Cell-CAM105 isoforms with different adhesion functions are coexpressed in adult rat tissues and during liver development

Abstract

The rat hepatocyte cell adhesion molecule cell-CAM105 has recently been shown to be composed of at least two isoforms. Expression of the two isoforms in different tissues and during fetal liver development in rats was studied by RNase protection using a probe which could specifically and simultaneously detect both isoforms. This probe revealed protected fragments of expected lengths for the L-form and the S-form in RNA samples isolated from various adult rat tissues. High levels of the L-form and S-form messages were detected in liver and intestine, moderate levels were detected in lung, and weak signals were detected in muscle, kidney, and spleen. In liver development studies, the messages for cell-CAM105 showed a major increase on the first day after birth compared to the fetal stage, and both isoform messages were proportionally increased. These results indicate that both cell-CAM105 isoforms may have function(s) related to hepatocyte differentiation. To study the adhesion function of cell-CAM105 isoforms, full-length cDNAs for these isoforms were expressed in insect cells. The insect cells expressing the L-form cell-CAM105 were found to aggregate. However, expression of S-form cell-CAM105 did not support cell aggregation. These results indicate that L-form, but not S-form, cell-CAM105 directly mediates the cell adhesion function.

Full text

THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
0
1993 by The American Society for Biochemistry and Molecular Biology, Inc.
VOl.
268,
No.
9,
Issue
of
March
25.
,
pp.
6139-6146,1993
Printed
in
U.
S.A.
Cell-CAM105 Isoforms with Different Adhesion Functions Are
Coexpressed in Adult Rat Tissues and during Liver Development*
(Received for publication, June 23,1992)
Peter
H.
Cheung$, Nancy L. Thompson§, Karen Earley$, Ognjen Culic$, Douglas HixsonS,
and Sue-Hwa LinSll
From the $Department of Molecular Pathology, The University of Texas M.
D.
Anderson Cancer Center, Houston, Texas 77030
and the §Department of Medical Oncology, Rhode Island Hospital, Brown University, Providence, Rhode Island 02903
The rat hepatocyte cell adhesion molecule cell-
CAM105 has recently been shown to be composed of at
least two isoforms. Expression of the two isoforms in
different tissues and during fetal liver development in
rats was studied by RNase protection using a probe
which could specifically and simultaneously detect
both isoforms. This probe revealed protected frag-
ments of expected lengths for the L-form and the
S-
form in RNA samples isolated from various adult rat
tissues. High levels of the L-form and S-form messages
were detected in liver and intestine, moderate levels
were detected in lung, and weak signals were detected
in muscle, kidney, and spleen. In liver development
studies, the messages for cell-CAM105 showed a major
increase on the first day after birth compared to the
fetal stage, and both isoform messages were propor-
tionally increased. These results indicate that both cell-
CAM105 isoforms may have function(s) related to hep-
atocyte differentiation.
To study the adhesion function of cell-CAM105 iso-
forms, full-length cDNAs for these isoforms were ex-
pressed in insect cells. The insect cells expressing the
L-form cell-CAM105 were found to aggregate. How-
ever, expression of S-form cell-CAM105 did not sup-
port cell aggregation. These results indicate that
L-
form, but not S-form, cell-CAM105 directly mediates
the cell adhesion function.
The rat hepatocyte cell adhesion molecule cell-CAM105
(Lin
et
al.,
1991; Aurivillius
et
al.,
1990) has structural features
typical of the immunoglobulin superfamily members. We have
isolated a new clone that is a variant of the previously isolated
cell-CAM105, indicating the existence of at least two isoforms
of the cell-CAM105 molecule (Culic
et
al.,
1992). In addition
to
having a shorter cytoplasmic domain, this new isoform has
substitutions clustered within the first 130 amino acids of the
extracellular domain. These sequence differences may or may
not confer different functions on these isoforms.
Cell-CAM105 was originally isolated from rat liver plasma
*This work was supported by Grants GM43189 (to
S.
H.
L.),
CA42715 (to D. C.
H.),
and Core Grant CA16672 from the National
Institutes
of
Health. The costs
of
publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked
“advertisement”
in accordance with
18
U.S.C. Section 1734 solely to indicate this fact.
to the GenBankTM/EMBL Data Bank with accession number(s) 504963
The nucleotide sequence(s) reported in this paper
hus
been submitted
(L-form cell-CAMl05) and 212019 (S-form cell-CAMlO5).
1
TO
whom correspondence should be addressed Dept. of Molecu-
lar Pathology, Box 89, University
of
Texas M. D. Anderson Cancer
Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-794-
1559; Fax: 713-794-4672.
