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l
l
THE ROLE OF CARBOHYDRATES IN THE I:NTERCBLLULAR J~HES::rON
MEDIATBD BY CARCINOEMBRYONIC ANTIGBN USING CHO MUTANT CBLL
LINBS
by
Julie Charbonneau
A Thesis submitted to the Faculty of Graduate Studies and
Research, McGill University, in partial fulfillment of the
requirements for the degree of Master of Science
Department of Biochemistry
McGill University
Montreal, Quebec
CANADA
Julie Charbonneau Pebruary 1992
.
i
ABSTRACT
Carcinoembryonic antigen (CEA) is a highly glycosylated cell
surface glycoprotein which has recently been demonstrated to
behave as a Ca++-independent intercellular adhesion molecul~.
In order to study the effects of carbohydrates on the
intercellular adhesion funcl._on of CEA, we l,ave transfected
the functional cDNA of CEA into wilù type and glycosylation
mutant cells Lecl, Lec2, and Lec8. Aggregation assays of cells
in suspension were performed with stable CEA transfectants of
these cell lines and showed that aIl of the aberrant CHA
glycofo~s could still mediate adhesion, and that the
specificity of adhesion of these glycoforms was not altered.
Lecl transfectants did, however, show an increased speed and
final extent of aggregation, which suggests that, although
carbohydrates can modulate the strength of adhesion, they do
not determine the adhesion property; this property must
therefore reside in the CEA protein backbone itself •
-
RESUME
L'antigène carcinoembryonnaire
surface cellulaire, est glycosylé
(CEA) ,
à 60%
ii
exprimé à la
de son
moléculaire et possède 28 sites
l'asparagine.
de glycosylation
poids
liée à
Nous avons récemment démontré que CHA peut agir comme
molécule d'adhésion intercellulaire indépendemment de la
température et de la concentration en calcium. Afin d'étudier
l'effet des glycanes sur la fonction d'adhésion
intercellulaire de CHA, le cDNA de cn a été transfecté dans
les cellules CHO mutantes Lecl, Lec2, LeeS. Celles-ci sont
déficientes en enzymes impliquées dans la modification de la
structure des sucres des glycoprotéines. Des études
d'aggrégation en suspension, à l'aide des transfectants ci
haut mentionés, ont démontré que l' adhésion él~it encore
possible malgré les glycoformes abbérantes de CEA. De plus.
les essais d'aggrégation homotypiques et hétérotypiques ont
démontré que la spécificité d'adhésion n'est pas influencée
par les glycoformes de CHA. La comparaison des courbes
d'adhésion des différentes glycoformes de CZA, a révélé que
celle du transfectant Lecl (CEA) était nettement plus
prononcée, ce qui signifie une adhésion plus rapide et un
pourcentage d'aggrégation plus élevé pour ce transfectant.
puisque Lecl possède le glycane le plus siDriile par rapport aux
autres mutants, nos résultats sont en accord avec le
iii
modèle qui suggère que les glycanes empêchent l'adhésion
entre leu domaines protéiques.
Nous concluons donc, Que les sucres de CBA ne sont pas
essentiels comme tel, mais participent à la force d'adhésion
intercellulaire. Les éléments essentiels à l'adhésion
intercellulaire de CBA devraient donc être dU à la ~tructure
de la protéine, i.e., le code spécifique des acides aminés.
~-
r 1
iv
PRIU'ACE
The work presented here has been sent for publication
to the Journal of cell Biology. The expression vector p91023B
cOlltaining the CEA cDNA was provided by Sarita Benchimol, the
antibodies to CEA were provided by Abe Fuks, the ELISA assaye
were performed by Aurora Labitan. The rest of the work was my
own achievement.
1 :
Il' ,
f f. ~ , L Il
t il l, ~ .~~ . ~ ~
l ~
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(
TABLE OF CONTENT
Abstract
Résumé
Préface
Table of contents
List of tables
List of figures
List of abbreviations
Acknowledgemellts
CHAPTER 1 :INTRODUCTION
1.1 Cell Adhesion Molecules
1.2 C~inical Significance of Carcinoemb~onic
Ar,~igen (CHA)
1.3 Molecular Biology of CHA
1.4 CD Pamily
1.4.1 Holecular Biology
1.4.2 Normal and Tumor Localization of
CBA Family Hembers
1.5 Possible Punctions of CHA
1.6 Adhesion Function of CHA and pamily Hembers
1.7 Role of Sugars in CD Mediated Adhesion
1.8 Carbohydrates in Cancer
1.9 Glycosylation of Glycoproteins
1.10 Hethods for Neasuring Cellular Adhesion
i
ii
iv
v
vii
vii
ix
xi
1
2
6
7
8
8
12
12
15
17
19
20
21
v
'1 ~
~ 1
j
J
j
r vi
CHAPTBR II MATBRIALS AND MBTHODS 23
II.l Cell Culture and Transfections 24
II.2 Aggregation Assay 25
II.3 Cell Sorting Assay 26
11.4 Western Blot Analysis 27
II.5 FACS analysis 28
CHAPTER III RESULTS 29
III.l Isolation of Glycosylation Defective CEA
transfectants 30
111.2 Homotypic aggregation of tranfectants 35 " r III.3 Specificity of cn Adhesion 38 , , 1 t 111.4 Capacity of Adhesion 43 .
CHAPTER IV DISCUSSION 51
CHAPTBR V REFERBNCBS 62
<.
Table 1
FIGURB 1
FIGURB 2
FIGURB 3
FIGURB 4
FIGURB 5
FIGURB 6
(
LIST OF TABLES
LISTS OF FIGURES
CHA family
Bxpected carbohydrate structures
of glycoproteins produced by the
various wild type and Lee mutant
PAGE
42
11
cell lines 32
Western blot analysis of CHA
transfectants 34
Kinetics of aggregation of
transfectants producing different
cn glycoforms
Speeificity of adhesion mediated by
different CHA glycoforms
Aggregation kineties for CHA
transfectants of different wild type
37
40
CHO cells 45
vii
a.a,
ASN, N
BGP
Cau
CEA
cDNA
CHO
DNase
ELISA
ER
FACScan
FBS
FITC
:t-CAM
:tg
:t.V.
kD
LecCAM
LFA-l
L-MAG
M
MAb
MEM
LIST OF ABBREVIATIONS
amine acid
asparagine
biliary glYCopI'otein
calcium
ix
carcinoembryonic antigen
complementary deoxyribonucleic acid
chinese hamster ovary
deoxyribonuclease
enzyme-linked immunosorbent assay
endoplasmic reticulum
fluorescence associated cell scan
fetal bovine se~
fluorescein isothyocyanate
intercellular cell adhesion molecule
immunoglobulin
int:ta venous
kilodalton
lectin cell adhesion molecule
lymphocyte function-associated
antigen 1
myelin associated glycoprotein
molar
monoclonal antibody
Bagle's minimum essential medium
NCA
N-CAM
NH2
PBS
PSG
sns PAGE
THR
X
x
Magnesium
non-specifie cross-reacting antigen
neural celI adhesion molecule
amino
phosphate-buffered saline
pregnancy-specifie glycoprotein
sodium dodecyl sulfate polyacrylamide
gel electrophoresis
threonine
any amino ae id
xi
ACKNOWLEDGBMENTS
1 would like to express my deepest gratitude to my
supervisor, Cliff Stanners, whom 1 greatly respect for his
love of research and hio shrewd scientific vision, for his
sup~ort and guidance through my gradua te studies. Thank you
for your patience and for giving me the freedom to develop my
skills and discover the true ups and doWDs of research, and
especially for showing faith in me, and making me realize that
l was the limiting factor of my achievements.
1 am sincerely grateful to my coworkers, who have made me
feel more a part of a family than a member of the laboratory.
A special thanks to Wendy Hauck, Hua Zhou and Mercedes Rojas,
my big sisters in the lab, for their friendship, honesty,
constant encouragement and help, and for many pleasant
analytical discussions, and to Chris Ilantzis, the man of the
lab, for his challenging research discussions, technical
advice, helpful literature references, and his immediate
attention in times of personal crisis. l thank Sarita
Benchimol, for her help, generosity, care and diplomacy
through the good and the bad times.
1 would also like to thank Nicole Beauchemin, Mireille
Cartier, Frank Bidelman, Karin sadoul, Prance Garnier, Claire
Turbide, ~lriel Chamoux, Philippe Gros and André Veillette
xii
for their support and resources, and the McGill Biochemistry
department for a good academic program and an outgoing staff
and student group.
