From the
The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a
mucin-like glycoprotein carrying the carcinoma-associated sialyl-Lewis
a carbohydrate epitope. In an attempt to identify its apoprotein, an
NH
Mucins are a heterogeneous group of secreted or membrane-bound
glycoproteins characterized by a high carbohydrate content (>50% by
weight) caused by the attachment of O-glycans to serine or
threonine residues in more or less repetitive amino acid
sequences
(1) . Mucins were first isolated as secreted oligo- or
polymerized molecules from the gel-like mucus layer covering epithelial
surfaces
(2) . Later, however, the term mucin has also been used
to describe integral membrane glycoproteins present on the apical
surface of various epithelial cells
(3) and even on leukocytes
and other non-epithelial cells
(4) . Increased abundance and/or
abnormal glycosylation of mucins are often observed in
carcinomas
(5) .
We have previously described the isolation of
two mucins carrying the carcinoma-associated sialyl-Lewis a
(Si-Le
In this paper we demonstrate
that the apoprotein of L-CanAg is identical to that of CD43
(leukosialin, sialophorin) or, more likely, to an
NH
The rabbit anti-CD43 antiserum
(17) and the Si-Le
L-CanAg is a relatively small (150-300 kDa)
Si-Le
In order to confirm that the CD43 detected was
indeed derived from the L-CanAg that was produced by the COLO 205 cells
and not a result of some contamination of the samples,
immunoprecipitation experiments were performed on lysates of
metabolically labeled COLO 205 cells. Antiserum against
HF-deglycosylated L-CanAg was found to precipitate a band with an
apparent molecular mass of 61 kDa; a band of identical size was found
with an antiserum against CD43. Identical results were obtained with
both antisera in immunoprecipitation from lysates of the
CD43-expressing promyelocytic cell line HL-60. The conclusion that the
band that had precipitated with the anti-L-CanAg/HF antiserum was
indeed the CD43 precursor protein was further strengthened by the shift
in electrophoretic mobility upon tunicamycin treatment and the
pulse-chase kinetics observed, because similar characteristics have
been reported for the CD43 precursor
(7, 17) . However,
whereas the antisera against leukocyte CD43 also recognize the mature
glycoprotein (apparent M
The expression of
CD43 by the COLO 205 cells was also established by Northern blot
hybridization, showing the two-transcript pattern typical of CD43 mRNA
(26) both in COLO 205 and HL-60. Normal large and small
intestine RNA were weakly hybridized in a Multi-Tissue Northern blot
(data not shown); however, this result could be explained as a
contribution from dispersed intestinal lymphatic tissue.
CD43 has
hitherto been described as a membrane-bound sialoglycoprotein present
on leukocytes and some other hematopoietic
cells
(9, 27) . Most of the present knowledge about the
properties of CD43 derives from studies of leukocytes or leukocyte cell
lines. Leukocyte CD43 carries 1 N-glycan and approximately 80
O-glycans on its extracellular NH
L-CanAg shows several novel
features when compared with the forms of CD43 that have been
characterized earlier. First, although a few cases of binding of
anti-CD43 mAbs to cancer cells of non-blood cell origin have been
reported
(34, 35) , this is the first unambiguous
identification and characterization of a CD43 molecule expressed
outside of the hematopoietic cell lineage. The three other colon
carcinoma cell lines tested for CD43 expression in Northern blots and
immunoprecipitation were all negative (data not shown). It is
noteworthy that the cell line SW1116, which secretes a mucin-like
molecule similar to L-CanAg in size, does not express CD43.
Second,
in contrast to leukocyte CD43, which generally has been described as
being released only upon activation, L-CanAg appears to be a
constitutively secreted molecule. It seems likely that L-CanAg in fact
consists only of the extracellular domain of CD43, released by
proteolytic cleavage at the extracellular/luminal side of the molecule.
The hypothesis that L-CanAg is a fragment of CD43 is supported by the
observation that the anti-L-CanAg/HF antiserum seems to react only with
the extracellular domain of CD43, indicating that other parts of the
molecule were missing from the L-CanAg used for immunization. Moreover,
the amino acid composition of L-CanAg matches that of extracellular
CD43 more closely than that of total CD43
(6, 9) . A
comparison of the amino acid yields in the peptide sequencing with the
total protein amount analyzed also indicates that the apoprotein of
L-CanAg cannot be as large as that of the entire CD43 (data not shown).