membrane (Ocklind and Obrink, 1982). With the availability
of antibodies, it was subsequently shown that this protein is
also present in various epithelia, platelets, and granulocytes
(Odin
et al.,
1988). Using indirect immunofluorescence and
immunogold labeling, we have recently shown that cell-
CAM105 is also expressed in specific segments of the renal
proximal tubules and the plasma membrane of endothelial
cells (Sabolic
et al.,
1992). However, using a monoclonal
antibody (mAb362.50) against cell-CAM105, this protein can
be detected only in the liver and kidney (Hixson and Mc-
Entire, 1985). These seemingly contradictory results presum-
ably arise from heterogeneities of these proteins in different
tissues. Consistent with this notion is the observation that
the anti-cell-CAM105 polyclonal antibodies react with pro-
teins with different apparent molecular weights in different
tissues (Odin
et al.,
1988). It has also been shown that the
biochemical properties of intestinal cell-CAM105 are signifi-
cantly different from those of liver cell-CAM105 (Hansson
et
al.,
1989). These observations together suggest that there are
tissue-specific differences between the cell-CAM105 mole-
cules. These differences could be due to variations in protein
modifications or protein sequences. Both glycosylation and
phosphorylation of cell-CAM105 have been observed (Odin
et
al.,
1986). According to the sequences derived from the cell-
CAM105 cDNA clones, each isoform has 16 potential
N-
linked glycosylation sites. The calculated molecular masses
based on the sequences derived from cDNA are 57 and 52
kDa for long form (L-form) and short form (S-form), respec-
tively, whereas the proteins purified from liver have apparent
molecular masses of 110 and
105
kDa, respectively. This
indicates that both isoforms are heavily glycosylated and that
about half of the apparent molecular masses are contributed
by the sugar moieties. Since liver cell-CAM105 sequences are
the only ones available
so
far, it is not clear whether cell-
CAM105 molecules from other tissues have protein sequences
identical to those of liver cell-CAM105. Studies on the expres-
sion of cell-CAM105 in different tissues may give insights
into the functional roles of this adhesion molecule
in
vivo.
In
addition, the relative expression of isoforms in different tis-
sues may provide clues to their functional roles.
Cell-CAM105
is
highly homologous to the carcinoem-
bryonic antigen (CEA)’ family of proteins, which includes
CEA, nonspecific cross-reacting antigen (NCA), biliary gly-
coprotein
1
(BGPl), and pregnant-specific glycoproteins
(PSG) (Lin
et
al.,
1991). Isoforms have also been found for
these other molecules (Thompson
et
al.,
1989a; Barnett
et al.,
1989; Thompson
et
al.,
1987; Oikawa
et
al.,
1987; Neumaier
et
al.,
1988; Tawaragi
et
al.,
1988; Zimmermann
et
al..
1989).
The abbreviations used are: CEA, carcinoembryonic antigen;
NCA, nonspecific cross-reacting antigen; BGP1, biliary glycoprotein
1; PSG, pregnant-specific glycoproteins; nt, nucleotide.
6139

6140
Function and Expression
of
Cell-CAM105
Isoforms
Because such a large number of highly homologous molecules
is
present in tissues, specificity cannot be guaranteed when
Northern blot analysis is used to detect cell-CAM105 expres-
sion. Moreover, it is difficult to distinguish between the
expression of isoforms with Northern RNA blot analysis. The
cell-CAM105 and its human homologue, BGP1, are unique
among the CEA family members in that they are the only
molecules that contain cytoplasmic domains (Barnett
et al.,
1989; Hinoda
et
al.,
1988). Probes generated from the cyto-
plasmic domain may be able to distinguish cell-CAM105
family members from other CEA family members, allowing
the expression of cell-CAM105 to be studied. In addition, use
of RNase protection instead of Northern blot analysis can
further enhance specificity. RNase protection is sensitive and
can quantify expression of specific sequences under investi-
gation. As a result, the relative amount of message for each
isoform can be measured if probes are designed from regions
of unique sequences.
The recently published nucleotide sequences of cell-
CAM105 isoforms (Lin
et
al.,
1991; Culic
et
al.,
1992) differ
in the sizes of their intracellular COOH-terminal tails (71
amino acids
uersm
10
amino acids). The cytoplasmic domain
of the L-form contains several potential phosphorylation sites
which are not present in the S-form.
In vitro
phosphorylation
study showed that both isoforms were phosphorylated, though
the L-form was phosphorylated to a significantly higher ex-
tent (Culic
et
al.,
1992). Whether this differential phosphoryl-
ation contributes to the mechanisms of regulation of isoform
functions is not known. Although the extracellular NH2-
terminal domains of L- and S-form cell-CAM105 are of the
same length (389 amino acids), subtle differences have been
identified in the primary structures of these extracellular
domains. The functional consequences of these differences
need to be determined before the physiological significance
for the presence of isoforms can be addressed. In this paper,
we report results from RNase protection studies of the tissue-
specific expression of cell-CAM105 isoforms in adult rats and
the expression of cell-CAM105 isoforms in rat livers during
development. In addition, the role of cell-CAM105 isoforms
in cell adhesion function was studied by expression of indi-
vidual cell-CAM105 isoforms in insect cells using a baculoviral
vector.
MATERIALS AND METHODS
Plasmid Construction
and
Preparation of RNA Probe-A 219-base
pair BglII-NsiI fragment localized near the
3'
end of cell-CAM105
cDNA was excised from full-length L-form cDNA (Fig.
L4)
and
inserted into the BamHI-PstI sites of Bluescript plasmid vector
(Stratagene, La Jolla, CA) to produce the plasmid pBS-BglII/NsiI.
Orientations of the inserts were determined by double-stranded
di-
deoxy sequencing with T3 and universal primers using the T7 Se-
quenase kit (United States Biochemical Corp.) according to the
procedures provided by the manufacturer.
To synthesize antisense RNA probes, plasmid pBS-BglII/NsiI was
linearized with restriction enzyme XbaI. Radiolabeled RNA probes
were synthesized from the linearized DNA template using T7 (pBS-
BglII/NsiI) polymerase in the presence of [3ZP]UTP as described by
Melton et
al.