1 cannot thank my family and Michael Adelman enough for
their constant encouragement throughout my studies. Their
presence and understanding has, and will always be, a major
driving force in my life.
In addition, 1 wish to thank Dr. Shore for the use of his
computer, Dr. Frojmovic of the Physiology department for the
use of the FACScan apparatus as well as Truman Wong and Aurora
Labitan for technical assistance. The open-door policy of the
Biochemistry department and the Cancer centre was very
beneficial to me. 1 feel that 1 have made many friends in the
different laboratories, and was able to develop new techniques
from the experts themselves.
This work was supported by an International Studentship
from the Paculty of Medicine of McGill university.
:2
1.1 Cell Adhesion Molecules
Cell adhesion molecules are cell surface receptors which
function to form and maintain tissue structure through either
of two different mechanisms, Ca++-dependent or Ca++
independent. Thus far, four families of widely accepted
adhesion molecules have been described: the integrin family,
the immunoglobulin (Ig) supergene family, the cadherins and
the selectins.
Integrins consist of a family of highly versatile cell surface
adhesion receptors which can transmit signaIs into and out of
cells. Twenty integrins have been characterized thus far in
this rapidly growing field (see 1, for a review). Integr:f.ns
~re believed to be the major receptors by which cells attach
to extracellular matrices, and some particular integrine also
play a role in cell-cell adhesion. AlI integrins are composed
of an a and a 8 subunit which come together to form a non
covalently associated heterodimer. The a subunits have
putative divalent cation binding motifs and require Ca·· or
Mg" for their function (2). The N-terminal domains of the Cl
and 8 subunits are believed to confer the ligand binding site.
Tbis binding site is connected by the two stalks to the
transmembrane domains, and tbus to the cytoplasmic tails
wbich interact with the cytoskeleton (3). Integrins can be
classified into three subfamilies according to tbeir shared 8
•
(
3
subuni t • These subf ami 1 ies are known as 81, 82, and 83
integrins. 81 integrin subfami1y includes receptors which bind
to extracellular matrix components such as fibronectin,
laminin and collagen and are expressed on leukocytes as weIl
as non-haemopoietic cells (4}. 82 integrins are expressed only
on white blood cells and are important for endothelium binding
of neutrophils and monocytes prior to extravasation through
blood vessel walls at sites of inflammation (5). 82 integrin
LFA-l (lymphocyte function-related antigen) which is expressed
on T-Iymphocytes can interact with antigen presentiûg cells
through direct interactions with Ig superfamily members I-CAM
1 or I-CAM 2 (intercellular adhesion molecule) and thus
mediates cell-cell adhesion (6).83 integrins are distributed
on endothelial cells and on platelets and are involved in
substrate binding auch as fibronectin, fibrinogen and
thrombospondin by cross-linking to Arg-Gly-Asp (RGD) sequences
on the substrate (7).
The immunoglobulin supergene family includes a wide range of
cell surface glycoproteins involved in cellular recognition
and adhesion (8). These molecules can interact homophilically
or heterophilically with other Ig family members, usually on
opposing cell surfaces. Ig molecules share primary sequence
homology as weIl as structural homology such as disulfide
bonds at conserved cysteine residues, Ig variable regions (V)
and Ig conserved regions (C). Ig molecules are either anchored
4
to the membrane through a transmembrane domain followed by a
cytoplasmic domain or via a phosphatidylinositol glycan. The
I~ family can be further subdivided into Cl-set and C2-set
molecules. The Cl-set subgroup includes Molecules involved in
cell-cell recognition proceBses of the immune system such as
TcR, CD4, CD8 and the C2-set subgroup includes moiecules
involved in Ca++-independent:. homophilic intercellular adhesion
such as N-CAM and MAG which are important for tissue
organization during development (9).
Cadherins are another family of cell-cell adhesion receptors
which can mediate selective homophilic intercellular adhesion
in a Ca++ and temperature dependent fashion. The cadherin
family is divided into Many subclasses that show different
tissue distribution patterns, e.g., E-cadherin (epithelial
cadherins or uvomorulin), p-cadherin (placental cadherins), N
cadherin (neural cadherins) and L-CAM (chicken liver cell
adhesion molecule) (10). Populations of cells expressing
different subclassed of cadherins, when mixed together, sort
out into homotypic aggregates, which suggests that cadherins
are invoived in celi adhesion selectivity (11). The
intracellular portion of cadherins has been demonstra~ed to
interact with the cytoskeleton, and this interaction appears
to be essential for the cell-cell adhesion function of
cadherins (12). The activity of cadherins influence the
formation of junctional complexes snch as t ight, gap and
l 5
desmosomes (13) and may also be involved in establishing
cellular polarity (14). Furthermore, the regulation of
cadherin expression, i.e., the on and off switching of
expression, may be involved in the segregation of cell layers
during development (15).
Selectins or Lec-CAMs (lectin cell adhesion molecules) are a
family of three related adhesion molecules including Mel 14
(homing receptor, LAM-1) , endothelial leukocyte adhesion
molecule (ELAM-1) and platelet activation dependent granule-
external membrane protein (PADGBM, CD62, GMP-140), which are
involved in leukocyte binding to the endothelium at
inflammatory sites (16). AlI selectins have a specifie N-
terminal lectin-like domain with homology to the calcium-
dependent carbohydrate binding family (17), an epidermal
growth factor domain (egf), and a variable number of short
('-,nsensus repeats (csr) also found in members of the
complement regulatory protein family, followed by a membrane
anchor and a cytoplasmic domaine The lectin-like domain ie
believed to directly mediate a Ca++-dependent prote in
carbohydrate interaction for specifie cell adhesion to
endothelium during various types of inflammation. More
specifically, selectins are thought to mediate the initial
unstable interaction of leukocytes to ftndothelium, preceding
the activation of 82 integrins which mediate the stronger
adhesions necessary for leukocyte arrest and extravasation
6
(18). Mel 14 is expressed on lymphocytes and la invo1ved in
lymphocyte··endothe1iwn binding (19). ELAM-1 is transient1y
expressed on activated endothe1ium and is be1ieved to mediate
the rapid influx of neutrophi1s at the site of inflammation
(20). PADGEM is expressed at the surface of activated
p1atelets and endothe1ia1 ce11s (21) as .) response to products
of the clotting cascade such as thrombin, and bind to
neutrophils and monocytes (22).
X.2 Clinical Significance of Carcinaa.bryonic ADtigen
Carcinoembryonic antigen, first described by Gold and Freedman
in 1965 (23), is a cell surface glycoprotein of 180 kD present
in elevated amounts on the surface of tumour cells as well as
in the blood of patients with colon, breast and 1ung cancer
(24). CHA was first be1ie7ed to be expressed during
embryogenesis and reappear specifically in colon cancers as an
oncofetal antigen and seemed an ideal candidate as a tumor
marker. It was soon realized however, that CAA overexpression
was not perfectly correlated with cancer. First of a11, with
the use of more sensitive immunoassays, it was demonstrated
that elevated CHA blood concentrations were detected in other
malignancies and rlon-ma1ignant diseases such af} a1coho1ic
cirrhosis, gastrointestinal inflammatory diseases and in
smokers (25, 26). Furthermore, only 60% of colonic tumors were
7
found to give elevated levels of blood CHA, depending on the
state of differention of the carcinoma (24). It is now known
that CBA iu normally expressed early during embry~genesis in
tissues derived from aIl three germ layers and persists mainly
in entodermal tissues in adult life, e.g., on the brush border
of colon:1.c epithelial cells (27). CD is nevertheless the most
widely used tumor marker, as a diagnostic tool and for the
clinical management of colon carcinoma. Thus CHA levels are
measured in the blood of cancer patients ta monitc,r their
progress following chemotherapy; arise :lll CHA blood
concentrotion above the normal 5 ng/ml concentration post-
operatively can represent an early indication of tumor
recurrence or metastasis formation.
