, MUC5B, MUC2, and
actin cDNAs. Note that the pattern of expression of the MUC5AC
transcripts is very similar to that of L31
mRNA.
Large and polydisperse transcripts of L31 clone
(from 12 kb to
1 kb) were observed in normal stomach (antrum
and fundus) (Fig. 9), but not in stomach cancers (data not
shown). No hybridization signal was detectable in samples from normal
colon, duodenum, or gallbladder (Fig. 9) or from uterus, ovary,
or thyroid (data not shown). The same pattern of tissue expression was
obtained with JER58 probe (MUC5AC) (data not shown). MUC6, a mucin cDNA
isolated from a gastric expression library
(15) , was found to be
strongly expressed in the stomach and in gallbladder, unlike L31 (data
not shown).
Figure 9:
Northern blot analysis of tissus. Total
RNA was extracted from normal antrum (lanes1 and
2), fundus (lanes3 and 4), colon
(lane5), duodenum (lane6), and
gallbladder (lane7). The same result was obtained
with the L31 or MUC5AC cDNAs. The arrows correspond to the
position of 28 and 18 S RNAs.
Southern Blotting Analysis
Southern blot analysis
was conducted on human genomic DNA from at least 4 individuals using a
variety of restriction enzymes. In all cases the membranes were probed
with L31 and the MUC5AC probe JER58. Particular attention was paid to
enzymes that do not cut within the L31 sequence. L31 detected a single
ScaI band of 9.5 kb, which was quite distinct from the single
band of greater than 18 kb detected with JER58 (Fig. 10). In
contrast, with EcoRI, HindIII, and XbaI the
single large band detected in each case was of the same mobility with
both probes and corresponded to fragments of 20-30 kb for
EcoRI (Fig. 10) and approximately 20 kb for
XbaI and HindIII (data not shown).
Figure 10:
Southern blot analysis. Four human
genomic DNAs were digested with EcoRI and four with
ScaI. The same two membranes were probed with L31 and JER58
(MUC5AC) cDNAs. The same single EcoRI fragment is detected
with the both probes, whereas two distinct ScaI fragments
hybridize with clone L31 and MUC5AC.
and COOH
terminus
(7, 8) . In this work, we have isolated
``unique'' mucin sequences using an antiserum prepared
against a gastric mucosal fraction containing native mucins.
20 kb) produced by digestion with three different
restriction enzymes. The combined results suggest strongly that clone
L31 represents the 3` end of the MUC5AC gene.
20-kb fragment. The possibility that these differences result from
rearrangement or splicing is thus currently being considered. The idea
of alternate 3` end splicing is also suggested by the report of another
candidate 3` end clone, JER51
(13) . In this clone, the stop
codon interrupts the TTSTTSAP repeat domain and yields a
carboxyl-terminal peptide with a very different overall structure.
Guyonnet Duperat et al. have proposed that different carboxyl
termini might exist in the case of MUC5AC
(13) . However, JER51
could also be located internally in the transcript. More work is
necessary to understand the relationship between the NP3a, L31, and
JER51 clones.
(
)
MUC5AC transcripts have been detected in 12-week-old fetal
colon but were no longer detectable later.
The decrease of
MUC5AC expression in colon is consistent with the disappearance of
mucins with gastric immunoreactivity during
development
(40) .
(
)
Another feature of MTX
cells is that mucin mRNAs are detectable by Northern blot analysis as a
single major large transcript
(25) , whereas large polydisperse
transcripts are detected in tissue
samples
(1, 6, 11, 12) . Although it has
not been confirmed that the multiple transcripts result from
alternative splicing, the presence of high levels of a single
transcript in HT29-MTX cells should simplify studies on the mechanisms
of regulation at the transcriptional level and facilitate the isolation
of the complete cDNA corresponding to a single transcript. The 5` ends
of mucin genes have so far been difficult to obtain, in part due to the
large size of mucin transcripts. The antiserum used here may facilitate
the isolation of 5` clones from a random primed cDNA library.
/EMBL Data Bank with accession number(s) Z48314.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.