(1984). DNA templates were digested with RNase-free
DNase
I
(Promega, Madison, WI), and the labeled probes were
purified on a 4% polyacrylamide, 7
M
urea gel or used directly for
RNase protection experiments.
Preparation of RNA from Different Tissues-For studies of tissue
specificity, adult rats were anesthesized and tissues were excised and
immediately homogenized in a guanidine isothiocyanate solution.
RNA was then isolated by the method of Chomczynski and Sacchi
(1987). The integrity of RNA was determined by formaldehyde aga-
rose gel electrophoresis followed by ethidium bromide staining and
UV
transillumination.
Northern RNA Blot Analysis-Aliquots of
10
pg of total RNA were
separated by agarose/formaldehyde gel electrophoresis. Gels were
then equilibrated in
1
M
ammonium acetate and transferred to Nytran
(Schleicher and Schull) as described previously (Thompson et
al.,
1989b). An RNA ladder (Bethesda Research Laboratories) was elec-
trophoresed together with tissue RNA to serve as size markers. The
219-base pair BglIIINsiI fragment was labeled for use as a probe by
incorporation of [32P]dCTP (Du Pont-New England Nuclear; 3000
Ci/mmol) using a random primer labeling kit (Boehringer Mann-
heim). Hybridization and washing of blots were carried out at 65 "C
according to the procedure of Church and Gilbert (1984). Blots were
wrapped in plastic wrap, and x-ray film was exposed to them (Kodak)
at
-70
"C in the presence of intensifying screens.
RNase Protection Assay-Ten micrograms of total RNA was hy-
bridized with
2
X
lo5
cpm of 32P-labeled antisense RNA probe and
subjected to an RNase protection protocol (Zinn et
al.,
1983). The
protected fragments were analyzed on an
4%
polyacrylamide, 7
M
urea gel. Gels were dried and analyzed by autoradiography. Auto-
graphs from RNase protection assays were scanned with a Zeineth
Soft Laser Scanning Densitometer (Biomedical Instruments, Fuller-
ton, CA) connected to a personal computer (Leading Edge) with a
software program purchased from the manufacturer. The band as-
signed to each given isoform transcript was quantified.
Preparation of RNA from Fetal and Neonatal Rat Livers-Timed
pregnant Sprague-Dawley rats were obtained from Charles River
(Wilmington, MA) and maintained on standard chow and water
ad
libitum. Fetuses were removed at gestational ages 15-21 days from
rats sacrificed by methoxyflurane (Metofane) overanesthesia and
were placed on ice in sterile 0.9% saline. Livers were dissected free of
other tissue with the aid of a dissecting microscope and sterile
instruments, and the majority of the tissue was flash-frozen in liquid
nitrogen for extraction of RNA. All livers from the same brood were
pooled. Neonatal animals were sacrificed at 1,
4,
8, or 19 days after
birth by Metofane anesthesia followed by decapitation. Livers were
dissected free of other tissue, pooled, and frozen as described above.
To repeat the developmental study, livers were harvested from a
second independent group of animals at 17 or 20 days' gestation or 1,
5, or 19 days after birth. Livers from normal adult (8-10-week-old)
Sprague-Dawley rats served as control samples. Total RNA was
prepared by homogenization of frozen tissue in guanidine isothiocy-
anate followed by centrifugation over a cesium chloride cushion as
described previously (Chirgwin et
al.,
1979).
Cloning of the Full-length Cell-CAM105 cDNAs into Buculoviral
Expression Vectors-The full-length L-form cell-CAM105 cDNA was
excised from pBS/full plasmid using XbaI and NsiI digestion (Lin
and Guidotti, 1989). The XbaI/NsiI fragment was then inserted into
plasmid pVL1393 baculoviral transfer vector utilizing the XbaI and
PstI cloning sites (Invitrogen, San Diego, CA). The full-length
S-
form cell-CAM105 cDNA in baculoviral transfer vector was con-
structed in a similar fashion by inserting the full-length S-form cDNA
(Culic et
al.,
1992) into pVL1393 between XbaI and PstI sites.
Recombinant baculoviruses carrying either the L-form or S-form
cell-CAM105 genes were generated, and
Sf9
(S.
frugiperda) cells were
infected and grown according to the procedures of Summers and
Smith (1987).
Adhesion Assays-Cell adhesion assays were performed using two
different methods. In the first method,
Sf9
cells in monolayer culture
were infected with L- or S-form recombinant virus or wild type
baculovirus. Formation of cell aggregates was examined at 96 h post-
infection. In the second method,
Sf9
cells in suspension were infected
with L- or S-form recombinant virus or wild type baculovirus. The
virus-infected
Sf9
cells were cultured in spinner flasks. At various
time intervals, aliquots of control
Sf9
cells
or
virus-infected
Sf9
cells
were removed and treated with 50 units/ml of DNase (Promega). The
number of single cells in these aliquots were counted using a hemo-
cytometer.
Immunoblot Analysis of Cell-CAM105 Isoforms Expressed in Sf9
Cells-Aliquots of
Sf9
cells with or without virus infection were boiled
in SDS sample buffer and analyzed by SDS-polyacrylamide gel elec-
trophoresis (Laemmli, 1970). Western immunoblotting using Ab669
was performed as described previously (Culic et
al.,
1992) except that
alkaline phosphatase-conjugated goat anti-rabbit antibody was used
as the second antibody. Color development was achieved using nitro
blue tetrazolium and
5-bromo-4-chloro-3-indolyl
phosphate.
