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J. Biol. Chem., Vol. 276, Issue 26, 23757-23762, June 29, 2001
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,,,,, and¶
From the INSERM U76, Institut National de la
Transfusion Sanguine, Paris 75015, France and § CNRS
UMR7590, Universités Paris 6 et Paris 7, Paris 75005, France
Received for publication, April 4, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Lutheran (Lu) blood group antigens and the
basal cell adhesion molecule antigen reside on two
glycoproteins that belong to the Ig superfamily (IgSF) and carry
five Ig-like extracellular domains. These glycoproteins act as widely
expressed adhesion molecules and represent the unique receptors for
laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF
domains responsible for binding to laminin. In plasmonic resonance
surface experiments, only recombinant Lu proteins containing the
N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to
inhibit binding of laminin to Lu-expressing K562 cells. Mutant
recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of
the extracellular domains except domain 3. Altogether, these results
indicate that IgSF domains 1-3 are involved in laminin binding and
that a specific spatial arrangement of these three first domains is
most probably necessary for interaction. Neither the RGD nor the
N-glycosylation motifs present in IgSF domain 3 were
involved in laminin binding.
Lutheran (Lu)1 blood
group antigens and the basal cell adhesion molecule antigen (a marker
of epithelial ovarian cancers) are carried by two glycoprotein (gp)
isoforms of 85 kDa (Lu) and 78 kDa (Lu(v13)) that are translated from
spliceoforms of a single pre-mRNA (1, 2). The Lu gps have a wide
tissue distribution with a predominant expression in the basal layer of
the epithelium and the endothelium of blood vessel walls (1). Lu and
Lu(v13) are type I membrane proteins of the immunoglobulin superfamily (IgSF) that share a similar extracellular domain composed of five Ig-like domains (two V-set and three C2-set) and a single transmembrane domain but differ by the presence or absence of a cytoplasmic C-terminal domain of 40 amino acids (59 versus 19, respectively) (2). The extracellular domain of Lu gps contains a
potential integrin binding site in the third IgSF domain as well as
five N-glycosylation consensus motifs, one in the third
domain and the others in the fourth domain. The 40 C-terminal residues
of the Lu gp cytoplasmic tail carry three characteristic motifs
involved in protein-protein interaction, polarization to plasma
membrane, and intracellular signaling pathways (3), such as (i) a
proline-rich motif for potential binding of Src homology 3 domains,
(ii) a dileucine motif responsible for the basolateral expression
of Lu gp in polarized epithelial cells (4), and (iii) potential phosphorylation motifs. These motifs are absent in the Lu(v13) gp
isoform which exhibits a nonpolarized expression in epithelial cells
(4). Recent studies have shown that the C-terminal intracellular domain
of Lu gps interacts with the membrane skeleton (5, 6).
Interestingly, the Lu gps share a significant sequence similarity with
the CD146 (MUC18) and CD166 (activated leukocyte cell adhesion
molecule) adhesion molecules found in human tissues (7, 8), and Lu and
Lu(v13) are themselves adhesion molecules that bind laminin with high
affinity (9, 10). Laminins are heterotrimeric extracellular matrix
glycoproteins (composed of one of each of the polymorphic Lu gps are expressed late during erythroid differentiation (14, 15) and
represent the unique receptors of laminin in normal and sickle red
blood cells as well as in erythroid progenitors (9, 10). Increased
expression of Lu antigens on sickle red blood cells correlates with an
increased adhesion to laminin (9, 10), which, together with other
adhesion molecules like CD36 and VLA-4, might contribute to the
reinforced adhesion of the sickle red blood cells to vascular
endothelium leading to vaso-occlusion crisis and strokes (16, 17).
