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(Received for publication, August 29, 1994; and in revised form, November 8, 1994) From the
Intercellular adhesion molecule 3 (ICAM-3, CD50) is a member of
the immunoglobulin superfamily and is a constitutively expressed ligand
for the leukocyte integrin LFA-1 (CD11a/CD18). ICAM-3 is expressed at
high levels by all resting leukocyte populations and antigen presenting
cells and is a major ligand for LFA-1 in the resting immune system.
ICAM-3 is a signal transducer and may play a key role in initiating
immune responses. Mutant ICAM-3 Fc-chimeric proteins were
quantitatively analyzed for their ability to bind COS cells expressing
human LFA-1. The LFA-1-binding site on ICAM-3 is located in the
N-terminal 2 Ig domains. Domains 3-5 do not significantly
contribute to adhesion. The binding site has been further resolved by
rational targeting of 14 point mutations throughout domains 1 and 2,
coupled with modeling studies. Within domain 1 a cluster of residues
(Glu Cell adhesion molecules (CAMs) ( A key receptor-ligand interaction that contributes significant
adhesion to all of these areas is the binding of the ICAMs
(intercellular adhesion molecules) to their integrin ligands, most
notably LFA-1 and Mac-1 (Pardi et al., 1992). The three known
ICAMs (ICAM-1, -2, and -3) are all members of the immunoglobulin
superfamily and have five, two, and five Ig domains, respectively
(Rothlein et al., 1986; Simmons et al., 1988;
Staunton et al., 1988, 1989; de Fougerolles et al., 1991, and 1993; Fawcett et al., 1992. Vazeux et
al., 1992). ICAMs 1 and 3 are the most closely related. The ICAMs
all bind to the leukocyte integrin LFA-1 (CD11a/CD18;
The different expression profiles support the
notion that the ICAMs serve different functions and are not
functionally redundant. ICAM-3 is expressed by antigen presenting
cells, specifically by Langerhans cells in the skin (Acevedo et
al., 1993). ICAMs 1 and 2 are either absent or expressed at very
low levels on these cells. In addition, on resting T cells, ICAM-3 is
the dominant ligand for LFA-1 (Campanero et al., 1993) and
provides a costimulatory signal for cell proliferation (de Fougerolles et al., 1994). Recent work shows ICAM-3 to be a signal
transducer. Cross-linking ICAM-3 produces a significant Ca The interaction of
ICAM-1 with LFA-1 is a multistep process, involving conformational
changes in the integrin, partly controlled by engagement of ligand
ICAM-1 (Cabanas and Hogg 1993). LFA-1 seems to have an intrinsically
low affinity for its ICAM ligands, and that affinity is increased by a
series of events that can be mimicked by a number of different stimuli
including: changes in bound divalent cations (Dransfield and Hogg 1989;
Dransfield et al., 1992), signal transduction agonists such as
phorbol esters (Dustin and Springer 1989); and a small number of mAbs
that induce or stabilize conformation changes (van Kooyk et al., 1991; Landis et al., 1993). However, apart from ligand
itself, the normal cellular regulators of this affinity change are not
yet known. The LFA-1-binding site in ICAM-1 has been mapped to the
N-terminal immunoglobulin (Ig) domain, and 2 residues, namely
Glu
COS-7 cells were obtained from the ICRF cell bank and grown in
Dulbecco's modified Eagle's medium, 5% fetal calf serum.
Activated T cells were expanded from peripheral blood mononuclear cells
as described (Cabanas and Hogg, 1993).
Recombinant chimeric-Fc plasmids were transiently
expressed in COS cells and secreted protein purified from tissue
culture supernatants with protein A-coupled Sepharose (Simmons, 1993).
In order to assess the activation state of LFA-1
expressed by COS cells, binding assays were performed onto ICAM-1 and
ICAM-3 in the presence or absence of activating stimuli.
LFA-1
Figure 1:
Regulation of LFA-1 activation state
in COS cells. COS cells transiently expressing LFA-1 (40% COS cells
LFA-1
Figure 2:
The
domain deletion series of ICAM-3-Fc chimeric proteins. Proteins were
purified as described under ``Materials and Methods.'' 5
µg of each protein were resolved by SDS-polyacrylamide gel
electrophoresis under reducing conditions and visualized with Coomassie
stain. Molecular masses deduced from electrophoretic mobilities are
ICAM-3(D1-2)-Fc 80 kDa (lane 1), ICAM-3(D1-3)-Fc
107 kDa (lane 2), ICAM-3(D1-4)-Fc 130 kDa (lane
3), ICAM-3(D1-5)-Fc 136 kDa (lane 4), and NCAM-Fc
(negative control) 153 kDa (lane
5).