RESULTS
Expression
of
Long- and Short-form
Cell-CAM105
in Differ-
ent
Tissues-To
study whether the cell-CAM105 isoforms are
differentially expressed in different rat tissues, total RNA

Function and Expression of Cell-CAM105 Isoforms
6141
FIG.
1.
A,
schematic diagram of the
L-
and
S-form
sequences and the region
used in the preparation
of
antisense
RNA
probe.
B,
sequence comparison of
the
L-
and S-form in the COOH-termi-
nal cytoplasmic region chosen for the
preparation
of
the antisense
RNA
probe.
The nucleotides were numbered accord-
ing
to
Culic
et
al.
(1992).
A
bp
1
300
600
900
1200
1500
t
800
I
I
1
I
I
I
I
I
I
I
I
L-form
B
L
1377
-
AGATCTCACA GAGCACAAAC CCTCAACCTC CAGCCACAAT
II
AT
S
1362
-
L
1417
-
CTGGGTCCTT CTGACGACTC TCCTAACAAG GTGGATGACG
S
1364
-
CTGGGTCCTT CTGACGACTC TCCTAACAAG GTGGATGACG
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
L
1457
-
TCTCATACTC TGTCCTGAAC TTCAATGCCC AGCAATCCAA
S
1404
-
TCTCATACTC TGTCCTGAAC TTCAATGCCC AGCAATCCAA
L
1497
-
ACGACCAACT TCAGCCTCTT CAAGCCCCAC AGAAACAGTT
S
1444
-
ACGACCAACT TCAGCCTCTT CAAGCCCCAC AGAAACAGTT
L
1537
-
TATTCGGTAG TCAAAAAGAA GTGACATTGT CTGTCCTGCT
S
1484
-
TATTCGGTAG TCAAAAAGAA GTGACATTGT CTGTCCTGCT
L
1577
-
GACTGCACCA GTGATGCAT
S
1524
-
GACTGCACCA GTGATGCAT
IIIIIIIIII IIIIIIIIII
IIIIIIIIO
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIIII
IIIIIIIII
12
3
45678910
7.5kb+
4kb+
2.4kb-
FIG.
2.
Northern blot
analysis
of cell-CAM106 messages in
tissues from various rat strains.
Lane
1,
liver (German Fischer);
lane
2,
liver (United States Fischer);
lane
3,
kidney (Sprague-Dawley);
lane
4,
kidney (German Fischer);
lane
5,
small intestine (German
Fischer);
lane
6,
small intestine (United States Fischer);
lane
7,
pancreas (Sprague-Dawley);
lane
8,
adrenal gland (Sprague-Dawley);
lane
9,
brain (United States Fischer);
lane
IO,
heart (United States
Fischer).
samples were isolated from various
rat
tissues. Northern blot
analyses of total RNA using
a
probe generated from the
COOH-terminal cytoplasmic domain showed that the cell-
CAM105 has
a
message size of around
4
kilobases (Fig. 2).
High levels of cell-CAM105 RNA were detected in liver and
small intestine from two different rat strains (German Fischer
and United
States
Fischer rats), whereas cell-CAM105 RNA
signal were not detectable in kidney, brain, heart, adrenal
glands, and pancreas. These results indicate that the cell-
CAM105 messages are more enriched in liver and small intes-
tine. There was no detectable difference in the expression of
cell-CAM105
at
the RNA level (Fig. 2) in two substrains of
Fischer rats
(ie.
United
States
and German), although differ-
ences in the expression of DPPIV protein were observed
previously (Thompson
et
al.,
1991).
Since Northern blot analysis does not provide information
on the relative expression of cell-CAM105 isoforms, RNase
protection studies were performed using probes generated
from the COOH-terminal cytoplasmic domain (Fig.
1).
The
cDNA sequences for both cell-CAM105 isoforms were derived
from
a
cDNA library made from Sprague-Dawley rat livers.
The sequences for the L- and the S-form in the COOH-
terminal cytoplasmic domains are shown in Fig.
1B.
The
COOH-terminal cytoplasmic domain antisense probe (305
nucleotides) spans nucleotides (nt) 1377 (BglII site) to 1595
(NsiI site) (Fig.
L4)
of the L-form and contains a segment
from the plasmid vector. Because a 38-nt sequence within the
219-nt BglII/NsiI fragment is deleted in the S-form, this probe
can detect a 181-nt fragment of the S-form transcript in
addition to a 219-nt fragment of the L-form transcript.
As
a
result, the antisense probe selected allowed the simultaneous
detection of both the L- and the S-form transcripts.
To quantify the relative expression of cell-CAM105 iso-
forms in different tissues, the 32P-labeled COOH-terminal
cytoplasmic domain antisense RNA probe was annealed to
total RNA prepared from different tissues of Sprague-Dawley
rats. After RNase digestion, 219- and 181-nt fragments of the
L-
and the S-form, respectively, should have been detectable
if the tissues had the same COOH-terminal cytoplasmic do-
main sequences
as
those of the liver. Fig. 3A shows that strong
signals of both 219 and
181
nt were detected in liver and
intestine, moderate signals were detected in lung, and weak
signals were detected for muscle, kidney, and spleen. Although
signals corresponding to both the L-f and the S-form were

6142
Function and Expression
of
Cell-CAM105
Isoforms
123456709
~.