The use of domain deletion mutants provided the first information on
the structure/function relationship by showing that at least one Lu
polymorphic blood group antigen is located on each of the five IgSF
domains (18). In addition, the membrane-proximal IgSF domain 5 was
shown to be the only critical domain for laminin binding (19). In the
present study, we further extended these studies by generating deletion
mutants of the NH2 extracellular domain of Lu gps and
mutants of the RGD and glycosylation motifs in order to better
characterize the binding site and protein motifs involved in the
interaction with human laminin. By the use of direct interaction
biosensor assays and inhibition assays with a flow cytometer, we found
that the first three N-terminal Ig-like domains are critical for
interaction with laminin. Moreover, neither the RGD nor the
N-glycosylation motifs of the third IgSF domain are involved
in this interaction.
Cell Culture, Transfection, and Flow Cytometry
Analysis--
Human K562 cells were obtained from the American Type
Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Stable transfected K562 cells
expressing Lub isoform were obtained as described (10).
Expression of Lub antigen on transfectant cell lines was
measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
using LM342 monoclonal antibody (gift of Dr. Robin Fraser,
Regional Donor Center, Glasgow, UK) as described (20).
Production of Soluble Recombinant Lu-Fc--
Recombinant
cDNAs encoding different extracellular domains of the Lu gp were
obtained by polymerase chain reaction using the cDNA GC-rich kit
(CLONTECH) and the Lub cDNA in
pcDNA3 vector as template (10). The polymerase chain reaction
products were subcloned in the pIgplus vector (pIg-Tail Expression
System; Ingenius; R & D Systems) and used for transient transfection of
COS-7 cells by the DEAE-dextran method (ProFection mammalian
transfection system, Promega). The transfected COS cells were grown 5 days in Iscove's modified Dulbecco's medium with Glutamax-1 and
immunoglobulin-depleted fetal calf serum. The cell culture supernatants
containing the secreted Fc proteins were applied to a protein
A-Sepharose column (Amersham Pharmacia Biotech), and the Lu-Fc proteins
were eluted as recommended by the manufacturer. Recombinant Fc protein
concentrations were assessed by colorimetric assay using a Bio-Rad
protein assay kit, and the integrity of the preparations was checked by
8% SDS-polyacrylamide gel electrophoresis and staining with silver nitrate.
Biosensor Laminin Binding Assay--
Biosensor studies were
performed on a Biacore X instrument (Amersham Pharmacia Biotech). All
experiments were performed at 25 °C using an HBS buffer, comprising
10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA. Rabbit anti-human Fc (Pierce) was diluted in a 10 mM acetate buffer, pH 4.5, and covalently coupled to a CM-5
or to an F1 biosensor chip by amine coupling, using an amino coupling
kit (Amersham Pharmacia Biotech). Lu-Fc and Fc control proteins were
diluted in HBS buffer and coupled to the anti-human Fc antibody.
Typically, a coupling level of 200-400 and 10-20 response units (RU)
were used for mapping and kinetic analyses, respectively. Different
concentrations of immunopurified laminin-10/11 (Life Technologies,
Inc.) or control fibronectin and merosin (laminin-2) proteins were
injected over the coupled Lu-Fc surface as well as a control protein
surface (ICAM-4-Fc) at a flow rate of 10 µl/min. A 25 mM
NaOH solution was used for regeneration of the immobilized surface
between different Lu-Fc injections. Sensorgrams obtained for the Fc
control protein were systematically subtracted from those obtained for
Lu-Fc proteins. For kinetic analysis, a range of six different laminin
concentrations were injected (5, 8, 10, 16, 20, and 30 nM)
at a flow rate of 30 µl/min with an Rmax not
exceeding 100 RU. Affinity constants were estimated by curve fitting
using BIA Evaluation 3.0 software assuming a 1:1 binding model.
In another series of experiments, laminin-2 and laminin-10/11 were
diluted in 10 mM acetate buffer, pH 3.3, and covalently coupled (9,000-10,000 RU) to flow cells FC1 and FC2, respectively, of
a CM-5 biosensor chip by amine coupling using an amino coupling kit
(Amersham Pharmacia Biotech). Lu-Fc and Fc control proteins were
diluted in the HBS buffer and injected over the laminin covered surface
at a flow rate of 10 µl/min. Sensorgrams obtained for laminin-10/11
were subtracted from those obtained for laminin-2.