It is not clear whether the putative cleavage event occurs
intracellularly or at the cell surface. It is interesting to note that
both the proteolytic cleavage of CD43 supposed to yield the plasma
galactoglycoprotein
(24) and the cleavage of the MUC1 mucin
apoprotein, which occurs during biosynthesis
(36) , have been
tentatively located to extracellular Phe-Arg sequences close to the
transmembrane domains of the respective glycoproteins.
Third, there
is a major difference between L-CanAg and leukocyte CD43 concerning
glycosylation. The predominant glycans of leukocyte CD43 have been
described as disialylated tetra- or hexasaccharides
(9) ,
depending on cell type. No individual glycan structure has been
determined for L-CanAg, but carbohydrate analysis has shown that its
sugar chains are considerably longer, with an average of about 17
sugars/chain, probably built on a polylactosamine backbone
(6) .
The abundance of fucose is also much higher in L-CanAg (14% of total
sugars) than in leukocyte CD43 (1.2% in HL-60 cells
(9) ), as is
the total carbohydrate content (85% (w/w) in L-CanAg, 67% in CD43 from
HL-60 cells
(9) ). The higher degree of glycosylation of L-CanAg
also explains its higher molecular mass as estimated by SDS-PAGE
(150-300 kDa
(6) ) when compared with leukocyte CD43
(115-135 kDa
(27) ). It has previously been shown that
different glycoforms of CD43 have markedly different mobilities in
SDS-PAGE
(37) .
Given all these differences between leukocyte
CD43 and L-CanAg, it does not seem surprising that the type of CD43
expression displayed by COLO 205 cells has escaped notice until now.
The standard method for detecting CD43 has been tissue immunostaining
or flow cytometry using glycosylation-dependent mAbs raised against
leukocyte glycoforms of CD43. One such mAb, Leu-22, was tested for
binding to L-CanAg in fluoroimmunoassay and showed no reactivity (data
not shown). This suggests the possibility that, if other carcinomas
also express L-CanAg-like CD43 glycoforms, they have not been possible
to detect with the anti-CD43 mAbs commonly used.
One might speculate
over the possible functional significance of CD43 expression in
carcinoma cells. CD43 has been described as being an anti-adhesion
molecule that prevents interactions between leukocytes (10, 38). The
expression of CD43 by a carcinoma cell might therefore facilitate tumor
dissemination. The observation that HeLa cells experience decreased
T-cell adhesion upon transfection with CD43
(39) strongly
suggests that CD43 expression may help malignant cells to evade immune
recognition. Recently, it has also been shown that L-CanAg can cause
Si-Le
We express our gratitude to CanAg Diagnostics AB for
providing monoclonal antibodies, to Dr. Eileen Remold-O'Donnell
for the gift of the anti-CD43 antiserum and for helpful suggestions,
and to Drs. Brian Seed and Jan Holgersson for sending the plasmid
containing CD43 cDNA.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal peptide sequence was obtained from purified
L-CanAg. In all interpretable positions, this sequence showed 100%
identity to the NH
-terminal of human CD43 (leukosialin,
sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only
identified in leukocytes and other hematopoietic cells. An antiserum
against deglycosylated L-CanAg and an anti-CD43 antiserum both
immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor,
from COLO 205 cells as well as from the known CD43-expressing cell line
HL-60. Results from immunoprecipitations following pulse-chase
experiments and tunicamycin treatments were in agreement with earlier
studies on the CD43 precursor. RNA blot analysis confirmed the
expression of CD43 by the COLO 205 cell line, whereas three other colon
carcinoma cell lines were negative. The glycosylation-dependent
monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to
bind L-CanAg, probably due to its much more extensive glycosylation. We
conclude that L-CanAg is the secreted extracellular domain of a novel
glycoform of CD43 and that CD43, if expressed in other carcinoma cells,
may have escaped notice in studies relying on glycosylation-dependent
monoclonal antibodies against leukocyte CD43.
;
NeuAc
2
3Gal
1
3(Fuc
1
4)
GlcNAc
1
)
(
)
carbohydrate epitope from
xenografts and spent culture medium of the colon adenocarcinoma cell
line COLO 205
(6) . The larger (600-800 kDa) mucin, named
H-CanAg, was predominantly a membrane-bound molecule and was shown to
have the MUC1 gene product as apoprotein. MUC1 mucins are
often abnormally expressed in carcinomas. The smaller (150-300
kDa) mucin, named L-CanAg, appeared to be a secreted molecule, and its
apoprotein could not be identified.