219 nt
-
L-form
181 nt
-
S-form
219 nt
-.
L-form
181 nt
-
S-form
C
detected in many tissues, the relative expression of the L- and
the S-form RNA varied among tissues (Fig.
3A).
In liver and
kidney, the S-form message was about twice that of the L-
form, whereas in intestine, the L-form message was slightly
more abundant than that of the S-form. Both isoform mes-
sages were expressed about equally in other tissues, including
lung and spleen. Brain, testis, and heart did not show detect-
able signals for either
L-
or S-form message. In addition,
different levels of isoform messages were observed in different
segments of intestine (Fig.
3B).
The small intestine had much
higher steady-state levels of isoform messages than did the
colon. Within the small intestine, the ileum had the highest
level of cell-CAM105 message, the jejunum the second highest,
and the duodenum the lowest level. Despite the differences in
the relative abundance of isoform messages, none of the
tissues examined expressed either the L- or S-form alone.
Expression of Cell-CAM105 in Different Rat Strains-
RNase protection experiments using the COOH-terminal cy-
toplasmic domain probe were also performed with rat liver
RNA samples prepared from two other strains. As shown in
Fig.
3C,
there was little variability between samples from
different strains,
ie.
Fischer, ACI, and Sprague-Dawley rats.
This result indicates that the liver cell-CAM105 isoforms of
these three rat strains have identical COOH-terminal cyto-
plasmic domain sequences and similar steady-state message
levels.
Expression of Cell-CAM105 Isoforms during Liver Develop-
ment-To
study whether the cell-CAM105 isoforms are dif-
ferentially expressed during liver development, total RNA
samples were isolated from dissected fetal, neonatal, and adult
219
nt
L-form
181
nt .
S-form
+
0.0
28s-
I
FIG.
3.
Expression of the
L-
and S-form cell-CAM105 iso-
forms in various rat tissues and in liver from various rat
strains
as
detected by RNase protection.
A, expression of the L-
and S-form transcripts in various rat tissues.
B,
expression of the L-
and S-form transcripts in different segments of small intestine.
C,
expression of the L- and S-form transcripts in livers from various rat
strains identified at the
top.
FIG.
4.
Analysis of
L-
and S-form cell-CAM105 expression
during liver development.
A,
autoradiograph showing the pro-
tected fragments of the L-form (219 nt) and the S-form
(181
nt)
transcripts in liver samples taken at various developmental stages.
10
pg of total RNA prepared from fetal and neonatal livers was used
for each developmental stage.
10
pg of tRNA was used as negative
control.
B,
ethidium bromide-stained RNA gel of samples correspond-
ing to those shown in
A.

Function
and
Expression
of
Cell-CAM105
Isoforms
6143
rat livers for RNase protection study.
As
shown in Fig.
4A,
the steady-state levels of RNA for both the L- and the S-form
were very low from day 15 to 20 of gestation.
A
dramatic
increase (10-fold) in both isoform messages was detected at
1
day after birth, and another 2-fold increase was observed
between
1
and 19 days after birth. Similar results were ob-
served with a second independent group of liver RNAs col-
lected at 17 or 20 days’ gestation as well as
1,
5, or 19 days
after birth (data not shown). In the second group, a 7-fold
increase was detected between day 17 of gestation to
1
day
after birth and the increase between birth to 19 days after
birth is about %fold. These results indicate that a dramatic
increase in the steady-state level of both the
L-
and the
S-
form RNA occurred within the first few days after birth. The
ratio of the
L-
to the S-form message was about 1:2, similar
to what was observed at the protein level in adult liver (Culic
et al., 1992). This ratio remained relatively constant through-
out development. These observations suggest that the expres-
sion of cell-CAM105 is developmentally regulated and that
both isoforms are expressed and regulated coordinately.
Immunoblot Analysis of
L-
and S-form Cell-CAM105
Ex-
pressed in Sf9 Cells-To study the roles of cell-CAM105
isoforms in cell adhesion function, individual cell-CAM105
isoforms were expressed in insect cells using a baculoviral
vector.
Sf9
insect cells were infected with recombinant bacu-
lovirus containing either L- or S-form cell-CAM105. As con-
trols, uninfected
Sf9
cells and cells infected with wild type
baculovirus were also examined. The cells were collected at
96 h post-infection and expression of the
L-
or S-form cell-
CAM105 proteins was detected by immunoblotting with pol-
yclonal antibody (Ab669) against cell-CAM105 (Lin et al.,
1991). Fig. 5A shows the expression of L- and S-form cell-
CAM105 gene products in
Sf9
cells. Immunoreactive proteins
with apparent molecular masses of 80 and 70 kDa were
detected in cells infected with L- and S-form recombinant
virus, respectively, whereas no immunoreactive protein was
detected in the uninfected or wild type baculovirus-infected
cells. Since the apparent molecular masses of the L- and
S-
form cell-CAM105 in liver are 110 and 105 KDa, respectively
(Culic et al., 1992), these immunoreactive proteins most likely
represent underglycosylated forms of cell-CAM105. Fig. 5B
shows that the L-form cell-CAM105 protein in the infected
Sf9
cells was detectable on day
2
and reached maximum on
day 4.