1.3 Molecular Biology of CHA
The gene and cDNA corresponding to CHA have recent ly been
cloned and characterized by our laboratory (28) and by others
(29, 30, 31). Sequence analysis revealed a processed leader
sequence of 34 amine acids (a.a.), followed by an amino
terminal domain of 108 a.a., three 178 8.a. internaI domains
with 68-72.5% amine acid sequence homology to one another,
each containing 4 cysteine residues bound by disulfide
bridges, followed by a short carboxy-terminal tail of 27 a.a.;
the latter domain is posttranslationally processed, to he
, 8
rep1aced by a phosphatidy1 inosito1 glycan 1inking CBA to the
cytoplasmic membrane. There are 28 potential asparagine-linked
glycosy1ation sites (ASN-x-Thr/Ser) in the protein, 2 in the
N-termina1 domain, 11 in the first internaI domain, 7 in the
second internaI domain and 8 in the third internaI domaine The
predicted molecular weight of the mature protein is 72.8 kD
and the predicted total glycosylation weight ia approximately
100 kD for a biantennary complex carbohydrate structure, which
is in reasonable agreement with the experimental molecu1ar
weight obtained on SDS polyacrylamide gel electrophoresis
(PAGE) of 180 kD.
1 .4 CHA Paaily
1.4.1 Molecular Biology
Nine CEA-1ike genes have been dlacovered, aIl of which are
clustered on human chromosome 19 (32, 33). Fig. 1 shows the
proteins which have so far been characterized. The CEA family
is divided into two subfamiIiea including the CHA subgroup,
containing CHA itself, Non-specific Cross-reacting Antigen
(ReA), different splice variants of Biliary Glycoprotein
(BGP), and CGM6; and the pregnancy specific glycoprotein
subgroup containing at least Il different gene products
(PSGs). These molecules all share a processed leader sequence,
-
9
an NHa-terminal domain of 108-110 amine acids, followed by 2
to 6 repeated Ig-like C2-set domains, consisting of either 92
or 96 a.a. (A domain) or 86 a.a. (B domain) and each
containing at least 2 cysteine residues forming a disulfide
bridge. Carboxy-terminal tails of CD, NCA and CGM6 are
processed to leave the molecules membrane-bound by a
phosphatidyl inositol anchor (34, 35, 36). AlI BGP splice
variants have transmembrane domains and either a short or long
cytoplasmic tail (37, 38). PSGs do not have a membrano anchor
and are secreted from the plasma membrane.
CEA FAMILY
1 CEA Subgroup
L N Al BI A2 B2 A3 B3 M
CEA ~t e e e e c e e e e e e e • '.1 l')f: ("j'L 86 92 86 92 86 26
L N A B M NCA l@ e CGM6
e e e • 34 loe 92 86 24/29
L N Al BI A2 TM CYT
BGPa @ e e le ccl c c ~ 34 108 02 86 96 43 18 49
L N Al BI ,.\2 TM CYl
BGPc 34 108 92 86 96 43 5
L N Al BI TM CYr
BGPb IZ], " e e le cc ~
34 108 (~2 86 43 18 49
L N Al BI TM CYr
BGPd E?ttfi"il e c le cc = 34 108 92 86 43 5
PSG Subgroup
PSG lb L N Al A2 B2 c PSG la ~ • C c e c e c 1 PSG le PSG Id 34 108/110 92 92 86 2/4111/13
L N Al 62 e
PSG 2
34 110 92 86 13
1 L N A2 B2 e
PSG 5 c c c c 1 34 110 92 86 13
1 12
I.4.2 Normal and Tumor Localization of CHA Faaily Meabers
As weIl as having a normal site of expression in the body,
each CEA family member has been shown to be associated with
malignancy. CEA is normally expressed at low levels on
intestinal mucosa and reappears on colon, breast and lung
tumor cells (24). NCA is norme.lly expressed in lung (39),
spleen (40), colon mucosa, granulocytes and monocytes (41),
and has been detected in breast and colon cancer cells (42).
BGP can normally be detected in bile canuliculi (43) and
mucosa of gal1 bladder (44), and transcripts can be found in
colorectal cell lines (38). CGM6 has been detected in normal
peripheral leukocytes (36) and in blast cells in peripheral
blood of patients with leukocytes of chronic myelogenous
leukemia (45, 42). PSGs are usually expressed in placental
tissues (46), and have been used as markers for
choriocarcinoma (47).
l . 5 POflsible Punctions of CHA
sequence analysis of CEA has also revealed a degree of
homology with the immunoglobulin (Ig) supergene family. Ig
family members can be classified according to their content of
constant-like 1 (Cl) or constant-like 2 (C2) domains. Cl-set
like molecules are involved in recognition processes between
{
13
free molecules whereas C2-set like molecules are involved in
recognition processes occuring at the cellular membrane. The
C2-set subgroup includes neural cell adhesion molecule (R
CAM), intercellular adhesion molecule (I-CAM), mye 1 in
associated glycoprotein (L-MAG) and CD2. (8). CHA family
members showed sequence homology to the Ig C2-set domains,
which suggested a possible role for CHA in intercellular
adhesion. Cell adhesion assays were designed in our laboratory
based on the work of Brackenbury et al. (48) and Orushihara et
al. (49), to study the function of CHA in vitro. To this
effect, LR-73, a line derived from Chinese hamster ovary (CHO)
cells, were transfected with the functional cDNA of CHA.
Positive transfectants in our adhesion assays demonstrated
Ca++-independent homotypic intercellular adhesion (50). These
results have since been confirmed by others (51). Adhesion
molecules are believed to be involved in cell-cell and cell
substrate interactions during development, and therefore play
an important role in tissue organization during embryogenesis
and tissue regeneration (9, 15, 52). Hence, CHA expression
during embryogenesis could participate in the organization of
cells of the gastro-intestinal tract. Furthermore, CHA
reappearance in tumor cells could generate new associations
between cells and thus cause disruption of normal tissue
architecture (50). Evidence for homotypic adhosion of CHA in
vivo has been provided by Hostetter et al. (53). They reasoned
that CHA being cleared from the blood by the uptake of Itupffer
"
14
ce1ls in the liver, and thus present at the surfaces of
Kupffer cells and hepatocytes, could act as a homing receptor
for metastatic cells disseminating trom a primary CBA
producing colonie tumor to the liver, via the portal
circulation. Their results showed that increased CBA blood
concentrations produced by 1:. V. injections of mice with
purified CHA correlated with an increased metastatic potential
of CEA producing tumor cells to the liver (53).
A second function for CHA was later proposed by Leusch et al..
(54). This group suggested a potential role for cn family
members in the recognition of bacteria and the regulation of
bacterial colonization of the human intestine. This hypothesis
stems from the observation that certain strains of bacteria
bind to immobilized purified preparations of CHA, NCA and BGP
on nitrocellulose. Furthermore, bacterial binding to CEA
family members appears to occur in a carbohydrate-specific
manner by interactions between oligosaccharides on CHA, NCA-55
and BGP-8S (specifie glycoforms of NCA and BGP) with lectins
on the bacteria. A comparative study between two NCA molecules
originating from the same cDNA but differing in their
carbohydrate structure showed different binding affinities to
bacteria (Leusch et al., personal communication). Thus NCA-SS
which contains 30-50% high-mannose type carbohydrate
structures readily binds bacteria, whereas TBX-7S which
contains only a few high-mannose type structures binds at a
1
-
15
much lower affinity. Binding of these molecules to bacteria
can be competed out using various a-glycosidases of D-mannose,
which suggests that the bacterial lectin recognizes the
mannose residues on carbohydrate structures of CHA family
members.
A third function for CHA has been proposed by Pignate1li et
al. (55). This group suggested that CBA plays an accessory
role in the in vitro binding of a human colon carcinoma cell
line (SW1222) to type 1 collagen matrix of collagen coated
culture dishes. This work has yet to be confirmed.
1 .6 Adhesion functioD of CBA aDd f_ily I1811bers
Subsequent to the f inding of the role of CD in homotypic
intercellular adhesion, similar adhesion experiments were
performed on other CHA family members. The cDRA of ReA was
transfected into LR-73 cells as previously described and was
also shown to act as a Ca++ - independent, homotypic
intercellular adhesion molecule. CBA and RCA transfectants
were tested for their specificity of adhesion by mixing
experiments. As a negative control, LR-73 parental cells were
mixed with either CHA or NCA transfectanta and were
demonstrated to segregate from homotypic aggregates of CD or
r r t' ~
1
1 f ~
16
NCA transtactants, indicating that the intercellular adhesion
is specifically due to CW',,-CEA or NCA-NCA interactions and not
to CU or NCA interactions with other cell surface molecules.