In order to
assess the effect of the C-terminal domains of ICAM-3 on the
interaction of LFA-1, the set of domain deletion mutants was used in a
binding assay with LFA-1
Figure 3:
The
first two amino-terminal domains of ICAM-3 (D1-2) contain the
LFA-1-binding site. LFA-1
Figure 4:
Sequence alignment of ICAM-1 and ICAM-3.
Sequence alignment of the first two domains of ICAM-1 and ICAM-3. The
predicted location of the
Figure 5:
Effect of ICAM-3 mutations on LFA-1
binding. COS cells transiently expressing LFA-1 (15% COS cells
LFA-1
COS cells expressing LFA-1 bound to the domain 2 mutants (Fig. 5). Interaction of LFA-1 with P158A and E143A was not
affected whereas binding to D166A, R127A, and R127WE/AAA was perturbed.
This indicates that domain 2 of ICAM-3 may have a role in the
interaction with ligand LFA-1. In order to distinguish the most
important binding area, LFA-1 expressed in COS cells was fully
activated with a combination of PMA and anti-
Figure 6:
Effect of ICAM-3 mutations on binding of
fully activated LFA-1. COS cells transiently expressing LFA-1 (25% COS
cells LFA-1
To define relationships between the epitopes of the mAbs,
competitive inhibition assays were performed (data not shown). CH3.1,
CH3.2, and CH3.3 recognize overlapping regions of ICAM-3 that are
distinct from the epitopes of BY44 and CG106. In addition, CAL3.10,
CAL3.38, and CAL3.41 are defined within a separate cluster. Mabs BY44,
CG106, CAL3.10, CAL3.38, and CAL3.41 are also functionally distinct
since they block the interaction of COS LFA-1 with ICAM-3(D1-D5)-Fc
(Bossy et al., 1994). All of the domain 1 mAbs recognize the
domain 1 mutants indicating the overall conformation of this Ig domain
to be preserved (Table 1). As the blocking mAbs BY44, CG106,
CAL3.10, CAL3.38, and CAL3.41 bind to all of the mutant proteins, this
suggests that there may be further regions to be discovered that are
important for the interaction of ICAM-3 with LFA-1.
KS128 bound to
the mouse-human chimeric protein, although binding was 50% of that to
ICAM-3(D1-D5)-Fc. This suggests that domain 2 and domain 1 of ICAM-3
contribute to the epitope of KS128. In addition, when screened on the
mutant panel, KS128 bound poorly to both the domain 2 mutants R127A,
R127WE/AAA, and D166A and the domain 1 mutants P12A and E43A. Thus the
epitope for KS128 appears to span both N-terminal domains of ICAM-3 and
this is consistent with another report (Klickstein et al.,
1993). Three further mAbs, CAL3.1, CAL3.4, and CAL3.16, also appear to
be influenced by domain 1 and domain 2. Like KS128, the binding of
these mAbs is decreased by mutations in both domain 1 and domain 2. An
explanation for this is that Pro The mAbs
CAL3.1, CAL3.4, CAL3.16, and KS128 do not recognize all of the domain 2
mutants (Table 1). Since binding of each domain 1/domain 2 mAb is
perturbed to R127A, R127WE/AAA, and D166A, it must be considered that
these mutations are destabilizing. In contrast, the majority of the
domain 1/domain 2 mAbs recognize E143A and P158A suggesting that these
mutants maintain structural integrity. In addition, each domain 2
mutant is recognized by all of the domain 1 mAbs, and this confirms the
conformation of the first domain in each case.
