From the Division of Hematology and the Duke
Comprehensive Sickle Cell Center, Department of Medicine, Duke
University Medical Center, Durham, North Carolina 27710, the
¶ School of Engineering, Duke University, Durham, North
Carolina 27710, and the
Glasgow and West Scotland Blood
Transfusion Service, Glasgow G2 5UA, Scotland
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ABSTRACT |
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Basal cell adhesion molecule (B-CAM) and Lutheran
(LU) are two spliceoforms of a single immunoglobulin superfamily
protein containing five Ig domains and comprise the sickle (SS) red
cell receptor for laminin. We have now analyzed laminin binding to murine erythroleukemia cells transfected with various human B-CAM/LU constructs. B-CAM and LU bound equally well to laminin, indicating that
the longer cytoplasmic tail of LU is not required for binding. However,
binding of soluble laminin did require the presence of the
membrane-proximal fifth immunoglobulin superfamily (IgSF) domain of LU,
while deletion of IgSF domains 1, 2, 3, or 4 individually or together
did not abrogate laminin binding. Under flow conditions, MEL cells
expressing B-CAM, LU, and LU lacking domains 1, 2, 3, or 4 adhered to
immobilized laminin with critical shear stresses over 10 dynes/cm2. However, MEL cells expressing LU lacking
domain 5 bound to laminin poorly (critical shear stress = 2.3 dynes/cm2). Moreover, expression of only IgSF domain 5 of
LU was sufficient to mediate MEL cell adhesion to immobilized laminin
(critical shear stress >10 dynes/cm2). Finally, Scatchard
analysis showed that SS red cells had an average of 67% more B-CAM/LU
than normal red cells, and low density red cells from sickle cell
disease patients expressed 40-55% more B-CAM/LU than high density SS
red cells. B-CAM/LU copy number thus may also play a role in the
abnormal adhesion of SS red cells to laminin.
Vaso-occlusion in sickle cell disease patients is a complex
process involving not only the mechanical obstruction of vessels by
misshapen and nondeformable sickle
(SS)1 red cells but also
cellular adhesion and the activation of coagulation (1). In addition,
sickle cell disease is characterized by endothelial damage, some of
which may result from the effects of a variety of cytokines on
endothelial cells, resulting in increased circulating endothelial cells
and, potentially, exposure of subendothelial matrix to flowing
blood (2, 3).
Among the subendothelial matrix molecules to which SS cells adhere,
laminin is the one to which these cells adhere most avidly (4). Laminin
is a family of at least 11 heterotrimeric proteins, each containing an
B-CAM was first described as an IgSF protein expressed along the basal
surface of epithelial cells (7). Later, Lutheran (LU) protein was
characterized as a spliceoform of B-CAM (8, 9). The extracellular
portions of B-CAM and LU are identical and contain five IgSF domains:
two variable-type, or V, domains and three constant-type 2, or C2,
domains (Fig. 1A). B-CAM differs from LU in that it lacks
the last 40 amino acids, including a putative SH3 binding site, of the
cytoplasmic tail and thus has a molecular mass of about 78 kDa,
compared with a mass of 85 kDa for LU. Both proteins are expressed by
normal and SS red cells.
Other IgSF proteins have also been identified as laminin receptors,
including members of the integrin subfamily (10-12). However, no
consensus motif for laminin binding has been identified, possibly because different laminin receptors bind to different sites and different isoforms of laminin. Previous studies of IgSF domain activity
in cell adhesion involving other ligands have identified the N-terminal
IgSF domain as the most frequent major binding domain, as in
ICAM-1/LFA-1 and ICAM-3/LFA-1 interactions (13, 14). The N-terminal
IgSF domain of ALCAM, which has two V and three C2 domains similar to
B-CAM/LU, also mediates ALCAM's interaction with CD6 (15). In some
cases, the second IgSF domain plays a less critical but important role
(16, 17), such as in E-cadherin/integrin In the present study of B-CAM/LU, we have measured the B-CAM/LU
copy number on SS and normal red cells, as well as on density fractionated SS red cells, in order to determine if increased expression of B-CAM/LU is likely to be a factor in the observed increased adhesion of SS red cells to laminin. We have also used a
number of recombinant LU proteins from which various IgSF domains have
been deleted to demonstrate that the membrane proximal IgSF domain
(domain 5) alone is critical for laminin binding, and that the
cytoplasmic tail of LU, which is absent from B-CAM, does not appear to
affect laminin binding.