Heterotypic adhesion between CHA and ~CA transfectants was
demonstrated as well as cell sorting between either CEA or NCA
transfectants and transfectants of other adhesion molecules of
the Ig family such as N-CAM or L-MAG. thus showing adhesion
specificity between cn family members but not between Ig
supergene family members (56). CGN6 has been reported to be
incapable of homotypic adhesion and yet was capable of
performing heterotypic adhesion with NCA (57), suggesting
binding interactions between these two molecules. Chimeric
mo].ecules have been constru(!ted using N-CAM cDNA and CU cDNA,
and LR-73 transfectants of these chimeric molecules have Deen
tested in adhesion experiments to define the domains of cn
involved in binding. CD homotypic adhesion was deduced to
depend on two essential binding sites, involving the N-
terminal domain of one molecule binding to one of the three
homologous internal domains of another molecule (Zhou, H. et
al., in preparation). BGP adhesion, unlike CD and NCA, was
demonstrated to mediate CaH-dependent homotypic adhesion (58)
as seen for another non-J:g class of adhesion molecules. the
cadherins (15), and in preliminary heterotypic adhesion
assays, BGP transfectants segregated fl"Om either NCA or cn
transfectants (Rojas and Stanners, unpublished), thUB
suggesting different mechanisms of adhesion between certain
17
CHA family members.
1.7 Role of SUgara in CHA llediated Adhesion
As described above, cn is a highly glycosylated molecule with
60% of its molecular weight due to oligosaccharidee.
Glycosylation analysis has been achieved using purified CBA
preparations (rom liver metastases deriveil from primary tumors
of colon and breast origins. Glycosylation was deduced to be
mostly asparf.\gine linked with 80% tetra-antennary structures,
15 tc, 20% tri-antennary structures and 5 to 10% diantennary
structures (59). Microheterogeneity of CBA carbohydrate
composition has been observed from one tissue preparation to
another. The above study revealed the presence of only complex
type carbohydrates, while "lnother study demonstrated 10% high
mannose type structures combined with 90% complex type
structures with various levels of sialylation and varying
percent age of branched structur\!s ( 60) •
Sugars have been shown to be important for protein stability,
for protein conformation and as a 1180rting signal/l for
cellular targeting of proteins to cellular organelles (61).
Recent evidence has invo1ved sugars in cellular compaction (
(62) and direct binding of glycoprotein through fts
18
carbohydrate structure to cell surface receptors (63). In
addition, cell adhesion molecules known as LecCAMS were
recently shown to bind to carbohydrate ligands (64, 65) using
lectin-like N-terminal domains (4, 66). Other evidence of
carbohydrate participation in adhesion has been demonstrated
for N-CAM embryonic (E) and adult (A) forme In this system,
the B-form which is heavily polysialy1ated is 1ess adhesive
than the A-form \'Ihich contains two thirds less sia1ic acid
residues. These N-CAM differences in adhesion abilities have
been attributed to ste rie hindrance and charge repu1sion
through sialic acid residues (67). N-CAM carbohydrates were
a1so demonstrated to assist the homophilic binding of L1 cell
adhesion moleculetl on one cell surface, to other L1 molecules
on an opposing cell surface (68). I-CAM-1, another member of
th~ 19 family, has been demonstrated to perform heterotypic
adhesion with LFA-1 and Mac-1 integrins. Interestingly, the 1-
CAM-l/Mac-l interaction is modulated by the level of I-CAN-l
glycosylation (69). The unusually high level of glycosylation
on CHA glycoprotein compared to other 19 family members has
led us to investigate a role for carbohydrates in the function
of this molecule, namely, homotypic intercelluldr adhesion.
(
19
1.8 Carbohydratea in Cancer
Correlations have been demonstrated between altered
glycosylation structures on cell surface glycoproteins and
malignant transformation. The more common sugar alterations
include increased branching, sialylation and fucosylation of
N-Linked carbohydrate structures (61). High levels of
polyslalylatlon and high levels of CBA at the cell surface
have b~en demonstrated to enhance the metastatic potential of
colorectal carcinoma cells injected Into the spleen of rodents
(53). In assaying the role of carbohydrates in CBA-mediated
adhesion, we hope to better understand the role of CBA in
development and carcinogenesis. If sugars are demonstrated to
be important for specificity and strength of CBA-mediated
adhesion, then it would imply that the adhesion function of
CHA has another level of regulation, i.e. in its glycosylated
structure. :It is thus possible that the various CBA glycoforms
with different physical and biochemical properties may each
have a unique set of biological activities important for
tissue architecture during development, tumorigenesis and
metastases formation.
1 20
1.9 GlycoaylatioD of GlycoproteinB
ASN-linked Glycosylaticn of proteins occurs in the rough BR as
proteins are translated and secreted through the ER membrane.
On the luminal side of the ER, oligosaccharyltransferase
enzyme tranafers a nine unit carbohydrate structure endhlg in
dolichol phosphate to the ASN residue of the proteine Three
basic oligosaccharide structures have been characterized for
N-linked glycoproteins: high-mannose, hybrid, and complex
types (for a review see 70). In the hybrid and complex type,
there are alternate branching possibilities such as bi, tri
and tetra antennary as weIl as bisecting structures. To this
level of complexity is added the options of fucosylation,
polylactosamination and polysialylation, ail of which are
regulated by the various processing enzymes in the BR and the
different biochemical compartments of the Golgi. apparatus.
Different glycoforms of the same molecules expressed in
different tissues or at different developmental stages have
been reported (71). These slight variations of carbohydrate
structures on the same molecules but in different environments
could be an efficient way to modulate the function in a subtle
fashion to bring more functional divecsity.
CHO mutant cell lines have been generated which are deficient
for certain enzymes in this processing pathway (72). In
transfecting these cells with the functional cDNA of CHA, we
(
1(
21
were able to produce CHA molecules bearing specifically
truncated oligosaccharide structures. Osing these
transfectants, we assessed the role of carbohydrates in CHA
mediated adhesion. (Jar results show that sugars have no
influence on adhesion specificity but do, however, ne~atively
modulate the speed and extent of CHA-mediated adhesion.
1.10 Methods for Measuring Cellular Adhe8ion
Different assays for measuring cellular adhesion have been
described over the year8. One 8uch assay con8ists of removing
cells from a monolayer culture with a mild treatment of
trypsin, and forcing the suspension through a syringe to break
up any remaining cellular aggregates in order to get a single
cell suspension. Single cell suspensions are then incubated at
37°C for a two hour period with stirring, and aliquots are
taken at various time points and assayed for the percent age of
single cells relative to the total number of cells. Rates and
extent of aggregation reflect the adhesion properties of the
cell line (50).
A variation of this type of experiment measures the adhesion
of a radioactively labelled cell sU8pension directly on a
confluent monolayer of cells (51). In this assay, the cell
mixture is incubated 15 to 80 min to allow the two cel1
22
populations to come into contact by gravity. The radioactivity
remaining after washing the cells is counted by liquid
scintillation methods to measure the degree of adhesion
between the two cell populations. Washing away the unbound
cells is a very critical step in these experiments as
undesired shear forces Can make the results poorly
reproducible and thq force of adhesion impossible to measure.
McClay et al. (73) have introduced a centrifugaI force-based
assay which Can quant if y the forces of adhesion between cells
and their substrates. In this assay, microtiter plates are
coated with substrates and a radioactively labelled cell
suspension is added to the chambers which is then sealed with
a second microtiter plate. The microtiter plates are
centrifuged briefly to bring the cells into contact with the
substrate and then immediately centrifuged in the opposite
direction to separate unbound cells at different centrifugaI
forces. Both substrate coated (bound) and opposing wells
(free) are clipped and counted by liquid scintillation
methods. The strength of adhesion between the cells and their
substrates is provided by the centrifugaI force required to
separate them.
Another group has modified the McClay assay to measure cell
cell adhesion. This assay is basically the same except that
the microtiter plates are initially coated with a confluent
monolayer of cells (74).
24
II.1 Cell CUlture and Tr~ectioD8
Wild type chinese hamster ovary (CHO) cell lines LR-73 (7S)
and Pro-S, and CHO glycosylation deficient mutants Lec1, Lec2
and Lec8 cells (72) were grown in monolayer in (X-MEN (76)
supplemented with 10% fetal bovine serum (FBS) at 37°C in a
humidified, S% carbon dioxide atmosphere. CHA transfectants
were obtained by the lipofection technique of BRL (Bethesda
Research Laboratories, Life Technologies, Inc. Gaithersburg),
except for the use of dioleoylphosphatidyl
ethanolamine/dioleoyloXY-3(trimethylammonio)propane, chloride
salt liposomes (a generous gift from Dr. John Silvius, McGill
university), or by calcium phosphate mediated coprecipitation
(77). For lipofect ions , cells were incubated for 16 hours in
10 ml of (X-MEN + 10% FBS plus a li90some-DNA mixture composed
of 40 ~g liposomes, 10 ~g of the P9l023B expression vector (R.