Laminin Binding Inhibition Assay--
Laminin binding inhibition
assay using secreted chimeric Lu-Fc fragment was performed as follows.
500-ng aliquots of laminin were incubated with different dilutions of
the Lu-Fc fragment or a control Fc fragment for 1 h at 37 °C in
PBS, supplemented with 0.5% bovine serum albumin. The suspensions were
then added to 5 × 105 K562 cells expressing the
Lub isoform and incubated for 45 min at room temperature.
After two washes with PBS, cells were incubated with the anti-laminin
monoclonal antibody (anti-A chain) (Roche Molecular
Biochemicals) for 30 min at 4 °C. The cells were then washed twice
with PBS and incubated with phycoerythrin-conjugated anti-mouse
IgG (Immunotech, Marseille, France) for 20 min at 4 °C. After
another washing step, 0.1 ng of TO-PRO-1 iodide (Molecular Probes,
Inc., Eugene, OR) was added, and positive cells (dead cells) were
excluded from analysis.
Sequence Analysis, Secondary Structure Predictions, and Homology
Modeling--
Sequences of the five IgSF domains were searched against
the SMART domain data base (21) for alignment against V-set and C2-set
profiles. The Protein Data Bank was also searched using PSI-BLAST (22)
and FUGUE2 for predicting the
Lu domain's secondary structures relative to experimentally solved
structures of Ig-like domains. Homology modeling of Lu IgSF domain 3 was performed using one of the fibroblast growth factor receptor 2 C2
domain (Protein Data Bank identifier 1EV2, chain E, position 150-254)
as template with the Modeller4 program (23).
Interaction of the Recombinant Lu12345-Fc Fusion Protein with
Laminin-10/11--
The interaction of the common extracellular region
of Lu and Lu(v13) gps with laminin was first analyzed by plasmon
resonance experiments using a Biacore X instrument. This region of 518 amino acids is organized in five IgSF domains, noted Lu12345
thereafter. The sequence coding for Lu12345 was fused to that coding
for the Fc fragment of a human IgG, and the soluble chimeric Lu-Fc
fusion protein was expressed in COS-7 cells (see "Experimental
Procedures"). After purification on Protein A-Sepharose, the chimeric
polypeptide migrated as an expected single band of 106 kDa (Fig.
1). For biosensor assays, the rabbit
anti-human Fc antibody was immobilized directly to a carboxylated
dextran matrix chip, and the Lu12345-Fc fusion protein was captured on
the matrix and tested for interaction with laminin. As shown in Fig.
2, protein laminin 10/11 bound to
Lu12345-Fc in a dose-responsive fashion in the range of 5-30 nM laminin. The specificity of the binding interaction was
determined by showing that laminin did not bind to the immobilized
adhesion molecule ICAM-4-Fc, which contains two IgSF domains (23, 25). In further control experiments, it was found that neither
laminin-2 (
Recent studies indicated that commercial human laminin preparations
contain laminin 10 ( Interaction between Lu gp Mutants and Laminin-10/11--
In order
to identify the IgSF domain(s) that interact(s) with laminin-10/11, 11 IgSF domain deletions (three small deletions of five amino acids and
two single amino acid mutants) were generated and expressed as Fc
fusion proteins. The schematic structure of these mutants is shown on
Fig. 3. To generate entire domain
removal, deletion sites were chosen in the interdomain sequences
deduced from the putative structure of the Lu gp (1). The Lu12345-Fc protein containing the five extracellular IgSF domains was used as a
positive control when analyzing the interaction between the different
mutated proteins and laminin-10/11. The four deletion constructs
Lu1234-Fc, Lu123-Fc, Lu12-Fc, and Lu1-Fc were first analyzed by
SDS-PAGE (Fig. 1), and among these, we found that only Lu1234-Fc and
Lu123-Fc bound to laminin (Fig.