Cell Line and Antibodies--
The MEL cell line was obtained
from Dr. B. Haynes, Duke University Medical Center, and maintained
under standard tissue culture conditions in RPMI 1640 with 10% fetal
calf serum. The anti-Lub mAb LM342/767:31 was produced by
Dr. Robin Fraser, Glasgow and West Scotland Blood Transfusion Service,
UK. mAb 4F2 was generated by immunizing a mouse with MEL cells
expressing rB-CAM and rLU and recognizes the fifth IgSF domain of
B-CAM/LU.2 Human anti-Lu8,
which recognizes an epitope on the second IgSF domain of B-CAM/LU (20),
was provided by the Immunohematology Laboratory of the Duke University
Medical Center Transfusion Service. Rabbit anti-human laminin was
obtained from Life Technologies, Inc. Horseradish peroxidase-linked
second antibodies and FITC-conjugated antibodies were obtained from
Jackson Immunoresearch (West Grove, PA).
Quantitation of B-CAM/LU Sites per Cell--
Purified
anti-Lub mAb was radiolabeled with 1.0 mCi of
Na125I (Amersham Pharmacia Biotech) per 1.0 mg of protein,
using glass tubes coated with IODO-GEN (Pierce) (21). The iodinated mAb was separated from free Na125I by repeated dialysis in
phosphate-buffered saline, resulting in mAb with a specific activity of
0.3-0.5 µCi/µg of protein, with >99% of the radioactivity
precipitable by cold 10% trichloroacetic acid.
Aliquots (100 µl) of serially diluted radiolabeled mAb with
concentrations ranging from 30 ng/ml to 60 µg/ml were added to equal
volumes of red cells suspended at 2 × 108/ml in
phosphate-buffered saline, pH 7.4, with 1 g/dl bovine serum albumin,
and incubated at room temperature for 1 h. Red cells used were all
homozygous Lu(a
In order to separate low density (reticulocyte-enriched) and high
density (older) red cells, a variable density solution composed of low,
intermediate, and high density arabinogalactan layers was prepared
according to the manufacturer's directions (Cellsep Protocol, Larex
Inc., St. Paul, MN). These layers had densities of 1.085, 1.090, and
1.095 g/ml, respectively. One ml of packed SS red cells was carefully
laid on the top of the density solution without disturbing the layers,
and the tube was then centrifuged for 45 min at 50,000 × g at room temperature. This procedure resulted in separation
of red blood cells into three distinct bands; the top and bottom layers
of cells were extracted and used as the low and high density cells, respectively.
Vector, cDNAs, and Expression of Recombinant Proteins--
A
partial human LU cDNA was provided by D. Anstee (International
Blood Group Reference Laboratory, Bristol, UK) and expanded to include
the entire coding region or modified to replicate the previously
described B-CAM cDNA by PCR. This construct was inserted into the
eukaryotic expression vector pcDNA3.1(
A complete LU cDNA constructed and inserted into the
pcDNA3.1(
All cDNAs were subcloned into the pcDNA3.1( Flow Cytometric Assays and Cell Sorting--
MEL cells
(107/ml) transfected with LU
Stably transfected MEL cells were assayed for their ability to bind
soluble laminin by incubation with 3 µg/ml human laminin (Life
Technologies, Inc.) at 37 °C for 1 h, followed by staining with
rabbit anti-laminin and FITC-conjugated secondary Ab, as described
previously (6). Flow cytometric analysis of soluble laminin binding was
performed on an Orthocytoron Absolute flow cytometer (Ortho Diagnostic
Systems, Inc., Raritan, NJ). Each transfected cell line was tested for
its ability to bind laminin a minimum of three times, and data shown
are representative of the results obtained.