-terminal part of this molecule. CD43, which was
originally isolated from leukocytes
(7, 8) , is an
integral membrane sialoglycoprotein with a mucin-like extracellular
domain
(9) . Its expression has until now been described as
restricted to the hematopoietic cell lineage
(10) .
Cell Culture
The COLO 205 colon adenocarcinoma
cell line (ATCC CCL 222) was cultured in Iscove's medium (Life
Technologies, Inc., Paisley, Scotland) containing 10% fetal calf serum
and supplemented as described
(11) . The promyelocytic cell line
HL-60 (ATCC CCL 240) was maintained in RPMI 1640 medium (Life
Technologies, Inc.) containing 2 mM glutamine and 10%
complement-inactivated fetal calf serum.
Purification of L-CanAg
Partially purified L-CanAg
was prepared by trichloroacetic acid precipitation and Superose 6 gel
filtration as described
(12) . This procedure yielded the
material used for HF treatment and immunization (see below). For
peptide sequencing, the sample was further purified with affinity
chromatography using immobilized Si-Le-reactive monoclonal
antibody (mAb) C241 and anion exchange chromatography in 8 M
urea, both as described
(6) . Finally, the purified L-CanAg was
passed over a 1-ml column of protein A-Sepharose (Pharmacia,
Sollentuna, Sweden) to adsorb possible residual immunoglobulin from the
affinity column; the flow-through fraction was then applied to a
Superose 6 HR 10/30 gel filtration column (Pharmacia) and eluted in 0.1
M NH
Ac, pH 7.0. Fractions were tested for
Si-Le
content by fluoroimmunoassay. Fractions from the
L-CanAg peak were collected and lyophilized. When finally purified,
L-CanAg was subjected to peptide sequencing.
Peptide Sequencing
Highly purified L-CanAg
corresponding to approximately 11 µg of protein was sequenced by
Edman degradation (13) using an Applied Biosystems 477A pulsed liquid
phase sequencer with on-line analysis of phenylthiohydantoin
derivatives
(14) .
Antibodies, Peptides, and DNA Probes
Partially
purified L-CanAg was deglycosylated by HF treatment at room temperature
overnight
(15) and was used to immunize rabbits as
described
(16) . Approximately 5 µg of protein was used for
each injection. The obtained antiserum will be referred to below as
``anti-L-CanAg/HF.''
-reactive mAb C241
(18) were gifts generously provided by Dr. Eileen
Remold-O'Donnell (Center for Blood Research, Boston, MA) and
CanAg Diagnostics AB (Göteborg, Sweden), respectively. The
CD43-reactive mAb Leu-22 was purchased from Becton Dickinson (San
José, CA). The plasmid pCD43TkHyg cfi
containing CD43 cDNA was a kind gift from Drs. Jan Holgersson and
Brian Seed (Harvard Medical School, Boston, MA). The CD43 insert was
excised by digestion with NotI and HindIII and
purified using the QIAEX DNA gel extraction kit (QIAGEN, Hilden,
Germany).
Cell Labeling
COLO 205 cells or HL-60 cells were
starved in methionine-free minimal essential medium (Life Technologies,
Inc.) supplemented with 2 mM glutamine and 5% fetal calf serum
1 h prior to labeling. In some experiments, tunicamycin (Calbiochem, La
Jolla, CA) was added to the methionine-free medium to a final
concentration of 20 µg/ml (for COLO 205) or 5 µg/ml (for
HL-60). Cells were labeled by the addition of a mixture of
S-labeled methionine and cysteine (Pro-Mix, Amersham
Corp.) in an amount corresponding to 200 µCi of
[
S]methionine/6-cm Petri dish. In pulse-chase
experiments, the chase was initiated by the addition of unlabeled
methionine and cysteine (both 100 µg/dish). The incorporation was
stopped by putting the dishes on ice. Cells were washed twice with
ice-cold 10 mM sodium phosphate, pH 7.2, 0.15 M NaCl
and then lysed by brief sonication in 1.25 ml of the same buffer
containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1%
Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1
µg/ml aprotinin, 25 µg/ml leupeptin, 0.7 µg/ml pepstatin,
and 2 µg/ml calpain inhibitor I (the last two were from Boehringer
Mannheim).