4A). The lack of interaction
between laminin and Lu1-Fc and Lu12-Fc proteins was not due to
defective chimeras, since these proteins were able to bind to
monoclonal antibody LM342, which recognizes the Lub
epitope localized in the first IgSF domain in Biacore experiments (data
not shown). Kinetic analysis also revealed that Lu123-Fc and Lu12345-Fc
had similar binding affinity for laminin 10/11 (KD = 9.8 nM versus 10.8 nM), and this
result, together with those reported above, suggests that the laminin
binding site(s) of Lu gps includes the third IgSF domain and that
domains 4 and 5 neither enhance nor decrease laminin binding affinity.
Accordingly, deletion mutants Lu4-Fc, Lu5-Fc, and Lu45-Fc did not
bind to laminin 10/11 (Fig. 3 and 4A).
These results are in general agreement with a recent study published
when this work was being written (6). However, our studies further
extended this analysis, since a panel of nine other Lu chimeras
carrying different Ig domain deletions, small deletions, and single
point mutations into IgSF domain 3 (see Fig. 3) were investigated.
The laminin binding capacity of Lu123-Fc but not of Lu12-Fc constructs
pointed to the important role of domain 3 of Lu gp for ligand binding.
To confirm this result, we first generated a mutant protein lacking
domain 3 only. As shown in Fig. 4A, this Lu1245-Fc chimera
did not bind to laminin-10/11. To test whether the N-glycan
of domain 3 is important for laminin binding, asparagine 321 was
mutated into alanine to abolish the unique potential
N-glycosylation site of the first three IgSF domains, which
is well exposed at the beginning of strand E according to the
structural model proposed in Fig.
5B. The LuN321A-Fc construct
directed the synthesis of a soluble protein with an apparent molecular
mass of 103 kDa versus 106 kDa for the wild type protein
(Fig. 1). This indicates that the Asn321 glycosylation site
is used, and the loss of about 3 kDa is compatible with the removal of
a single N-glycan chain. Arginine 292 belonging to a
potential RGD integrin binding site of domain 3 was also mutated into
alanine (construct LuR292A-Fc), since the RGD motif is commonly
involved in cell adhesion and is crucial for some integrin binding and
for the binding of many proteins to cells, such as fibronectin and
transferrin to their cognate receptors (31, 32). As shown in Fig.
4B, the sensorgram of the interaction between laminin-10/11
and LuN321A-Fc and LuR292A-Fc constructs was similar to that obtained
with Lu12345-Fc, indicating that neither N-glycosylation nor
the RGD motif of domain 3 were involved in the interaction with laminin
10/11. This conclusion is also supported by the finding that the mouse
homologue of Lu gp binds human laminin but does not contain any RGD
motif (6, 28). Moreover, as shown on the model of the Lu IgSF domain 3 structure (Fig. 5B), the RGD sequence is not included in a
loop, as observed for fibronectin RGD sequence (33) but lies extended
in the C-terminal part of strand
Next, three different small deletions of five amino acids were
introduced into the third IgSF domain of the Lu12345-Fc protein (see
Fig. 3). All deleted amino acids are conserved between human, mouse,
and rat (Fig. 5A). These are predicted to be localized in
three regions included in distinct secondary structures, namely the B-C
loop and the beginning of the C strand (Lu
To confirm these results, we performed a reverse series of biosensor
assays in which laminin-10/11 was covalently coupled to the sensor
chip, and several dilutions of Lu-Fc chimeras were injected. Laminin-2
was used as a negative control. Lu12345-Fc, Lu1234-Fc, Lu123-Fc,
LuN321A-Fc, and LuR292A-Fc showed typical specific binding curves with
laminin-10/11, whereas the other Lu-Fc chimeras did not bind to the
immobilized laminin (data not shown).