Western Blots--
Red cell membrane proteins were prepared as
previous described (21, 25, 26). MEL cell transfectants were lysed in
Tris-buffered saline with 1% Triton X-100, 5 mM EDTA, and
1 mM phenylmethylsulfonyl fluoride (Sigma). All membrane
protein lysates were boiled in non-reducing SDS-PAGE sample buffer for
5 min. Five µg of SS red cell membrane proteins and 10 µg of MEL
transfectant membrane lysates were separated by SDS-PAGE and
transferred to nitrocellulose (27). A combination of mAbs 4F2 and
anti-Lub was used as primary antibody, since no single
antibody recognized all the LU deletion mutants expressed. Blots were
developed using the ECL system according to manufacturer's directions
(Amersham Pharmacia Biotech).
Flow Chamber Assay of Adhesion to Immobilized
Laminin--
Studies of intact cell adhesion to immobilized laminin
were performed using a flow chamber system in which a graduated chamber height allowed variable shear stresses to be produced along the length
of a single slide, as described previously (6). In brief, 3 µg/ml
human placental laminin (Life Technologies, Inc.) was coated onto a
glass slide, which was then mounted into the flow chamber. Following a
15-min static incubation with MEL cell transfectants containing a LU
construct or vector only and quantification of cells in each of seven
fields along the length of the slide, flow was applied at 20 ml/min for
6 min. After flow, the adherent cells were again counted at the same
seven areas of the slide, corresponding to shear stresses ranging from
0.2 to 10 dynes/cm2. Results were calculated as the
percentage of cells adherent after flow, compared with the number of
cells initially seen in each field after the static adherence
phase. Critical shear stress is defined as the shear stress at which
50% of the cells become detached. Each transfected cell line was
examined at least twice, with concordant results, by this method.
B-CAM/LU Copy Number on the Surface of SS and Normal Red
Cells--
As previous results have suggested that the B-CAM/LU
proteins are overexpressed by SS red cells (6, 28), we measured the
direct binding of radiolabeled anti-Lub mAb to determine by
Scatchard analysis the copy number of B-CAM/LU expressed by both SS and
normal red cells. Red cells from six randomly selected normal blood
donors expressed 1550, 1750, 1800, 1820, 1900, and 1970 molecules of
B-CAM/LU per cell, with an average of 1798 ± 144 molecules per
cell. The red cells from six patients with sickle cell anemia expressed
2500, 2600, 2700, 2980, 3080, and 4300 molecules of B-CAM/LU per cell,
with an average copy number per cell of 3027 ± 662 (p < 0.005 compared with red cells from normal
donors). Thus, on average, SS red cells expressed about two-thirds more
B-CAM/LU molecules than did normal red cells.
Similar studies using density fractionated red cells were also
performed on red cells from four sickle cell disease patients (Table
II) selected to represent a range of
B-CAM/LU copy numbers per cell. Although the copy number of B-CAM/LU
per cell for low density red cells enriched in reticulocytes was about
40-55% more than that seen in the high density fraction, the B-CAM/LU
copy number of unfractionated SS red cells was only 2-9% more than that of the high density fraction (Table II), demonstrating that the
presence of reticulocytes with higher numbers of B-CAM/LU molecules per
cell affected the overall expression of B-CAM/LU by circulating red
cells very little. Furthermore, the binding affinities of the
radiolabeled anti-Lub mAb to B-CAM/LU on normal and SS red
cells varied only slightly among red cells donors. The range of
dissociation constants (Kd) observed was 1.0-5.6
pM. In addition, the antibody also showed similar
affinities when reacted with high, low, and unfractionated SS cells
(data not shown), suggesting that the conformation of the B-CAM/LU
Lub antigen was similar among cells of different ages.