Figure 7:
Model
for domains 1 and 2 of ICAM-3. The
The location of the 14 mutations is
indicated on the molecular model (Fig. 7). The residues critical
for the association of ICAM-3 with LFA-1 (Glu There are seven potential N-linked glycosylation sites in
the two N-terminal domains of ICAM-3, and their position is relevant
when considering the LFA-1-binding site. In a previously solved x-ray
crystal structure of an Fc antibody fragment, an N-linked
oligosaccharide covered hydrophobic residues on one face of the Ig fold
(Diesenhofer, 1981). The oligosaccharide from this Fc fragment was
clipped and placed on each of the potential glycosylation sites on the
molecular model of ICAM-3. Freely rotatable bonds of the
Asn-oligosaccharide complexes were then adjusted to remove steric
clashes and to cover exposed hydrophobic residues. The general
conclusion drawn from this exercise is that the CC`FG face on the first
domain is relatively free from oligosaccharide cover, and the
oligosaccharides at positions four and five impinge only on the edges
of this region (Fig. 7). To study the binding site for LFA-1 on ICAM-3, we measured
the adhesion of COS cells expressing LFA-1 to a series of mutant ICAM-3
proteins. This model system had two significant benefits. First, by
using COS cells to present LFA-1, we avoided the influence of
interleukocyte LFA-1/ICAM binding events that may affect the binding
avidity of LFA-1 on leukocytes (Cabanas and Hogg, 1993). Therefore, COS
cells expressing LFA-1 permitted a direct analysis of ICAM-3 binding.
Second, all adhesion assays were normalized for the amount of wild-type
or mutant protein presented to the LFA-1 Here we show that LFA-1 expressed by a
non-leukocytic cell type, COS cells, can exist in a variety of
activation states with different affinities for ligand. The avidity
state of COS cell LFA-1 can be regulated both by
``outside-in'' signals (mediated by mAb KIM185) and
``inside-out'' signals (mediated by the protein kinase C
agonist, PMA). In a separate study, COS cell LFA-1 was activated with
anti-CD11a mAb MEM83 and binding to ICAM-1 was enhanced (Landis et
al., 1994). This ability to regulate COS cell LFA-1, differs from
a previous report, in which LFA-1 expressed in COS cells was shown to
be constitutively active (Larson et al., 1990). This
discrepancy may reflect differences in the level of expression of LFA-1
on the COS cells. Larson et al.(1990) achieved a higher
transfection efficiency (50% of LFA-1 Domains 1 and 2 of ICAM-3 are necessary and sufficient
for interaction with LFA-1. There are no protein recognition sequences
for LFA-1 within domains 3-5. These domains may, however, have a
role in presentation of the binding site to LFA-1 when ICAM-3 is
expressed at the cell surface in the context of the cell glycocalyx.
This appears to be true for the interaction of ICAM-1 with its receptor
on Plasmodium falciparum infected erythrocytes (Berendt et
al., 1992). The ICAM-1-binding site for malaria-infected
erythrocytes maps to the first two domains, yet adhesion to a shortened
version of ICAM-1 containing only these domains is dramatically reduced
presumably due to steric hindrance. In the present work, domain-deleted
and wild-type chimeric proteins were presented equally to LFA-1 via
anti-Fc antibodies so eliminating the issue of binding site
accessibility. Having established the importance of domains 1 and 2
of ICAM-3 for interaction with LFA-1, site-directed mutants were
generated to dissect the binding site. Mutations were targeted onto the
CC`FG face and ABE face of the first domain of ICAM-3. The CC`FG face
of ICAM-3 was of particular interest since the equivalent region of
ICAM-1 contributes to LFA-1 binding (Berendt et al., 1992). We
show that glutamine at position 37 of ICAM-3 is essential for LFA-1
binding. ICAM-3 E37 is equivalent to ICAM-1 Glu A second
key residue for the ICAM-3/LFA-1 interaction is Gln The fact that ICAM-3 binding was eliminated by mutations at
Leu The ICAM-3 D1 ABE face mutations had
less dramatic effects than most of the CC`FG face substitutions. The
mutant D27A bound ligand to the same extent as wild-type ICAM-3, and
this residue is equivalent to ICAM-1 S24 which also is not involved in
LFA-1 binding (Staunton et al., 1990). The two other ABE face
mutations, Pro Alignment of ICAM-1 with ICAM-3 reveals
that the highest degree of sequence homology occurs in the second
domain (77% amino acid identity), suggesting functional importance for
this region of the ICAMs. Because of lack of reagents (mAbs and
mutants), only limited analysis of ICAM-1 domain 2 has been reported.
To date, only the F-G loop of ICAM-1 D2 polypeptide is known to
contribute to LFA-1 binding (Berendt et al., 1992; Staunton et al., 1992). To determine the extent of interaction of LFA-1
with D2 of ICAM-3, domain 1 proximal and distal mutations were made.