Immunoprecipitation
Labeled cell lysates were
incubated overnight with 25 µl of nonimmune rabbit serum (NRS) at 4
°C followed by the addition of 150 µl of a 10% (w/v) suspension
of fixed Staphylococcus aureus (Immunoprecipitin, Life
Technologies, Inc.). After 30 min at room temperature, the
Immunoprecipitin was removed by centrifugation at 14,000 g for 2 min. Precipitation with NRS was repeated once, and the
Immunoprecipitin from the second NRS precipitation was saved and used
as a nonspecific control. Specific antiserum (12 µl for anti-CD43,
25 µl for anti-L-CanAg/HF) was added and precipitated as described
above. The Immunoprecipitin was washed 5 times with 1 ml of 10
mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1% Triton X-100,
and 0.1% SDS and eluted by incubation at 95 °C with 50 mM
Tris-HCl, pH 6.8, 10% glycerol, and 2% SDS.
SDS-PAGE and Autoradiography
Samples were applied
to 1.5-mm-thick SDS-polyacrylamide gels
(19) with 3% stacking
gels and either 10% homogeneous or 3-15% gradient separation
gels. C-Methylated Rainbow high molecular weight marker
proteins (Amersham) were used as size references. Gels were fixed,
dried, and exposed as described
(6) .
Northern Blot Analysis
Total RNA was prepared from
COLO 205 and HL-60 cells by the guanidinium thiocyanate-guanidinium
chloride method
(20) . Extraction of polyadenylated RNA and
Northern blot analysis were performed as
described
(21, 22) . A Multi-Tissue Northern membrane
(Clontech, Palo Alto, CA) was also probed with CD43 cDNA.
NH
Seventeen rounds of Edman degradation of intact, highly
purified L-CanAg yielded the sequence
XXXAVQXPXXGEPLVXX, where X represents a cycle in which no amino acid could be identified. The
glutamate in position 12 was regarded as ambiguous. A computer search
of the EMBL data base using the TFASTA algorithm of the GCG program
package
(23) showed that the identifiable amino acids had a 100%
identity with the NH-terminal Peptide Sequence of
L-CanAg
-terminal of human CD43 (leukosialin,
sialophorin) in its mature form (signal peptide removed). As shown in
Fig. 1
, the unidentifiable positions in the L-CanAg sequence all
coincided with Ser or Thr in the CD43 sequence. Because glycosylated
amino acids are lost in the Edman analysis upon solvent extraction of
the derivatized amino acid before chromatography, it seems likely that
the blanks in the L-CanAg sequence were due to O-glycosylation
of serines and threonines.
Figure 1:
The NH-terminal amino acid
sequence of L-CanAg, as determined by Edman degradation, compared with
the CD43 sequence. Cycles in which no amino acid could be determined in
the analysis of L-CanAg are marked by x. The lowercase e in position 12 of the L-CanAg sequence indicates that this
identification was ambiguous. In the CD43 sequence, the site of
cleavage of the hydrophobic signal peptide (26) is indicated by an
arrow.
Detection of CD43 mRNA in COLO 205
Cells
Poly(A) RNA from the colon carcinoma cell
line COLO 205, which produces L-CanAg, and from the promyelocytic cell
line HL-60, which is known to produce CD43, was examined using a CD43
cDNA probe in a Northern blot hybridization. In both HL-60 and COLO
205, two hybridizing bands of 2.0 and 8.3 kilobases were found
(Fig. 2). The amount of CD43 mRNA appeared to be substantially
lower in COLO 205 than in HL-60, because the amounts of total RNA used
were the same for both cell lines. None of the colon carcinoma cell
lines SW1116, LoVo, or LS174T contained detectable levels of CD43 mRNA
(data not shown).
Figure 2:
Northern blot showing
poly(A) RNA from the COLO 205 and HL-60 cell lines
hybridized with a CD43 cDNA probe. Equal amounts of total RNA from COLO
205 and HL-60 were subjected to poly(A)
RNA extraction
and Northern blotting. The positions of RNA marker bands are indicated
by the lines. kb,
kilobases.
Immunoprecipitation of the L-CanAg Precursor
Protein
Rabbit antisera against HF-deglycosylated L-CanAg
(anti-L-CanAg/HF) and against CD43 were used in immunoprecipitation of
cell lysates of metabolically labeled COLO 205 and HL-60 cells. In COLO
205, the anti-L-CanAg/HF antiserum and the anti-CD43 antiserum both
precipitated a single band with an apparent molecular mass of 61 kDa
(Fig. 3A). Upon tunicamycin treatment, the band
precipitated by anti-L-CanAg/HF was shifted in apparent size to 58 kDa.