Role of Lutheran Cytoplasmic Domain on Laminin Binding--
In
order to determine whether the presence or absence of the cytoplasmic
tail lacking from B-CAM affects laminin binding, stably transfected MEL
cells expressing B-CAM or LU were tested for their ability to bind
soluble laminin by flow cytometric assay (Fig.
1B). Recombinant B-CAM and LU
proteins were easily detected by anti-Lub on the
transfectants containing B-CAM or LU cDNAs, but not on cells
containing vector alone. Likewise, soluble laminin bound equally well
to both B-CAM and LU transfectants but not to the cells transfected
with vector alone. This indicates that the cytoplasmic region has
little or no effect on laminin binding.
Expression of Lutheran Protein Deletion Mutants--
To determine
the domain of B-CAM/LU that contributes to laminin binding, we
expressed cDNA constructs containing LU cDNA from which had
been deleted individual regions encoding each of the five IgSF domains
(Fig. 2A). Stably transfected
MEL cells expressing these constructs were then examined by immunoblot
with anti-Lub and 4F2 (anti-domain 5) mAbs (Fig.
2B). These studies demonstrated double protein bands arising
from expression of all the Lutheran deletion constructs expressed in
MEL cells; in each case, the lower band was 62-70 kDa and the higher
band was 75-85 kDa. We hypothesize that these multiple bands arise
from varying degrees of glycosylation in MEL cells. The lower protein
bands of the LU Binding of Soluble Laminin to Recombinant LU Proteins Lacking
Individual IgSF Domains--
Stably transfected MEL cells expressed
all the LU domain deletion constructs to a variable but easily
detectable degree (MFC = 106.5-165.3, versus
MEL/vector MFC = 59.8) when reacted with either
anti-Lub mAb or a human anti-LU8 antibody in flow
cytometric assays (Fig. 3A). LU8 recognizes the second
IgSF domain of LU (20) and was therefore used for detecting the
expression of LU protein lacking IgSF domain 1 (LU
The observed ability of LU proteins without IgSF domains 1, 2, 3, or 4 to bind soluble laminin was variable, most likely in large part due to
the different levels of protein expression achieved. Nevertheless, MEL
cells expressing the recombinant proteins LU
In order to demonstrate that Lutheran domain 5 mediates laminin
binding, we deleted from LU cDNA sequences encoding the first four
IgSF domains. This new construct (LU5) was translated into a 25-kDa
protein by MEL cells and was detected by immunoblot using the
anti-domain 5 mAb 4F2 (Fig. 2B, lane 9). As shown
in Fig. 4, mAb 4F2 bound significantly to
MEL/LU5, as did soluble laminin, when examined by flow cytometry.
Adhesion of Transfected MEL Cells Expressing Recombinant LU
Proteins to Immobilized Laminin--
We also studied the ability of
various mutated LU proteins expressed by stably transfected MEL cells
to mediate intact cell adhesion to immobilized laminin using a flow
chamber assay. The MEL/vector control cell line began to detach at a
shear stress of 0.3 dyne/cm2, and 50% of cells were
detached at a shear stress of 0.7 dyne/cm2 (critical shear
stress). In contrast, cells expressing LU or LU deletion constructs
LU Scatchard analysis of 125I-labeled
anti-Lub mAb binding to SS and normal red cells allowed us
to evaluate red cell expression of B-CAM/LU. Although the expression of
B-CAM/LU on SS and normal red cells varied among individuals, on
average, SS red cells expressed 67% more molecules of B-CAM/LU than
did normal red cells, consistent with our earlier observations using
flow cytometric assays (6). The mechanism of B-CAM/LU overexpression on
SS cells is not known. While the expression of B-CAM/LU by
reticulocytes from sickle cell disease patients was 40-50% higher
than that of unfractionated or high density red cells from sickle cell
disease patients, expression of B-CAM/LU by dense SS red cells was only
minimally different from that of total circulating cells, indicating
that overexpression of B-CAM/LU is a characteristic of the total
circulating SS red cell population, not only of the reticulocytes. This
is also consistent with our previous results using two-color
immunostaining and flow cytometry to characterize B-CAM/LU expression
on reticulocytes.3 The
affinity of 125I-labeled anti-Lub mAb binding
to B-CAM/LU on either unfractionated or fractionated SS cells, as well
as on normal red cells, did not vary significantly, making it unlikely
that the Lub epitope of IgSF domain 1 is activated or
altered in sickle cell anemia. However, this does not rule out the
possibility of conformational change elsewhere in B-CAM/LU of SS cells,
especially at the laminin binding site.