Inhibition of Lu-Laminin Binding by the Lu-Fc Soluble
Proteins--
Previous adhesion and flow cytometric analyses have
shown that K562 cells expressing Lu gps bind laminin (9, 10) and that
the Lu12345-Fc chimera protein dose-responsively inhibited the
laminin-Lu gp interaction (10). To further confirm the BIAcore results
(see above), we have used the different Lu-Fc chimeras to inhibit the
interaction of laminin with K562.Lu cells. Increasing amounts of each
recombinant protein were preincubated with 500 ng of laminin-10/11
prior to the binding assay with K562.Lu cells. Among all the mutated
proteins, only Lu123-Fc, Lu1234-Fc, LuN321A-Fc, and LuR292A-Fc
molecules dose-responsively inhibited the binding of laminin to K562.Lu
cells, as did the control Lu12345-Fc protein (Fig.
6). More than 80% inhibition was
obtained with 10 pM of these mutated constructs. The
inhibition assays largely confirm the BIAcore results and indicated
again that laminin binding to K562.Lu cells requires the three first
Ig-like domains of Lu gp.
All these results suggested that the interaction between laminin-10/11
and Lu gps involves sequences present in the three extracellular
domains 1, 2, and 3, which might be arranged together so that they
provide a specific interaction surface. Lu gps share sequence
similarities with cell adhesion molecules belonging to the IgSF
(e.g. neural cell adhesion molecule, axonin, etc.), which are similarly organized (i.e. with an N-terminal string of
IgSF domains (5 and 6 for neural cell adhesion molecule and axonin, respectively). Interestingly, the first four IgSF domains of axonin (this is also true for hemolin) adopt a U-shaped arrangement, forming a
compact four-domain module with contacts between domains 1 and 4 and
between domains 2 and 3 (34). By analogy, as the IgSF domain 4 of Lu gp
does not appear critical for laminin binding (see Fig. 4A),
it is suggested that the first three IgSF domains of Lu gp could
constitute a three-domain module having a specific spatial arrangement
with domains 1-3 playing a key role in the interaction properties.
Similarly, the interaction between laminin and activated leukocyte cell
adhesion molecule apparently requires the first two IgSF domains only
(35).
Independently, two groups have reported the characterization of the
laminin binding site on the fifth (19) or the first three (6) IgSF
domains of the Lu gps. Here, we have shown by two different techniques,
using purified chimeric proteins and Lu-expressing cells, that the
N-terminal domains 1-3 are critical for laminin-10/11 interaction,
since neither domain 1, domains 1 + 2, domain 3, domains 1 + 3, nor
domains 2 + 3 were able to mediate laminin interaction. In addition,
the present study demonstrates that the structural integrity of the
IgSF domain 3 is necessary for laminin-10/11 interaction and that
neither N-glycosylation nor RGD motifs of this domain were
involved in laminin-10/11 binding. Our results and those recently
reported (6) apparently conflict with those from Zen et al.
(19) that have assigned the laminin binding domain to the fifth IgSF
domain of Lu gp. Although different techniques of investigation have
been used, and much fewer mutants analyzed compared with the present
report, the reason for this discrepancy is not clear, but we cannot
exclude the possibility that Lu gps may have two distinct binding sites
for laminin.
Further investigation should document the critical residues of Lu gps
involved in laminin interaction and reciprocally those involved in the
laminin
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, 
, and
chains) that are found in all basement membranes and are involved
in cell differentiation, adhesion, migration, and proliferation
(11-13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
211) nor
fibronectin at 20 nM bind to either immobilized Lu12345-Fc or ICAM-4-Fc fusion proteins (data not shown). For kinetic analysis, small amounts of Lu12345-Fc were captured on the anti-human Fc surface
in order to not exceed an Rmax of 50-100 RU
when injecting saturating concentrations of laminin. After injecting a
range of six different concentrations (5, 8, 10, 16, 20, and 30 nM), kinetic analysis revealed that Lu12345-Fc bound
laminin with high affinity, with a dissociation equilibrium constant
KD of 10.8 nM (kinetic constant
ka = 2.45 × 106
M
1 s1).
This value correlates well with recently reported independent studies
(6).
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Fig. 1.
SDS-PAGE analysis of chimeric protein
mutants. The wild type Lu12345-Fc and mutant proteins (Lu1234-,
Lu123-, Lu12-, Lu1-, and R292A-Fc) were purified from the culture
medium of COS cells on a Protein A-Sepharose column, and samples were
analyzed by SDS-PAGE as described under "Experimental Procedures."