Mutation of Arg LFA-1 interacts with key residues on the CC`FG
face of domain 1. For members of the immunoglobulin superfamily, the
faces of the Amino acids Glu In conclusion, we have identified the CC`FG face of
ICAM-3 to be an important binding region for LFA-1. Residues critical
for interaction with LFA-1 are present on this face of domain 1. Some
of these essential residues may form the basis of a common motif for
all IgSF-integrin interactions.
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 877-884
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Leu
, Ser
, and
Gln
), that are predicted to lie on the CC`FG face of the
Ig fold, play a dominant role in LFA-1 binding.
)play a key role in
stabilizing and strengthening cell-matrix and cell-cell interactions.
Leukocyte CAMs provide antigen-nonspecific recognition and are involved
in a wide range of intercellular interactions, including those between
helper T cells and antigen presenting cells, cytotoxic T cells, and
natural killer cells and their targets (Springer 1990). In addition to
these interleukocyte activities, CAMs are also involved in mediating
leukocyte adhesion to and transmigration across vascular endothelium. 
,), and ICAM-1 has been shown to bind
an additional integrin Mac-1 (CD11b/CD18;
,) (Diamond et al., 1991).
While the integrins are only expressed on hematopoietic cells, the
ICAMs have very different expression profiles. ICAM-1 is expressed at
low levels under normal conditions and is strongly up-regulated by
cytokines on many cell types, including all leukocytes, endothelial
cells, keratinocytes, and fibroblasts. ICAM-2 is constitutively
expressed by all leukocytes, and endothelial cells, but at low levels.
ICAM-3 is constitutively highly expressed by leukocytes and appears to
be inducible on vascular endothelium in certain disease states,
especially in lymphomas and myelomas (Doussis-Anagnostopoulou et
al., 1993). flux and association with tyrosine kinases (Juan et al.,
1994). A consequence of signaling through ICAM-3 is increased cell
adhesion via integrin
1 and 2 pathways (Campanero et
al., 1993; Cid et al., 1994). Thus, ICAM-3 appears to be
the major ligand for LFA-1 in the primary or initiating phases of
immune responses (Hernandez-Caselles et al., 1993) and plays a
role in cell adhesion and signal transduction. and Gln
, form a key part of that site
(Staunton et al., 1990). Domain 1 is an Ig C2 fold, consisting
of seven
strands arranged on two surfaces, an ABE face and a
CC`FG face. A model of the structure of the domains 1 and 2 of ICAM-1
has been produced which places these key residues on the CC`FG face;
Glu is in the C-C` loop, and Gln
is placed in
the F to G loop (Berendt et al., 1992). These residues are
conserved in ICAM-3 (Fawcett et al., 1992; Vazeux et al., 1992; de Fougerolles et al., 1993). We have previously
shown that the N-terminal two Ig domains of ICAM-3 are competent to
bind LFA-1 (Fawcett et al., 1992) and now report a detailed
analysis of the LFA-1-binding site in ICAM-3. In addition to those
residues predicted to be critical for interaction with LFA-1, we
identify 2 amino acids on the F strand of D1 that are also essential
components of the LFA-1-binding site. Based on this mutagenesis screen,
we propose a model for domains 1 and 2 of ICAM-3 and discuss the
critical area for interaction with ligand LFA-1.
Monoclonal Antibodies and Cell
Culture
Anti-LFA-1 subunit monoclonal mAb 38 has been
described (Dransfield et al., 1989). Anti ICAM-3 mAbs CH3.1,
CH3.2, and CH3.3 were obtained by the fusion of spleen cells from a
BALB/c mouse immunized with ICAM-3(D1-D2)-Fc. Anti-ICAM-3 mAbs CAL3.1,
CAL3.4, CAL3.10, CAL3.16, CAL3.38, and CAL3.41 were obtained by the
fusion of spleen cells from a BALB/c mouse immunized with
ICAM-3(D1-D5)-Fc (Bossy et al., 1994). Other mAbs were
provided as follows: anti-CD11a MHM24, anti-CD18 MHM23, and anti-ICAM-3
mAbs KS128, BY44, and CG106 by Dr. D. Mason (Nuffield Department of