Our previous data had suggested that soluble laminin binding to both
normal and SS red cells was proportional to B-CAM/LU copy number (6).
However, under conditions of flow, adhesion of SS red cells to
immobilized laminin was at least 3-fold stronger than that of normal
red cells. Since our current data indicate that the magnitude of the
increased expression of B-CAM/LU is less than 2-fold, the increased
laminin binding of SS red cells is likely to be due at least in part to
factors other than simple overexpression of the laminin receptor.
Possible explanations include that increased adhesion might be
partially due to an activation process or that increased B-CAM/LU
protein expression markedly enhances homotypic aggregation or
protein-protein interaction, leading to receptor activation and much
stronger cell adhesion.
This study also confirms that both B-CAM and LU proteins can mediate
laminin binding. We had previously shown that on Western blot both
B-CAM and LU proteins from normal and SS red cells bound soluble
laminin and that rB-CAM expressed by MEL cells mediated strong adhesion
to immobilized laminin (6). We have now shown that both B-CAM and LU on
intact cells mediated both binding of soluble laminin and adhesion to
immobilized laminin equally well. Thus, presence or absence of the
cytoplasmic tail of LU does not appear to affect laminin binding. The
role of the putative SH3 binding domain or of possible serine/threonine
phosphorylation of the cytoplasmic domain of LU, as well as the
potential functional differences between B-CAM and LU, thus require
further study.
Expression of LU cDNAs by MEL cells resulted in double bands on
Western blot. For the complete LU cDNA, the higher band
demonstrated a molecular mass of 95 kDa, even higher than the 85-kDa LU
band of red cells. LU deletion mutants also gave two bands. We
hypothesize that this phenomenon results from differences in the
glycosylation process. Since all five putative
N-glycosylation sites are within domains 3 and 4 (8),
deletion of which does not affect laminin binding, it is therefore
likely that laminin binding to LU is glycosylation-independent.
Studies designed to identify the laminin binding domain of LU by both
flow cytometry and flow chamber assays revealed that LU IgSF domain 5 is both critical and sufficient for laminin binding. Only deletion of
domain 5 (MEL/LU So far, structural studies identifying the functional domains of other
IgSF proteins, especially IgSF adhesins, have shown the N-terminal IgSF
domain(s) as the major heterotypic binding site(s) (13, 14). However,
our work shows that a membrane-proximal IgSF domain can also be a major
heterotypic binding site. Laminin, a family of heterotrimeric molecules
with molecular masses up to 800 kDa, might be imagined to have
difficulty accessing such a membrane-proximal domain. Thus, although LU
domain 5 alone could bind to both soluble and immobilized laminin
directly, it may not be the first contact point for laminin in
vivo. A non-critical site might exist elsewhere in B-CAM/LU and
act as a preliminary docking site for laminin, thereby facilitating the
attachment of laminin to LU domain 5.