Lane M, molecular mass marker proteins. Apparent molecular
mass of mutant proteins (kDa) was calculated using Sigma-Plot software
from Jandel GmBH (Germany).
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Fig. 2.
Binding of laminin-10/11 to Lu12345-Fc
coupled to an anti-Fc antibody immobilized on the surface of a plasmon
resonance sensor chip. A rabbit anti-human Fc was immobilized
(8,000 RU) on a carboxylated dextran matrix chip before coupling
ICAM-4/LW-Fc (280 RU) and Lu12345-Fc (300 RU) in flow cells FC1 and
FC2, respectively. Laminin-10/11 was injected at 5, 8, 10, 16, 20, and
30 nM over the recombinant Fc protein surface at the flow
rate of 10 µl/min. Curves represent subtracted sensorgrams
(FC2 
FC1) normalized as to the base line of 0 RU.
511)
and some laminin 11 (521) but no laminin 1 (26). Since 5 chain is present only in
laminin 10 and 11 isoforms (27) and Lu gps bind neither to murine
laminin 1 (111) nor to
laminin 2 (211) (9, 10,
28), it was postulated that Lu gps bind to laminin-10/11 via the
specific 5 chain (Ref. 6 and this report), which is also
in agreement with other studies showing that sickle red blood cells
adhere to laminin-10/11 isoforms (29). Furthermore, the laminin
5 chain is expressed in bone marrow and by several
developing epithelial cells, and a majority of epithelial cells might
use laminin-10 rather than laminin-1 as an adhesive protein in
vivo (26, 30).
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Fig. 3.
Schematic representation of the deletion and
point mutation mutants of the Lu gp extracellular domain fused with the
Fc portion of a human IgG. The soluble chimeric proteins are
presented schematically. The Fc portion represented as a
hatched box was fused to the carboxyl-terminal
end of the IgSF domains shown as loops. Numbers
1-5 indicate the respective IgSF domains, and the
intradomain disulfide bonds are shown as S S. Black boxes indicate deletions within the IgSF
domain sequences.
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Fig. 4.
Binding of laminin-10/11 to Lu-Fc mutants
coupled to an anti-Fc antibody immobilized on the surface of a plasmon
resonance sensor chip. A rabbit anti-human Fc antibody was
immobilized (8,000 RU) on a carboxylated dextran matrix chip before
coupling ICAM-4/LW-Fc (280 RU) and Lu-Fc mutants (200-300 RU) in flow
cells FC1 and FC2, respectively. Laminin-10/11 was injected at 20 nM over the recombinant Fc protein surfaces at a flow rate
of 10 µl/min. Curves represent subtracted sensorgramms
(FC2 FC1) normalized as to the base line of 0 RU. A,
proteins Lu1-, Lu12-, Lu13-, Lu3-, Lu23-, Lu1245-, Lu-4, Lu5-, and
Lu45-Fc did not bind laminin, whereas those containing intact IgSF
domains 1, 2, and 3 (Lu123- and Lu1234-Fc) bind similarly to the wild
type protein Lu12345. B, LuN321A- and LuR292A-Fc bind
laminin as the intact chimera Lu12345, whereas proteins Lu-
LNVNL-,
Lu-RVEDY-, and Lu-PSPEY-Fc exhibit abnormal binding curves.
B, just after the cysteine residue
implicated in disulfide bonding with the strand F cysteine, and
therefore has a different presentation as compared with canonical
RGD.
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Fig. 5.
Lu gp IgSF domain 3 and model of secondary
protein structure. A, sequence alignment of IgSF domain
3 of Lu gp from human, mouse, and rat. Identical amino acids are in
boldface type. The protein motifs discussed in
this report are boxed. B, Molscript
representation of the model of the Lu IgSF domain 3 three-dimensional
structure, using as template one of the C2 domains of fibroblast growth
factor receptor 2 (Protein Data Bank identifier 1EV2, chain E). Strands
are labeled A-G. The Asn321 and Arg-Gly-Asp
sequence (RGD positions 292-294) are shown as ball-and-stick
models, whereas the three deleted segments are shaded
black. The disulfide bridge (Cys positions) linking strands B and
F is also indicated.