Morphology of Sf9 Cells Expressing Cell-CAM105 Iso-
forms-To test for a possible intercellular adhesion function
of cell-CAM105,
Sf9
cells in monolayer culture were infected
with
L-
or S-form recombinant virus, or with wild type
baculovirus, respectively. The morphologies of these cells at
96 h post-infection are shown in Fig. 6.
Sf9
cells infected with
L-form recombinant viruses showed significant aggregation,
whereas the S-form recombinant- and wild type baculovirus-
infected cells did not. The aggregation phenomenon of L-
form-infected cells was completely inhibited by anti-cell-
CAM105 antibody (Ab669) in
1
to 50 dilution but not by
nonimmune serum (data not shown). These results indicate
that the cell aggregation observed was due to the expression
of the L-form cell-CAM105 protein.
To better quantify the cell aggregation,
Sf9
cells grown in
suspension were infected in separate experiments with L- and
S-form recombinant virus or wild type virus. Significant ag-
gregation was observed only in the L-form recombinant virus-
infected
Sf9
cells after 48 h (Fig. 7A), when synthesis of the
L-form cell-CAM105 protein was detectable in Western im-
munoblot analysis (Fig. 5B). The appearance of cell aggrega-
tion was correlated with the disappearance of single cells (Fig.
A
1234
228k-
110k-
70k-
44k-
28k-
B
a
Q
>I
-
:;5zz
E;
12345
6
7
110-
70-
44-
FIG.
5.
Expression
of
L-
and
S-form
cell-CAMiO5 gene
product
in
Sf9
cells.
A,
immunoblot analysis of cell lysates from
control
Sf9
cells
(lane
I),
cells infected with wild type baculovirus
(lane
2),
cells infected with L-form recombinant virus
(lane
3),
and
cells infected with S-form recombinant virus
(lane
4).
Each lane
contained 5
pg
of total protein. Ab669 in
1
to 1000 dilution was used.
B,
time course of the expression of the L-form cell-CAM105 protein.
Lane
1,
uninfected
Sf9
cells;
lane
2,
wild type baculovirus-infected
cells at 96 h post-infection;
lane
3-7,
L-form recombinant virus-
infected cells at
3,
24,
48, 72,
and 96
h
post-infection, respectively.
7B). No aggregation was observed in control
sf9
cells or
s-
form recombinant virus- or wild type baculovirus-infected
cells. These results indicated that expression of L-form cell-
CAM105 in
Sf9
cells in suspension caused aggregation similar
to that observed in monolayer culture.
Localization of
L-
and S-form Cell-CAM105 Expressed
in
Sf9 Cells-The lack of aggregation phenotype in S-form cell-
CAM105-expressing cells may have resulted from failure of
the insect cells to target S-form cell-CAM105 to the cell
surface. To determine whether L- and S-form cell-CAM105
expressed in
Sf9
cells were localized on the cell surface,
immunofluorescence studies were performed with
Sf9
cells
infected with L- and S-form recombinant virus, respectively,
using Ab669. The cells were fixed with
3%
formaldehyde and
treated with or without 0.1% Triton X-100 before immunoflu-
orescence staining. Approximately 85-90% of the cells were
impermeable to trypan blue after formaldehyde fixation, in-
dicating that the formaldehyde fixation alone did not signifi-
cantly permeabilize the cells. After staining the cells with
Ab669 followed by fluorescein-conjugated goat anti-rabbit
secondary antibody, strong fluorescence staining was seen on
the plasma membrane of both L- and S-form recombinant
virus-infected cells with or without detergent permeabiliza-
tion (Fig. 8,
A
and B). In a control experiment, we found that

6144
Function and Expression
of
Cell-CAM105
Isoforms
'
v+-T
-.<+fy#y7
.
.-
81
i
FIG.
6. Morphology of cell cultures
at
96
h
post-infection.
A,
uninfected
Sf9
cells;
B,
wild
type
baculovirus-infected
Sf9
cells;
C,
L-form recombinant virus-infected
Sf9
cells;
D,
S-form recombinant
virus-infected
Sf9
cells.
24h 48h
72h 96h
,-:
i'
I.
..
no
vlrur
wild
type
virus
V
0
S-form
virus
0
s
I,,,\
,,_,,_(
"0
0
20 40
60
80
100
time after infection
(hr)
FIG.
7.
Aggregation study in suspension culture.
Sf9
cells
cultured in suspension were infected with wild type virus
(wiM
type),
L-form recombinant virus
(L-form),
S-form recombinant virus
(S-
form),
or
no
virus.
At
various
time
intervals, aliquots of samples were
removed for microscopic examination
(A)
and
cell
number determi-
nation
(B).
In
B,
the cell number used for infection
at
time
0
was
used as
100%.
an antipeptide antibody (anti-C1) (Lin
et
al.,
1991), which
recognizes the cytoplasmic domain of L-form cell-CAM105,
could stain L-form expression cells only after Triton X-100
permeabilization (data not shown). This observation suggests
that under our experimental conditions, formaldehyde fixa-
tion alone did not make cells permeable to antibodies, and
Triton X-100 treatment was required to make cell permeable.
Therefore, staining of
L-
and S-form cell-CAM105 by Ab669
without Triton X-100 solubilization suggested that both cell-
CAM105 isoforms were mainly expressed on the cell surface.