With the identification of the active laminin-binding domain of LU,
further studies are now feasible to determine the importance of red
cell adhesion to laminin in sickle cell vaso-occlusion. We anticipate
the generation of inhibitory antibodies or other ligands directed
against domain 5, followed by in vivo studies, perhaps in
current murine models of sickle cell disease, to determine the
physiologic effect of blocking red cell adhesion to laminin. If
adhesion to laminin should prove physiologically important in sickle
cell anemia, as we suspect, B-CAM/LU may become a new target for
therapeutic intervention in this disease. In addition, further
investigation into the mechanism of B-CAM/LU overexpression and
activation on SS red cells should offer additional opportunities to
understand and modulate the vaso-occlusion process.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
,
, and
subunit (5). We have reported previously that basal
cell adhesion molecule/Lutheran glycoprotein (B-CAM/LU), a member of
the immunoglobulin superfamily with two known isomers (B-CAM and LU),
is the major laminin receptor on SS red cells and promotes SS but not
normal red cell adhesion to immobilized as well as soluble laminin (6).
Expression of B-CAM/LU appeared to be increased on SS red cells, as
measured by flow cytometry, and the level of B-CAM/LU expression was
proportional to laminin binding among red cells from both normal donors
and individuals with sickle cell disease (6). However, the exact scale
of increased expression was not determined in that study. The role of
B-CAM/LU in adhesion to laminin was further supported by the binding of
transfected MEL cells expressing recombinant human B-CAM (rB-CAM) to
both soluble and immobilized laminin.
E
7 interaction. And in other instances,
such as in the interactions of CD4 with major histocompatibility
complex II and CD8 with major histocompatibility complex I, multiple
IgSF domains are involved (18, 19).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
b+), according to routine typing performed in the Duke
Hospital Transfusion Service. In order to determine nonspecific
binding, inhibition of the binding of 125I-mAb to red
cells was achieved by incubating the cells with 50 M excess
of unlabeled mAb prior to adding 125I-mAb. Quantitation of
antigen sites per cell was calculated as described elsewhere (22, 23),
with the assumption that each antigen site was capable of binding one
Ig molecule.
) (Invitrogen, Carlsbad,
CA), which was used for all studies involving expression of recombinant proteins.
) vector as described above was used as the template for
all further PCR reactions to create constructs encoding domain deletion mutants. The cDNA sequences encoding each IgSF domain were deleted individually from the full-length LU cDNA using deletional
oligonucleotide primers in overlapping extension PCR (24). The forward
primers used to produce the 3' cDNA fragments of each construct are
listed in Table I. Complementary reverse oligonucleotide primers were utilized to generate the 5' fragments of each construct. A first round
of PCR reactions was performed using one of the deletional oligonucleotide primers (forward or reverse sense) and a corresponding vector primer under the following cycling conditions: 1 cycle at
94 °C for 3 min; 4 cycles at 94 °C for 1 min, 42 °C for 1 min, 72 °C for 1.5 min; 30 cycles at 94 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min; and finally, 1 cycle at 72 °C for 10 min.
The two overlapping cDNA fragments were then used to prime each
other for 4 cycles (94 °C for 1 min, 66 °C for 1 min, 72 °C
for 1.5 min). The 5' and 3' vector primers were then added to the PCR reactions to amplify the entire mutagenized cDNA, and PCR was performed as follows: 30 cycles at 94 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min; and 1 more cycle at 72 °C for 10 min. A LU
construct containing only domain 5 (LU5) without domains 1-4 was also
generated using the same PCR method and primers as listed in Table I.
The amino acids expressed by each LU deletion construct are also listed
in Table I, and the structure of all constructs used is illustrated in Fig. 2A.
Construction of Lutheran Proteins with IgSF Domain Deletions
) expression
vector; restriction mapping and DNA sequencing confirmed that the vectors contained the desired constructs with deletions, and constructs were transfected into MEL cells by electroporation, as described previously (6). Transfected cells were grown in RPMI 1640 containing 10% fetal calf serum and 0.5 mg/ml Geneticin (Life Technologies, Inc.), and cells with strong expression of recombinant protein were
further selected by immunofluorescence-activated cell sorting.