PSPEY-Fc), the F strand
(Lu RVEDY-Fc), and the D strand and the beginning of the D-E loop
(LuLNVNL-Fc) (Fig. 5B). As shown in Fig. 4B, these small deletions within domain 3 drastically alter the association and dissociation curves and reduce the laminin binding property of the
extracellular region of the Lu gps. Disruptions of regular secondary
structures in which these segments participate and the nonobservance of
geometrical constraints probably impede the correct folding of domain
3. It is also worth noting that two of the deleted segments
(PSPEY-Fc and RVEDY-Fc) contain two amino
acids (underlined Y and V, respectively) whose positions are always
occupied by hydrophobic amino acids in the multiple alignment of C2
domains and are buried within three-dimensional structures (data not
shown). These amino acids are therefore predicted to be essential to
the maintenance of the IgSF fold, and their absence would thus
drastically impair the correct folding of domain 3. Although these
results confirm the importance of the third domain, it could not be
concluded whether domain 3 alone, albeit critical, was sufficient for
laminin binding. Indeed, it is often observed that the combination of several IgSF domains is necessary for interaction with partners, as
discussed below. To further address this issue, a mutant protein containing only domain 3 (Lu3-Fc) was generated. As shown in Fig. 4A, Lu3-Fc did not bind to laminin-10/11 even when large
amounts of laminin were injected, indicating that the third IgSF domain of Lu gps is necessary but not sufficient to mediate laminin-10/11 binding. Together with the positive binding of Lu123-Fc to laminin, these results indicated that domain 1 and/or 2 may also play a role in
the interaction process. To find out whether the interaction involves
domain 1 or 2, Lu13-Fc and Lu23-Fc chimeras were generated, but none of
these two chimeras were found to bind to laminin-10/11 (Fig.
4A).
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Fig. 6.
Inhibition assay by recombinant Fc
fragments. Laminin-10/11 aliquots (500 ng) were preincubated with
increasing amounts of the recombinant Lu-Fc proteins (0.625, 2.5, and
10 pM) before the addition to K562 cells expressing the Lu
gp isoform. The percentage of bound laminin is expressed as the
relative fluorescence intensity versus Fc fragment
concentration. Only chimeric proteins containing the first three IgSF
domains (Lu12345-, Lu1234-, and Lu123-Fc) are inhibitors. Lu1- and
Lu12-Fc do not inhibit laminin binding to K562.Lu cells.
5 chain counterpart more precisely. This information may be useful to develop inhibitors that block the Lu-laminin interaction and may reduce the increased adhesion of sickle
red cells to laminin, which is suspected to contribute to
vaso-occlusion in patients (16, 17). This will be of great importance
also, since Lu antigens are overexpressed in some epithelial cancer
tissues (36, 37) and since laminin is involved in cancer metastases
(38).
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ACKNOWLEDGEMENTS |
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We thank P. Hermand and P. Bailly (INSERM U76, Paris) for the gift of purified control ICAM-4-Fc protein and C. Rahuel (INSERM U76, Paris) for alignment analysis. We are indebted to R. H. Fraser (Regional Donor Center, Glasgow, UK) for supplying the LM 342 monoclonal antibody.
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FOOTNOTES |
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* This work was supported in part by INSERM and the Institut National de la Transfusion Sanguine.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: INSERM U76, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France. Tel.: 33 1 44 49 30 46; Fax: 33 1 43 06 50 19; E-mail: levankim@idf.inserm.fr.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M102978200
2 J. Shi, T. L. Blundell, and K. Mizuguchi, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Lu, Lutheran; gp, glycoprotein; IgSF, immunoglobulin superfamily; RU, response units; ICAM, intercellular adhesion molecule.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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