Furthermore, the membrane localization of S-form cell-
FIG.
8.
Immunofluorescence staining of L-form
(A)
or
S-
form
(B)
recombinant virus-infected cells.
Sf9
cells were in-
fected with
L-
or
S-form recombinant virus
for
72
h.
Cells
were fixed
with
3%
formaldehyde
and labeled
with
Ab669
(1:200
dilution).
Fluorescein isothiocyanate-labeled goat anti-rabbit antibody
was
used
as
second antibody
(1:250
dilution).
CAM105 in
Sf9
cells was studied by incubating the infected
cells with antibody Ab669 and fluorescein-conjugated goat
anti-rabbit antibodies without fixation. The viability of the
cells after staining was assessed by addition of 1% trypan
blue. It was found that more than 90% of cells were still viable
after staining with antibodies and significant amounts of cells,
which were not stained by trypan blue, were positive with
Ab669 staining. This result also supports the surface localiza-
tion of S-form cell-CAM105. The punctate "ring-like" stain-
ing pattern in S-form-expressing cells is typical for
Sf9
cells
due to their sphere shape. Similar phenomenon was seen with
other membrane proteins expressed in
Sf9
cells (Germann
et
al.,
1990; Burkhardt
et
al.,
1989; Niikura
et
al.,
1992). In the
aggregates of L-form-expressing cells, the plasma membrane
in the cell-cell contact regions appeared flattened, suggesting
the presence of strong membrane interactions between adja-
cent cells (Fig.
8A).
In contrast, S-form-expressing cells re-
tained the round shape of
Sf9
cells.
DISCUSSION
We report here results of RNase protection studies on the
expression of two cell-CAM105 isoforms in various tissues
and during liver development. In addition, the functions of
these isoforms were investigated by expressing them individ-
ually in insect cells using baculoviral vectors. The presence of
cell-CAM105 isoforms in liver raises questions concerning
their functions, mechanisms of regulation, and roles in em-
bryonic development. We have used RNase protection to
probe the expression of these isoforms, because this method
can clearly distinguish between the two highly homologous
isoforms of cell-CAM105.
RNase protection studies on RNA isolated from different
tissues showed that the probe, derived from the COOH-
terminal cytoplasmic domain, protected fragments of ex-
pected lengths for the L- and the S-form in several tissues,
indicating that the corresponding regions of the two cell-
CAM105 genes in these tissues have sequences identical to
those of the liver. We have recently isolated several cell-

Function and Expression
of
Cell-CAM105
Isoforms
6145
CAM105 clones from
a
rat intestinal cDNA library, and
sequence analysis of these intestinal clones indicated the
presence of both the L- and S-form RNA with sequences
identical to those of the liver cell-CAMlO5. Therefore, the
observed difference in protein mobility between liver and
intestinal cell-CAM105 molecules described by Hansson
et
al.
(1989) is most likely due to differences in posttranslational
modifications. Similarly, the variations in protein mobilities
in SDS-polyacrylamide gel electrophoresis observed for sev-
eral other tissues (Odin
et
al.,
1988) may also be due to
posttranslational modifications. A complete cDNA sequence
analysis will be needed for confirmation.
Differential expression of isoforms has been observed for
several cell adhesion molecules. In the case of N-CAM, at
least five forms are generated by differential splicing of
mRNA encoded by the single N-CAM gene. The alternative
spliced variants produce N-CAM molecules with different
modes of attachment to the cell membrane or different cyto-
plasmic domains (for review, see Edelman and Crossin
(1991)). In both brain and muscle, individual N-CAM iso-
forms differ in their spatio temporal pattern of expression
(Edelman, 1986; Walsh, 1988), suggesting that structural var-
iation in N-CAM may be important for the expression of its
function. In the case of cell-CAM105, it seems unlikely that
differential expression could be the major mechanism of reg-
ulating cell-CAM105 functions, since both isoforms are coex-
pressed in different tissues as well as in different stages of
embryonic development. Despite the differences in the rela-
tive level of these two isoforms, none of the tissues examined
expressed only the
L-
or the S-form. These results raise the
possibility that expression of both isoforms in the same tissue
may be necessary for the physiological function of cell-
CAMlO5. However, the exact function of the cell-CAM105
isoforms in normal liver remains to be elucidated.
The message levels for cell-CAM105 isoforms showed sig-
nificant differences among the several tissues tested (Fig.
3).
Intestine and liver had high levels of both isoform messages,
whereas smaller amounts of both messages were detected in
kidney, lung, muscle, and spleen. Since cell-CAM105 is pres-
ent in the endothelial cells and megakaryocytes (Odin
et
al.,
1988; Sabolic
et
al.,
1992), the small amount of message
observed in muscle and spleen may be contributed by cell-
CAM105 in endothelial cells and megakaryocytes, respec-
tively. The tissue distributions of cell-CAM105 messages we
found agree qualitatively but not quantitatively with organ
distributions of cell-CAM105 protein determined by radio-
immunoassay (Odin and Obrink, 1987). The discrepancy be-
tween RNA message level and protein expression level sug-
gests that both transcriptional and translational regulations
play a role in tissue-specific expression of cell-CAM105.
The predicted amino acid sequence of the liver cell-CAM105
is
highly similar to that of CEA (Oikawa
et al.,
1987; Beau-
chemin
et al.,
1987) and other related cell surface glycopro-
teins,
i.e.