2, LU
3, LU
4, or LU
5
constructs and grown for >2 weeks in medium containing Geneticin were
incubated with anti-Lub mAb at 4 °C for 1 h,
followed by washing with phosphate-buffered saline, pH 7.4, with 1 g/dl
bovine serum albumin, and incubation with FITC-linked secondary Ab, and
repeated washing. MEL cells expressing LU
1 were incubated with human
anti-Lu8. MEL/LU5 cells were incubated with mAb 4F2. Transfectants
expressing high levels of desired proteins were selected by sterile
sorting using a Becton Dickinson FACStar Plus at Duke University Cancer Center.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copy number of B-CAM/LU on density-fractionated and unfractionated SS
red cells
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Fig. 1.
B-CAM/LU expression and laminin binding by
transfected MEL cells. A, schematic representation of
B-CAM and Lutheran proteins. B-CAM differs from Lutheran only in that
it lacks the last 40 amino acids of the cytoplasmic tail. B,
the binding of anti-Lub mAb and soluble laminin
(LAM) to MEL cells transfected with B-CAM or LU cDNA
constructs or vector (VEC) alone.
1, LU
2, LU
3, and LU
5 mutated proteins were
expressed as dominant bands, while the upper ones were expressed
weakly; the higher band of MEL/LU
5 was barely detectable. However,
the two protein bands of LU
4 were equally expressed, and the
molecular weights of these were lower than that of the other four
deletion mutants, consistent with the fact that most of the
glycosylation sites are on domain 4 (7, 20). MEL cells transfected with full-length LU cDNA also showed two bands, with molecular mass values of approximately 80 and 95 kDa, suggesting that in MEL cells, LU
protein can be more extensively glycosylated than it is in human red
cells.
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Fig. 2.
Construction and expression of Lutheran
deletion mutants on MEL cells. A, schematic
representation of Lutheran deletion mutants. The extracellular two V
domains and three C2 domains of LU cDNA were deleted individually.
B, the mutagenized LU proteins were expressed by MEL cells
and detected by immunoblot using a combination of two anti-LU mAb (4F2
and anti-Lub). Lane 1, SS red cell
ghosts; lane 2, MEL cells transfected with
vector; lane 3, MEL cells transfected with LU
cDNA; lanes 4-8, MEL cells transfected with
LU IgSF domain 1, 2, 3, 4, or 5 deletion constructs; lane 9,
MEL cells expressing Lutheran domain 5, transmembrane domain, and
C-terminal tail (MEL/LU5).
1), because
anti-Lub mAb recognizes the first IgSF domain. Binding of
LU8 to MEL/vector followed by FITC goat anti-human second Ab resulted
in similar "background" fluorescence as that observed with
anti-Lub and FITC goat anti-mouse (data not shown).
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Fig. 3.
Flow cytometric analysis of LU deletion
mutants. A, the binding of anti-LU Ab to transfected
MEL cells; a human anti-LU8 was used to detect LU 1 expression, and
mAb anti-Lub was used to detect LU
2, LU
3, LU
4, and
LU
5 mutants. B, soluble laminin binding to MEL cells
expressing LU deletion constructs. Fluorescence intensity is presented
in log scale.
1, LU
2, LU
3, and
LU
4 all gave distinctively higher fluorescence when reacted with
soluble laminin (MFC = 126.6, 100.3, 110.8, and 120.7, respectively) than did MEL/vector (MFC = 84.7) (Fig. 3B). However, the recombinant LU protein without domain 5 (LU
5) bound laminin minimally, with MFC of only about 89.6, even
though the expression of LU
5 protein detectable by
anti-Lub mAb (MFC = 152.4) was much higher than those
of LU
2 (MFC = 106.5), LU
3 (MFC = 117.6), and LU
4
(MFC = 139.5). No laminin binding was detected when LU
5 was
expressed at levels similar in terms of MFC to those of MEL/LU
2 or
MEL/LU
3. These data, therefore, suggested that domain 5 of LU
protein is critical for laminin binding.