Kaufman, Genetic« Institute, Boston, MA) containing CHA cDNA,
and 1 Jlg of the dominant selectable marker, pSV2 -AS (78).
Cells transfected by the calcium phosphate method were
obtained by coprecipitation of S Ilg of CEA cDNA, O. S Jlg of
pSV2-AS and 4.5 Jlg of CHO-N3 (79) carrier DNA per 3xlO li cells.
Transfected clones were selected with 2mM albizziin in
asparagine free medium as previously described (78), picked
and maintained in selective medium. Clones producing levels of
CHA exceeding 50 ng/mg celi protein, as assessed by a double
monoclonal clinica1 assay for CBA (Abbott, Mississauga), were
25
selected for further experimentation. The numbers after each
transfectant designate the clone number. Control transfectant
clones which did not produce CEA were obtained by tranafection
with the same expression vector containing en oDNA in the
antisense orientation. Antisense clones are referred to, in
the text and figures, as "anti".
%I.2 AggregatioD Assay
Cells were removed from plastic surfaces with 0.06% trypsin
(Difco: Bacto trypsin) in phosphate buffered saline (PBS)
containing 15 mM sodium citrate and resuspended in (X-MBM +
0.8% FBS + 10 ~g/ml DNase I. Aggregation assays were carried
out in suspension at 10' cells/ml in 17 x 100 mm polypropylene
round bot tom Falcon tubes (Becton, Dickinson labware) at 37°C
with stirring (80-100 rpm) using a 3 x 10 mm stir bar, as
previously described (50). In aIl separate experiments, the
seme initial cell count was used and all cell populations
showed a single fluorescent peak as determined by their
FACScan profiles. 10 ~l aliquots were gently removed from
stirring cell suspensions at varioulY time points using a
pipetman and a 200 ~l plastic pipet tip (Sarstedt). The
percentage of single cells relative to the total number of
cells was counted on a haemocytometer and was plotted as a
function of time. Sinee the probability of aggregation
r 1
t 1
• 26
decreases as a function of the concentration of single cells
in suspension, the initial rates of aggregation are rapid and
the curves level off to a certain percent age within the l hour
incubation period.
1:1.3 Cell Sorting Assay
Cell sorting assays in mixtures of two transfectant cell
populations were carried out as previously described (50).
Brief1y, 2x104 cells of one ce11 population were labelled with
50 ~1 of a 0.5 mg/ml stock solution of fluorescein-
isothiocyanate (Sigma, St.uouis) in 100 ~l Puck's saline + 2%
FBS. Cells were washed 01'1: excess FITe by centrifugat ion
through a column of FBS prior to mixing with 2xlO li unlabelled
cells of another cell population in a final volume of 3 ml (l
MEIl + 0.8% FBS + 10 ~g/ml DNase I. Slidei! were made from
aliquots taken after a 75 min incubation at 37°e with stirring
and the aggregates were visualized under light and fluorescent
microscopy. FITe can 1eak out of cells after prolonged
incubation times, however, our experiments were done within
relatively short periods and as demonstrated by the negative
controis of Fig. 5 and Table 1, and the ability to visualize
mixed aggregates, FITC leakage was not a problem. FITC
analogues which get trapped within the cells are available and
shou1d be used in experiments with longer incubation periods.
(
27
II.4 We8tern Blot Analysis
Confluent cel18 were washed, removed with PBS containing 15 mM
sodium citrate, eentrifuged and resuspended in 0.5 ml PBS.
Celi suspensions were then sonicated at 4°C. The protein
content of the supernatant was measured using the Bio Rad
assay for protein determination (Bio-Rad laboratories,
Richmond, CA). 50 ~g of protein was boiled for 5 min in 0.4%
sodium dodecyl sulphate (SDS): 1% 28-mereaptoethanol sample
buffer, and electrophoresed on a 7.5% polyaerylamide gel
eontaining 0.1% SDS under standard conditions (80). The
separated proteins were transferred to a nitrocellulose
membrane and probed with a goat gamma-eut polyclonal anti-CBA
antibody (obtained from Dr. A. Puks, McGill University) and
subsequently probed with a rabbit anti-goat antibody
eonjugated to alkaline-phosphatase (Alkaline Phosphatase AP
F(ab')2 Pragment Rabbit Anti-Goat IgG (H&L), Jackson Immuno
Research Laboratories, Inc.). The CBA proteins were detected
by the reaetion of alkaline-phosphatase with its sub8trates,
5-bromo-4-ehloro-3-indolyl-phosphate and nitro blue
tetrazolium, in carbonate buffer (0.1 M NaHC03 - 1 mM "gCla• pH
9.6), (Promega, Madison) which produces a visible dark blue
band.
-
28
II.5 PACS ADalysis
Cell surface CD levels in transfectants were measured by flow
cell fluorimetry <:)f whole cells treated with fluorescent anti
CD antibody. Cell monolayers were rendered single cell
suspensions by a 2 min incubation with 0.06% trypsin in PBS
citrate, a treatment which did not affect the level or the
molecular weight of cell associated cn, as assessed by
Western blot analysis. 2.5xl05 cells were resuspended in 0.5
ml PBS + 2% FBS, labelled first with a mouse monoclonal anti
CD antibody, B18 (81), at 40 ~g/ml and second with an FITC-
conjugated goat anti-mouse antibody for 30 min at 4°C.
Labelled cells were washed with 2 ml PBS + 2% FBS, resuspended
in 0.75 ml of the same solution, and their fluorescence
intensity measured using the FACScan fluorimeter (Becton
Dickinson, Canada, Inc. ). Comparison of CBA levels for
different cn glyeoforms determined with one antibody assumes
an equal degree of labelling per molecule of CBA, independent
of the level of glycosylation. Since the same quantitative
relationship between the mean fluorescent labelling for
different Lee mutant transfectant clones was obtained using
two antibodies which recognize different epitopes, this
assumption seems justified.
30
III.1 Isolation of Glycoaylation Defe,~tive CHA 'l'ransfectanta.
To examina the effects of the carbohydrate structure on the
intercellular adhesion function of CHA, CHO derived cell lines
(LR-73, Pro'S, Lecl, Lec2, Lec8), which yield different
glycosylation structures, were transfected with the functional
cDNA of CHA. The CHO Lec mutants have been shown to be
defective in various steps of their N-glycosylation processing
pathway (72). Fig. 2 displays the predicted final
glycosylation states of glycoproteins produced by the
different cell lines. LR-73 cells and the direct parent of the
lec mutants, pro'S, do not have defects in their N
glycosylation pathways and thus are capable of producing
complete glycosylation structures terminating in sialic acid.
Lec2 cells, which are defective in the translocation of CMP
sialic acid across the gOlgi membrane, are believed to
terminate glycosylation with galactose. Lec8 cells, which are
defective in the translocation of UDP-Galactose into the golgi
apparatus, result in sugar structures ending with N
acetylglucosamine residues. Lecl cells are defective for the
enzyme N-acetylglucoaaminyltransferase-I and express
glycoproteins which bear 5 mannose residues as terminal
carbohydrate structures. Stable CHA cDNA transfectant clones
of these normal and glycosylation deficient cell lines
producing levels of CHA exceeding 50 ng per mg of cell prote in
were aelected for further study. In order to characterize the
.
31
FIGURB 2
Ibr:peCtad carbohydrate 8tructure8 of glycoprotelna
producad b.Y the .arlou8 .114 type aDd Lac .utant call
I1D88
<.'
1
, .-, "
f ~
r t
l .~
1 ~,
'.1'
SA SA
1 1 Gal Gal
1 GleNac GleNac
1 Man Man -- ......Man
(GleNAc) 2
1 ASN
1 WddType 1
Gal Gal
1 1 GleNac GleNac
1 1 Man Man -- ....Man
(GleNAc) 2
1 ASN
GleNac GleNac
Man Man -- ....Man
(GlcNAc)2
1 ASN
32
Man Man -- ......-Man Man -....... ......Man
(GJCNAc)~
1 ASN
33
PJ:GURB 3
Western blot aaalysis of CIA traDSfactaDts
CHA proteiD.8 were detected iD eell lysates from various
transfeetants by immunoblotting witb goat polyelonal
anti-CHA antibody. Tbe numbers after each transfeetant
designate the clone number. The molecular weights in
kilodaltoDs of marker proteiDS are given at the left.
l
_ nrn.~-~~~-:;-........ ~~.=-, .. "~ ... ,,...., .... ~.---., ,!"",.;r ... ~ ... "''"-'''- - .. ~~-- ,... .... ~ ... __ ~~_~ .. r ~ ... ,.- -,-- ..