Identical bands appeared in immunoprecipitation experiments with HL-60
cells, with both the anti-L-CanAg/HF (Fig. 3B) and the
anti-CD43 antisera (data not shown). A pulse-chase experiment with COLO
205 cells showed that the anti-L-CanAg/HF band remained at its position
until after 40 min of chase and then disappeared (Fig. 4).
Similar results were obtained in HL-60 cells (data not shown).
Figure 3:
Immunoprecipitation of lysates of
metabolically labeled COLO 205 (A) and HL-60 cells
(B) using antisera against deglycosylated L-CanAg and against
CD43. Cells were starved for 1 h with (+) or without (-)
tunicamycin (20 µg/ml for COLO 205; 5 µg/ml for HL-60) as
indicated and labeled with 200 µCi of
[S]methionine for 20 min. Lysates were
precipitated twice with nonimmune rabbit serum (the second precipitate
is shown in the lanes marked by NRS) before
immunoprecipitation using antiserum against HF-deglycosylated L-CanAg
(anti-L-CanAg/HF) or antiserum against CD43
(anti-CD43) (17). The samples were analyzed on a 10% SDS-PAGE
gel. The lines indicate sizes in kDa of molecular mass marker
proteins.
Figure 4:
Immunoprecipitation of lysates of
pulse-chase labeled COLO 205 cells using antiserum against
deglycosylated L-CanAg. Cells were starved for 1 h, labeled for 10 min
with 200 µCi of [S]methionine, and chased
with excess unlabeled methionine for the time periods indicated.
Lysates were precipitated twice with nonimmune rabbit serum (the second
precipitate is shown in the lanes marked by NRS)
before immunoprecipitation with antiserum against HF-deglycosylated
L-CanAg (lanes marked by Ab). Samples were analyzed
on a 3-15% gradient SDS-PAGE gel. The lines indicate
sizes in kDa of molecular mass marker
proteins.
-carrying mucin secreted by the colon adenocarcinoma
cell line COLO 205. These cells also produce a membrane-bound MUC1
mucin, H-CanAg. In the present study, we have in several ways
demonstrated that the apoprotein of L-CanAg is identical to at least a
part of the apoprotein of the leukocyte-associated membrane
sialoglycoprotein CD43. The amino-terminal peptide sequence of L-CanAg
was identical to that of CD43 in all positions that could be determined
(9 out of 17) (Fig. 1). Because the 8 unidentifiable positions in
the L-CanAg sequence all corresponded to Ser or Thr in the CD43
sequence, it seems most likely that all of these hydroxy amino acids
were glycosylated in L-CanAg, because glycosylated amino acids are not
recovered in the sequencing procedure used. A similar
NH
-terminal glycosylation pattern has been reported for
human plasma galactoglycoprotein, a fragment of CD43 present in normal
blood
(24) .
approximately 115 kDa),
which starts to appear after 10-20 min of chase in leukocyte cell
lines
(7, 17) , the reactivity of the anti-L-CanAg/HF
antiserum seemed to be restricted to the nonglycosylated precursor both
in COLO 205 and HL-60. It therefore seems likely that all epitopes
recognized by the anti-L-CanAg/HF antiserum were directed to the
extracellular, Ser/Thr-rich domain of the apoprotein and eventually
became masked by O-glycosylation upon maturation of the
glycoprotein. It should also be added that, although there is a wide
discrepancy between the apparent M
of the
immunoprecipitated bands (61 kDa) and the molecular mass deduced from
the cDNA sequence of CD43 (40 kDa), this highly aberrant
electrophoretic mobility of the CD43 precursor has been observed in
several other studies
(17, 25) .
-terminal
portion, the latter mostly occurring as tetra- or
hexasaccharides
(9) . The 123-amino-acid cytoplasmic domain is
constitutively phosphorylated and thought to be engaged in
transmembrane signaling
(28, 29, 30) . CD43 is
proteolytically cleaved from the surface of leukocytes upon activation
by binding of anti-CD43 mAbs
(31, 32) . Released
extracellular CD43 is found in normal blood as a ``plasma
galactoglycoprotein'' at a concentration of >10
µg/ml
(24, 33) .
-dependent inhibition of leukocyte binding to
E-selectin, an endothelial leukocyte receptor (12). Secretion of
Si-Le
- or Si-Le
-carrying glycoforms of CD43 may
therefore be advantageous to cancer cells by impeding leukocyte
recruitment to the tumor. The cell-activating properties of CD43, which
in some cases lead to proliferation
(29) , might also influence
the phenotype of a CD43-expressing carcinoma cell.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.