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Fig. 4.
Lutheran domain 5 expression and soluble
laminin binding by transfected MEL cells (MEL/LU5). A,
binding of mAb 4F2 to MEL/LU5 (thick line) and
MEL cells expressing vector alone (thin line).
B, binding of soluble laminin to MEL/LU5 (thick
line) and MEL/vector (thin
line).
1, LU
2, LU
3, or LU
4 all showed little detachment at shear
stress as high as 10 dynes/cm2 (Table
III). The critical shear stress for
LU
5 transfected MEL cells, however, was only 2.3 dynes/cm2 (Fig. 5), only
slightly above that seen with MEL/vector transfectants. In addition, we
also tested the adhesion of MEL/LU5 to immobilized laminin under
similar flow conditions. As shown in Fig. 5, the critical shear stress
of MEL/LU5 attachment to laminin was above 10 dynes/cm2,
indicating that LU domain 5 alone is sufficient to mediate strong cell
adhesion to immobilized laminin.
Adhesion to laminin by MEL cells expressing various rLU proteins
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Fig. 5.
Adhesion to immobilized laminin by MEL cell
transfectants. The x axis is the shear stress applied,
and the y axis is the fraction of cells that remained
immobilized on the laminin-coated slide. The MEL cell LU deletion 5 transfectants (MEL/LU 5) were detached at low shear stress (critical
shear stress = 2.3 dynes/cm2). MEL cells expressing
the LU domain 5 construct, lacking domains 1-4 (MEL/LU5), adhered to
immobilized laminin with a critical shear stress of >10
dynes/cm2.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5) significantly decreased soluble laminin binding
to transfected MEL cells, and MEL/LU
5 cells detached from
immobilized laminin easily at low shear stress (critical shear
stress = 2.3 dynes/cm2) under flow conditions.
Deletion of all of the other four IgSF domains did not abrogate laminin
binding. In addition, MEL/LU5 adhered strongly to immobilized laminin
under high shear stress, although, in flow cytometric assays, soluble
laminin did not bind to MEL/LU5 as strongly as it bound to MEL/LU. One
possibility is that deletion of all first four IgSF domains affected
the conformation of the remaining domain 5, altering its ability to
bind soluble laminin. A second possibility is that the level of
expression of LU5 achieved, which was lower than the level of
expression achieved for LU, resulted in lower laminin binding. A third
possibility is that additional LU domains may contribute to the
localizing of laminin to its binding site on domain 5.
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ACKNOWLEDGEMENT |
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We are indebted to Manisha Udani for technical assistance and many thoughtful suggestions.
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FOOTNOTES |
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* This work was supported in part by Grant RO1 HL 58939 from the National Heart, Lung and Blood Institute, National Institutes of Health, and by a Focused Giving Grant from Johnson & Johnson, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Box 2615, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-5638; Fax: 919-681-7688; E-mail: qinzen{at}acpub.duke.edu.
The abbreviations used are: SS, homozygous for sickle hemoglobin; B-CAM, basal cell adhesion molecule; LU, Lutheran glycoprotein; MEL, murine erythroleukemia; IgSF, immunoglobulin superfamily; Ab, antibody; mAb, murine monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; MFC, mean fluorescence channel; PCR, polymerase chain reaction; FITC, fluorescein isothiocyanate; ALCAM, activated leukocyte cell adhesion molecule (CD166).
2 M. Lacaze, Q. Zen, and M. Telen, unpublished data.
3 M. Udani, Q. Zen, and M. Telen, unpublished data.
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REFERENCES |
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