, ?
en ..... CD ..... en ...... 0)
1 1 1
•
N
8
or ..
,--~ , ... "<
1
LR-73
LR(CEA)-6
Pro -5 (CEA)-7
Lec2 (CEA)-3
LeeS (CEA}-4
Lec1 (CEA)-15
lec1 (anti)
w ...
--~--
1 35
CHA glycoforms produced by these transfectants, Western b10t
ana1ysis of total cellular proteins using po1yc1ona1 anti-CBA
antibody was performed (Fig. 3). The different migration
patterns seen for CHA produced by the various cell 1ines are
consistent with the predicted glycosy1ation deficiencies;
i.e., CHA with the apparent mo1ecu1ar weight (180 kD) of the
fu11y glycosy1ated mo1ecule was produced by wi1d type LR-73
transfectant ce11s, and a more heterogeneous and slight1y
lower mo1ecular weight CHA glycoform by wi1d type Pro-5
transfectant ce11s, while CBA with successive1y lower
mo1ecular weights down to 135 kD was produced by the Lec
mutants giving successive1y more truncated sugar structures.
III.2 H~ty'pic AggregatioD of Transfectants
To study the involvement of carbohydrate structures in the
interce11u1ar adhesion mediated by CHA, we performed homotypic
adhesion assays using the various transfectants. The resu1ts
are shown in Fig. 4. In each experiment, the kinetics of
aggregation of the transfected mutant were compared with its
antisense transfectant and to the fu11y glycosy1ated LR-73
(CHA) transfectant as negative and positive contro1s,
respectively. In every case the aggregation of the antisense
transfectant was minimal.
36
FIGURE 4
Kinetics of aggregatioD of transfectants producing different CHA glycoforms
Homotypic aggregation assays of mutant cells t.ransfected with the functional cDNA of CEA in the sense and antisense orientation. In all separate experiments, the kinetics of aggregation of a low (0) and a high (A) CEA producing transfected mutant clone are eompared to the antisense transfectant (D) and wild type LR (CEA) transfectant (+) as negative and positive controls respeetively. A, homotypic aggregation of suspensions of Lec2 (CEA)-7 (0) and Lec2 (CEA)-3 (A) low and high producers, respeetively. B, homotypic aggregation of suspensions of LeeS (CEA) -3 (0) and LeeS (CEA) -4 (~ low and high produeing transfectants, respectively. C, homotypic aggregation of cell suspensions of Lee! (CEA) -9 (0) and Lee! (CEA) -40 (A) low and medium produeing transfectants, respectively. Three sets of experiments showed similar kineties of a~gregation. These data represent results from a typieal experiment.
t
" i
-
37
1
100
80
60
40
.!!!
1f 20
j!
~ 1/)
ë 0 15 30 4S 60
~ ~ lime (min)
20
o~--~----~--~----~~ o 30 eo 10 120
Time(min)
(
38
Aggregation studies of the Lec mutant CHA transfectants
demonstrated homotypic adhesion of low and high producing
clones, with more rapid aggregation and lower final levels of
single cells for the high producers (Fig 4 A, B, and C).
The above aggregation studies thus show that CEA transfectants
with an altered glycosylation structure are capable of
homotypic adhesion and that this adhesion is direetly related
to the expression level of CEA by the transfeetant clones;
this suggests that carbohydrate residues are not directly
involved in intercellular adhesion mediated by CEA.
111.3 B.Pecificity of CHA Adhesion
To examine the question of specificity of adhesion mediated by
the different eBA glycoforms 1 we assessed the ability of mixed
populations of transfectants displaying CHA moleeules with
different earbohydrate structures to sort themsel ves into
homotypic aggregates. Specifie cell sorting has been observed
for transfectants bearing different adhesion molecules (56),
and would be expected if sugar structures were capable of
conferring adhesive specificity. Fig. 5 shows the results of
an aggregation experiment in which equal concentrations of LR
(CBA)-6 cells labelled with FITC were mixed with unlabelled
Leel (CHA) -40 cells in suspension. After a suitable incubation
39
PIGtJRB 5
s.pecificity of adbeaioD .adiated bY different CHA
glycofoD18
lqual amounts of cells of two different populations, one
labelled with PITe (*) and the other not labelled, were
mixed and incubated at 37°C for a 75 min periode Aliquots
were Acored for the percent age of labelled cells per
aggregate and plotted as a function of the total amount
of aggregates visualized. A, control experiment between
non-transfected wild type cella, LR-73, and Lecl (CHA) -40
shows cell sorting. B, heterotypic aggregation of PITe
labelled LR (CD) -6 cells with Lecl (CHA) -40 transfectant
cells is observed. Insert shows light and fluorescent
micrographs of the aggregates.
40
60~----------------------------------------,
70 "LR·73 vs Lee1 (CEA)-40
60
50
40
30
20
10
40 "LR (CEA)-6 vs Lee1 (CEA)-40 B
30
10
o 10 20 30 40 50 60 70 80 90 100
Percent labelled cells per aggregate
-
-
41
period, a1iquots were visua1ized under 1ight and fluorescent
microscopy and the number of aggregates with a given
percent age of 1abe11ed ce11s per aggregate p10tted as a
histogram. The resu1ts show that 90% of the aggregates were
heterogeneous in nature (Fig. 5 B). Control mixing experiments
between Lecl (CHA) -40 and the parental ce11s demonstrated that
the parental LR-73 ce11s remained as single ce11s (Fig. 5 A).
Thus the observed adhesion in Fig. 5 B was presumab1y due to
CD-CHA interaction and not to CD binding to some other
mo1ecu1e on the parental ce11s. Table l summarizes the resu1ts
of mixing experiments with a11 the transfectants. Results with
LS-180, a human colon cancer ce11 1ine which produces high
endogenous 1eve1s of CD are inc1uded and, from the
heterotypic aggregates observed, demonstrate that CD
mo1ecu1es with a human carbohydrate structure can mediate
adhesion with CHA mo1ecu1es with a hamster structure. A11 CBA
producing ce11s tested in these experiments, regard1ess of
their considerable differences in glycosy1ation, showed an
abi1ity to mediate heterotypic interce11u1ar adhesion. We
conclude that carbohydrates do not influence the specificity
of CD-mediated interce11u1ar adhesion. This specificity
therefore probab1y lies within the protein backbone itse1f.
, ,
42
Table 1. Specificityof Adhesion of Various CEA Glycoforms
Expressed on the Cell Surface of CHO Cells
Cell populations Aggregates visualized
FITC labelled Unlabelled Homotypic Heterotypic Homotypic unlabelled mixed labelled
% % %
LR-73 Lec1 (CEA) 82 17
LR (CEA) Lec1 (CEA) 3 90 7
LR (CEA) Lec2 (CEA) 7 90 3
Lec8 (CEA) LA (CEA) 14 86 0
Lec1 (CEA) Lec2 (CEA) 7 92
LA (CEA) LS-180 4 94 2
Summary of heterotypic experiments between two
populations of cells, one labelled with FITe and the
other unlabelled. Large percentages of mixed
aggregates indicate heterotypic adhesion whereas low
percentages suggest cell sorting.
( 43
111.4 Capacityof Adhesion.
previous experience with transfectants producing variable cell
surface amounts of various Adhesion molecules, including eEA
family members (CD and NCA), N-CAM and B-cadherin has shown
that the kinetics and final levels of aggregation provide a
semi-quantitative measure of the strength of intercel1ular
adhesion (56). Thus en transfectants producing increasing
levels of cn at the cell surface showed progresf ively faster
kinetics of aggregation and higher percentages of aggregated
cells (50, 56) • 'l'he kinetics of aggregation of CBA
transfectants of the two CHO derived wild type cell lines were
compared in Fig. 6, taking into account the cell surface CBA
production of each transfectant as measured by FACS. Under the
conditions of this experiment, the untransfected LR-73
parenta1 cell line was shown to aggregate to the same low
extent as a pra·s CD transfectant clone with a fluorescent
mean of 590, whereas the Pro-S parental cell line showed
virtually no aggregation. An LR-73 CD transfectant with a low
cell surface mean fluorescence of 128 also demonstrated a
greater level of aggregation than a second pro·S CBA
transfectant with a much higher cell surface mean fluorescence
of 798. These results clearly demonstrate that the cellular
background can influence intercellular adhesion; the extent of
Adhesion in cn transfectants of LR-73 cells is presumably
FIGURE 6
AggregatioD ld.neticl! for CM trlUl8fectant. of different
.i14 type CHO cella
Comparison of the kinetics of aggregation of non
transfected clnd CHA transfected wild type CHO cell line.