BGPl (Hinoda
et
al.,
1988), NCA (Neumaier
et al.,
1988), and PSGpl (Watanabe and Chou, 1988). It has been
reported that CEA, NCA, and BGPl function as intercellular
adhesion molecules
in
vitro
(Benchimol
et al.,
1989; Oikawa
et
al.,
1989; Rojas
et
al.,
1990). However, the physiological
function of these molecules are not clear. CEA was first found
to be present in colonic carcinoma and in fetal intestine, but
not in normal colonic mucosa (Gold and Freedman, 1965a,
196513; von Kleist and Burtin, 1969). Therefore, CEA was
proposed to be a marker for tumorigenesis and fetal develop-
ment. However, it was later shown that CEA was also present
in normal human plasma (Chu
et
al.,
1972), liver (Kupchik
and Zamcheck, 1972), and colon mucosa (Fritsche and Mach,
1977). More recently, with the availability of CEA cDNA, it
was further shown that the message levels for CEA are com-
parable in fetal, adult, and tumor samples (Kuroki
et al.,
1989). As
a
result, the role of CEA during tumorigenesis, fetal
development,
as
well as under normal physiological condi-
tions, is not clear. In contrast, our studies on the expression
of the cell-CAM105 isoforms during liver development using
RNase protection showed that expression of both isoform
messages is coordinated and that there is a dramatic increase
of the messages at birth. Message levels of cell-CAM105
isoforms remain fairly constant from
1
day after birth to
adulthood, suggesting that both cell-CAM105 isoforms are
secondary cell adhesion molecules that function at a relatively
late stage of development (Edelman, 1987). Furthermore, the
expression of cell-CAM105 is greatly decreased or completely
suppressed in primary hepatocellular carcinomas and trans-
plantable hepatoma cell lines (Hixson
et
al.,
1985; McEntire
et
al.,
1989; Hixson and McEntire, 1989). These observations
are consistent with selection for a less differentiated pheno-
type during carcinogenesis.
The sequence differences between the L- and S-form cell-
CAM105 suggest that these isoforms are derived from differ-
ent genes (Culic
et
al.,
1992). The observations that both cell-
CAM105 isoforms are coexpressed in different tissues
as
well
as at different stages of embryonic development raise intrigu-
ing questions concerning the regulation of cell-CAM105 iso-
form expression. It is possible that the regulatory regions of
both genes have similar sequences and respond to the same
regulatory factors with similar efficiency. Furthermore, such
a regulatory factor(s) may belong to a group of transcription
factors involved in cell type-specific transcription and cell
lineage commitment, since expression of these proteins is
tissue-specific and developmentally regulated. Genomic clon-
ing and analysis of the promoter regions of both cell-CAM105
isoforms should give insight into the mechanism of this reg-
ulation.
The functional roles for individual cell-CAM105 isoforms
were investigated using an insect cell expression system. This
system, which often produces large quantities of proteins, is
suitable for expressing cell-CAM105 and its modified forms
for structure and function studies. Although the extent of
glycosylation in the insect cell system was different from that
found in the mammalian cells, expression of L-form cell-
CAM105 caused significant cell aggregation, indicating that
the degree of glycosylation was sufficient for cell-CAM105 to
function. Since the L-form is 61 amino acids longer than the
S-form, the difference in molecular mass for the L- and
S-
form cell-CAM105 expressed in insect cells,
i.e.
80
versus
70
kDa, is mostly likely due to the difference in protein mass
rather than the extent of glycosylation. Furthermore, the
amino acid differences in the extracellular domains of the L-
and the S-form of cell-CAM105 do not change the number of
potential N-link glycosylation sites, and expression of the
first NHz-terminal Ig-domain of either form results in se-
creted glycoproteins of similar sizes (data not shown). These
results suggest that the differences in glycosylation are not
likely to account for the observed functional differences. Since
the structural differences between the L- and the S-form cell-
CAM105 reside in the first NHz-terminal Ig-domain and the
cytoplasmic domain, the observation that only the L-form,
but not the S-form, conferred cell adhesion function indicates
that the functional difference may be due to structural differ-
ences in either or both of these domains. Chimeric molecules
containing L-form extracellular domain and S-form cyto-
plasmic domain or vice versa have been constructed and will
be used to answer the question. The cytoplasmic domain of

6146
Function and Expression of
Cell-CAM105
Isoforms
the L-form contains several potential phosphorylation sites
which were not present in the S-form. It has also been shown
that the L-form was phosphorylated
in vivo
(Culic
et
al.,
1992). These observations may indicate that phosphorylation
of the L-form cytoplasmic domain may play a role in regulat-
ing the adhesion function, whereas the first Ig-domain may
play a role in regulating the specificity of recognition.
The coexpression of L- and S-form cell-CAM105 during
liver development and in different tissues suggests that both
isoforms may be required for coordinating the functions of
cell-CAM105
in uiuo.
This together with the observation that
the S-form did not support cell adhesion when expressed
in
vitro
leads to
a
hypothesis that the L-form cell-CAM105
directly mediates cell adhesion, whereas the S-form regulates
the L-form activity.
Acknowledgments-We thank Dr. Larry Teeter for help with
RNase protection experiments and Dr. Susan Henning for helpful
discussion and providing intestinal RNA prepared from various seg-
ments of intestines.
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