LR-73 and Pro·S. At the right. the PACS distributions.
relating the number of cells with a given fluorescence
and the mean fluorescence, are shawn. The dashed-line
distribution8 represent the anti8ense mutant clone
labelled in the 8eme way as the CHA tran8fected mutant
clone, and used a8 a negative control for fluorescence
measurement8. These data repre8ent 8ingle point8 from a
typical experiment.
45
1
1°Oi==~~&-~==~=====;======l Pro"S (MIl)
10 Ut-73
LR-73 Pnl"'5 (CEA)-20
!! B ..... tcf.N40 QI Ct · c: '. 581 iii , . ë Pro"'5 (CEA)-22 ' . , . ~
, · 1 • · QI ..... tcIA)oa Q.
LR (CEA)-7
· '. 1118 , . , . , . , • 1 • · 20 UttcEAt-7 . '. 128 '. " •• , . , . • • / 1 • \
0 0 ., 10 120 1r1 101 ~ttf
TimI(,,*,)
46
facilitated by the presence of other endogeneous molecules at
the cell surface. For quantitative compariBon of adhesive
strengths produced by the various CEA glycoforms it is thus
apparent that the Pro·5 wild type CHO cell line, the direct
parent of the Lee mutants, must be used.
To define a potential role for carbohydra":es in the ability to
recruit the maximal percentage of cells into aggregates, the
kinetics and final levels of aggregation were measured for
transfectants producing similar cell surface levels of various
cn glycoforms as determined by FACS analysis. The Lecl (CHA)-
6 transfectant, with the most severely truncated sugar
structures terminating in 5 mannose residues and a lower mean
fluorescence of 350, showed more aggregation than either Lec2
(cn) -5 lacking only sialic acid residues, Lec8 (CU)-4
lacking both galactose and sialic acid residues or a
transfectant of its direct parental cell line, Pro-5 (CEA) -22,
wit.h a complete carbohydrate structure, aIl at mean
fluorescence values of 800 and above (Fig. 7). Lec2, Lec8 and
pro·S transfectants, aIl at comparable levels of CHA
production show decreasing degrees of adhesion, in that order.
The rates of aggregation in Fig. 6 and 7 for the various
" . .. transfectants can be misleading in their similar appearances f:-T , f
when comparing profiles of single point experiments. However, t t k
when the profilt!s of the mean of three sets of experiments for
~ /~ . the various transfectants are compared, the initial rates of ~
l l ~
47
aggregation tend to be more pronounced for clones with greater
extent of aggregation. This order in capacity of adhesion:
Lec1 > Lec2 > LecS > pro·5 was repeatedly observed with
transfectants of mean fluorescence approximating 500 and 300
in value (data not shown). The Lecl (CD) carbohydrate
structure thus appears to favor CBA-mediated adhesion most,
perhaps due to the greatest exposure of binding sites on the
proteine Lec2 (CBA)-5 showed a better aggregation than LecS
(CD) -4 which suggests that adhesion is not necessarily
related to the number of carbohydrate residues removed, but
more likely related to the overall conformation of the protein
modulated by the carbohydrate structure. These results suggest
that carbohydrates negatively modulate the capacity of CBA
mediated adhesion.
48
PIGUR. 7
Ca.parativa capacities of adhesioD for different CBA
glycoforas
The kinetics of aggregation of transfectants bearing CHA
with different glycosylation structures are shown for
wi1d type Pro"S, Lec2 and Lec8 transfectants producing
simi1ar leve1s of CHA, and Lec1 transfectant with a lower
CHA 1evel of production. These data represent single data
points from a typical experiment. PACS profiles for each
transfectant cel1 population (solid line) are shown
together with profiles for populations of their
respective parental cell 1ine (dashed llnes).
49
(
PrG"51ŒA) -22
l' 1 I, 118
80 I, • 1 l ,
/ ' LeclIŒA)-4
Jll
1i 60 PIQ-S (CEA)-22 .' l ,
l ,
~ g' iii
• , Lec8 (CEA)-4
• \ 1 ,
E
~ Q.
40 Lec2 (CEA)·S
" " " " 1 1
• , 1 ,
LeelIŒAH
20 Lee1 (CEA)~
3110
0 104 0 30 60 90
Tune (min)
(
52
Carbohydrate structures on prote in molecules have enormous
potential for variability and therefore for the storage of
information (see introduction). Such information could be used
for molecular recognition as required, for example, in
specifie interactions between extracellular adhesion
molecules. The extremely high level of glycosylation of CHA
and the biological variability observed in the structure of
ite sugar chains (59) lend credence to the view that these
structures could play a significant role in the intercellular
adhesion function of CEA. The specificity of adhesion observed
between cells bearing CBA, 1. e., the failure of these cells to
bind to the parental cells or cells presenting other adhesion
molecules of the immunoglobulin supergene family (56), makes
it unlikely that en adhesion is simply due to the non
specifie stickiness of its carbohydrate chains. In addition,
highly glycosylated cn constructs lacking relatively short
regions of domains essential for adhesion do not mediate
intercellular adhesion (Bidelman and Stanners, unpublished
results) .
The results obtained in this work show that functional CHA
cDNA transfectants of mutant cells with different defects in
their ability to glycosylate proteins are still capable of
specifie aggregation. Even the most severely truncated
structures terminating in mannose residues do not affect the
ability of the CHA moleeules to mediate intereellular adhesion
53
and do not affect the specificity of adhesion. There is,
however, the question of effects of these structures on the
strength of adhesion, a parameter which can be important in
the function of adhesion molecules during development (82) and
which, under extreme conditions, can even produce cell sorting
of strongly adhering cells in the presence of weakly adhering
cells (83). OUr ::-esults suggest that the CHA carbohydrates may
be more important in modulating adhesion than in determining
its essential features. Thus the faster kinetics and increased
extent of aggregation of CHA transfecants of Lecl mutant cells
may in fact be due to a carbohydrate structure which changes
the secondary structure of the CHA molecule, a conformational
change which enhances the access to binding sites on the
prote in backbone. A reduction in charge, due to the absence of
sialic acid residues on CBA glycoforms produced by certain of
the Lec mutant cells, could remove a repulsive component in
intercellular interactions. This charge effect has been
suggested to account for the reduced adhesion observed for
polysialylated N-CAM relative to the adult less sialylated
glycoform (67). Since our desialylated mutant transfectants
were more adhesive than fully sialylated wild type
transfectants, our findings support the charge hypothesis; the
large differences in the capacity of adhesion observed for
transfectants bearing different desialylated sugar structures,
however, indicates that the role of carbohydrate structures in
CHA at least is more complex.
1, f, 1
54
The very different tinetics of aggregation of CHO wild type
Pro-S and LR-73 transfectants has demonstrated that the cel1
surface background can influence the analysis of cn function
in relation to its adhhesive capacity, Although both of these
wild type cell lines are deri ved from CHO cells, the LR-73
line shows a more normal flattened morphology in monolayer
culture (75). Presumably, the presence of higher levels of
other adhesion molecules in LR-73 relative to Pro-5 cells has
synergistic effects with the intercellular adhesion mediated
by CHA. Alternatively the Pro-S cells could express anti-
adhesive molecules on their surfaces. These considerations
should be taken into account in the Interpretation of results
in which the adhesive properties of transfectants of a CHO Lec
mutant are compared with those of a CHO wild type control
different from Pro-S, the direct parent of the mutants, aB in
the recent report for Po transfectantB of CHO cell lines (84) •
Although Pro-S cells would have been a better wild type
control than LR-73 cells in determining the role of
carbohydrates in the specif icity of CBA-mediated adhesion
(Fig. 5 and Table 1), our results suggest that LR-73 and LR
(CBA) transfectants were Buitable cell lines to assess this
question for the following reasons. Firstly, although the
above results are consistent with the suggestion that LR-73
cells were observed to express background endogeneous adhesion
molf/cules, when mixed with CHA transfectants, LR-73 cell.s
:cemained essent ially aingle cella while Lee (CHA)
( 55
transfectants for.med homotypic aggregates. Furthermore, in LR
(CD) transfectants, these putative background endogeneous
molecules did not Interfere with CBA-mediated Adhesion between
LR (CBA) and Lee (CHA) transfectants, as demonstrated by their
ability to form mixed aggregates.
In general, our results support the findings of others for
1ess extensively glycosylated adhesion molecules of the
immunoglobulin supergene family; Le. that the information for
the specificity of Adhesion does not reside in the
carbohydrate chains on glycoprotein adhes ion molecules but
that these structures can negatively modulate the rate and
extent of cell aggregation (67, 70).
An altered carbohydrate structure on glycoproteins is a weIl
characterized phenomenon in cancer cells. The most common
changes include increased fucosylation, increased sialic acid
content and increased branching (for a review see 61).
Preparations of CBA from serum of normal patients and patients
with benign disease showed carbohydrate structures of the
complex biantennary and hybrid type without internaI fucose,
whereas CD preparations from the serum of patients with
colorectal cancer showed more heterogeneity of glycoforDls with
increased branching and increased fucosylation (85). CD
preparations from liver metastases derived from primary
colonie carcinomas showed carbohydrate structures of mostly
56
complex-type with bi, tri, and tetra-antennary structures and
pOlys ialylat ion (59). Thus CHA carbohydrate structures
isolated from the serum of cancer patients and from liver
metastases of colon primary tumor are consistent with the
pattern observed in other malignancies. Recent studies
revealed that an increased blood concentration of CBA favors
metastasis formation to the liver possibly by homophilic
intercellular interactions between Kupffer cells of t,he Iiver,
which have bound circulating CBA, and CBA expressing tumor
cells (53). Furthermore, an increased sialic acid content in
the CBA on tumor cells increased the colonization process of
metastasis in the liver, presumably through an
oligosaccharide-lectin-type of recognition event (86).
OUr results have shown that sugars affect the capacity of cell
recruitment into aggregates in transfectants in vitro. During
embryogenesis and carcinogenesis, when CBA may play a role as
an intercellular Adhesion molecule, different glycosylated
structures could modulate these functional roles.
Glycoproteins have been shawn to have different glycoforms,
with distinct biochemical and biophysical properties leading
to functional diversi~y (71). Par example N-CAM sialic acid
content regulates its affinity ir! homophilic intercellular
Adhesion during development (67). Insulin shows reduced
binding affinity for Lecl cell- expressing insulin receptor
when compared to that of wild type cells, which suggests that
glycosylation is involved in the
hormone/receptor binding affinity (87).
57
regulation of
CHA, with its 28
potential glycosylation sites, has a tremendous potential for
glycoform variability, i.e., glycosylation at Any of the 28
sites coupled with heterogeneity of the carbohydrate
structures at each glycosylation site. Thus, the glycoform
diversities of cn found in metastasis preparations and in the
serum of cancer patients may effect subtle variations in the
conformation of the molecule leading to functional diversities
which are not present in normal tissues. It is thus possible
that the overexpression of CD, coupled to an abnormal
carbohydrate processing mechanism in the cell, could result in
the disruption of normal cellular interactions.
Our results have also shown that CHA retains its specificity
of Adhesion, regardless of the struC"ture of its carbohydrates,
but its Adhesive capacity is affected. In CHA expressing
tumors, where CHA is heterogeneous in its glycoforms,
intercellular Adhesion could differ depending on the CHA
glycoform expressed by the cells. Thus, subpopulations of
cells within the tumor may interact with more or less
strength, creating regions of stronger adhering cells and
weaker adhering cells. These subpopulations may break away
from the primary tumor and form metastases. As metastases were
found to express CHA with highly branched, fucosylated and
polysialylated carbohydrate structures which, by our results
, "
58
are predicted to form less Adhesive intercellular
interactions~ we would suggest that subpopulations of weakly
adhering cells dissociate from the primary tumor and form
metastases. Since less glycosylated carbohydrate structures
were shown to be more adhesive, it is possible that tightly
adhering cells of primary tumors could express more high
mannose-type CBA, whereas less adhering cells could express
more highly branched structures. Opdyke and Nicholson (88), on
the other hand, have predicted that metastasis is favored by
increased homotypic intercellular Adhesion. It is possible
that carbohydrate structures of fer a subtle variation in the
strength of Adhesion so that aggregates of cells bearing
complex structu~es in the primary tumor are weak enough in
their intercellular adhesion to break away from it, and yet
strong enough to remain in an aggregated form for a better
chance of survival in the circulation and for lodging and
growth in other organs. A comparison of glycoforms expressed
by cells of metastases and the primary tumor they arose from
may shed some light on this hypothesis. As our results suggest
that CHA Adhesion is dependent on its carbohydrate structure,
its level of expression and its cellular background, the role
of CBA glycoforma in carcinogenesis and metaatasea fo~tion
may be complex.
The role of carbohydrates in the intercellular Adhesion of
other CBA family members remains to be investigated. NCA
l 59
shares many common features with cn including sequence
homology, sites of asparagine-linked glycosylation and percent
carbohydrate content, and would be expected to behave like CBA
in these experiments. BGP, however, was demonstrated to
mediate intercellular Adhesion by a different mechanism than
that of CD and RCA, and might respond differently in
glycosylation experiments. BGP Adhesion, unlike that of. CBA
and NCA is calcium and temperature dependent and has a reduced
speed and extent of Adhesion (58). lt would be interesting to
study these particular features of BGP Adhesion i. e., Ca++ and
temperature dependence, with respect to the carbohydrate
structures on BGP using the Lec mutant celi lines.
Lecl cells which express glycoproteins with high-mannose type
carbohydrate structures could represent a good experimental
system for the bacterial binding experiments described by
Leusch et al. (54, and l.5). CHA, BGP, and RCA transfectants
of Lecl cells may be used to produce high-mannose type
molecules, or used directly in experiments to investigate the
importance of mannose residues on CD family members in the
interaction with lectins of type l-fimbriated bacteria.
Oitawa et al. (in press) have reported that the R-terminal
portion of CD is essential for homotypic intercellular
Adhesion. In these experiments 2/3 of the N-terminal domain of
CHA, including two R-linked glycosylation sites, were deleted.
, r
1 60
Transfectants of LR-73 cells with this CBA mutant (AN) were
demonstrated to lose their adhesive function (Kidelman et al. 1
in preparation). It would be interesting to evaluate the
importance of these glycosy1ation sites on the interce11u1ar
adhesion function of CBA by "knocking them out" using site-
directed mutagenesis.
OUr Lec (CBA) ce11 1ines cou1d a1so be used to verify our
hypothesis that aggregates of cells m,pressing CBA molecules
with complex type carbohydrates [Pro"S (CBA)] wou1d be favored
to metastasize from an aggregate composed of a mixture of
cells expressing CBA glycoforms of high-mannose type [Lecl
(CBA)] and complex type carbohydrates. To this effect, mixed
aggregates of CBA transfectants of Lec mutants and wi1d type
cells could be implanted into nude mice, as out1ined by
Hostetter et al. (53). If tumors develop and give rise to
metastases, then the cellular composition of the primary tumor
and derived metastases could be assayed with regards to the
CBA glycoforms expressed.
The work presented in this thesis has given us more insight
into the mechanisms of CBA mediated interce1lu1ar adhesion.
The carbohydrate strucure of CBA, which we predicted would be
important for its function, has been demonstrated to
negatively modulate the capacity to recruit cells into
aggregates but not the specificity of homotypic intercellular
( 61
interactions mediated by CBA. Although the measure of capacity
to recruit cells into aggregatos clearly suggests that
carbohydrates can modulate CEA-mediated adhesion function, a
more in depth characterization of the strength of adhesion
would be greatly beneficial to our understanding of the
mechanisms involved in cell-cell interactions during
development and carcinogenesis. McClay type assays described
in the Introduction cou),\! be usod to measure strength of
adhesions between CBA family membera with and without
molecular modifications such as glycosylation variations. To
reduce the effects of variable cell surface context, which can
Interfere with Interpretation of data, purified CHA molecules
could be coated onto plastic microtiter plates thus serving as
a direct a~esive subatrate for the various CHA transfectants
in these assaya. Alternatively, CHA transfectants could be
replaced by CBA-coated latex beads (89) and thus eliminate
non-specifie adhesion mediated by unknown adhesion molecules.
pinally, the Lee cell linea appear to represent a good system
for future functional experiments involving the members of the
CBA family.
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