Pathology, John Radcliffe Hospital, Oxford) and anti-CD18 mAb KIM185 by
Dr. M. Robinson (Celltech, Slough, United Kingdom). In adhesion assays,
purified mAbs KIM185 and 38 were used at 5-10 µg/ml and other
mAbs as 1/2 dilution of tissue culture supernatant final concentration. Fc-chimeras and Mutants
ICAM-1(D1-D2)-Fc and
ICAM-3(D1-D2)-Fc consisting of the first two extracellular domains
fused to human IgG1-Fc fragment have previously been described (Fawcett et al., 1992). ICAM-3(D1-D3)-Fc, ICAM-3(D1-D4)-Fc, and
ICAM-3(D1-D5)-Fc chimeras were generated by polymerase chain reaction
(PCR) amplification of ICAM-3 cDNA and cloning into Fc expression
vector pIG1 (Fawcett et al., 1992; Simmons, 1993). Primers
used for PCR amplification were CDM8 forward sequencing primer
5`-CTAGAGAACCCACTGCTTAAC-3` and 5`-AC AGATCT ACTTACCTGT GAA GCT AAA GAC
CGT CAA GTT-3` for ICAM-3(D1-D3)-Fc; CDM8 forward sequencing primer and
5`-AC AGATCT ACTTACCTGT TTT CCA TTT CAA GTG CTG GGG-3` for
ICAM-3(D1-D4)-Fc; CDM8 forward sequencing primer and 5`-AC AGATCT
ACTTACCTGT GTG GGA GCT CCC AGC CTC AAT-3` for ICAM-3(D1-D5)-Fc. The
mouse-human chimera was made by PCR ligation of domain 1 of mouse
ICAM-1 to human ICAM-3 domains 2-5 as an Fc chimera. Plasmid
encoding ICAM-1(D1-D5)-Fc was a gift from Dr. A. Craig, Institute of
Molecular Medicine, Oxford. The NCAM-Fc consisted of the entire
extracellular domain (five Ig related domains and two type III
fibronectin-related domains) of neural cell adhesion molecule fused to
human IgG1-Fc fragment. Mutant forms of ICAM-3(D1-D2)Fc were made by a
two-step PCR strategy (Higuchi, 1990). A list of mutagenic primers can
be provided on request. All constructs and mutants were sequenced in
their entirety.Transfection of COS Cells with LFA-1 and Cytofluorometric
Analysis
CD11a and CD18 cDNAs cloned into pcDNA/AMP (Invitrogen)
were transiently expressed in COS cells. The cDNAs (at 5 µg/ml
each) were transfected in the presence of 1 mg/ml DEAE-dextran and 100
µM chloroquine for 2-4 h at 37 °C followed by a
2-min shock in PBS, 10% MeSO. For cytofluorometric
analysis, COS transfectants were lifted 72 h post-transfection in PBS,
2 mM EDTA, washed in PBS, 2 mM EDTA, 2 mg/ml bovine
serum albumin at 4 °C, stained with primary mAbs (1:10 dilution of
tissue culture supernatants of anti-CD11a mAb MHM24 or anti-CD18 mAb
MHM23), washed, stained with fluorescein isothiocyanate-conjugated goat
anti-mouse IgG, washed three times, fixed in PBS, 2% formaldehyde, and
analyzed on a Becton Dickinson FACScan. COS cell transfected with LFA-1
were routinely 15-40% positive by FACScan analysis.Adhesion Assays
Immulon-3 96-well plates (Dynatech
Laboratories Inc., Chantilly, VA) were precoated with 1 µg/well
goat-anti-human-Fc Ig (Sigma, Poole, Dorset U.K.) overnight at 4
°C, blocked with PBS, 0.4% bovine serum albumin (Fraction V, Sigma)
for 2 h at room temperature, and then coated with chimeric proteins in
PBS for at least 2 h at room temperature. Prior to adhesion assay at
72-h post-transfection, COS cells were labeled for 12-24 h with 
[H]thymidine at 10 µCi/2 
10
cells and harvested prior to the assay with PBS, 2 mM EDTA. Cells were washed twice in assay medium, consisting of RPMI,
2.5% fetal calf serum. Stimulation with phorbol 12-myristate 13-acetate
(PMA, Sigma) was performed by addition of PMA to 50 ng/ml (80
nM), incubation for 30 min at 37 °C, and then removal by
washing in assay buffer. Cells were added to ICAM-Fc plates to give 3
10
/well in a final volume of 50 µl. Cells were
preincubated on ice for 20 min in the presence of antibodies as
appropriate, followed by a further 30-min incubation at 37 °C.
Cells that remained bound after two washes with prewarmed RPMI were
lysed in 1% SDS, scintillant added (Ready Safe, Beckman), and
incorporated radioactivity counted using a Beckman LS 5000 CE counter.
The percent of LFA-1-positive cells was calculated from the
incorporated radioactivity by taking into account the total cell input
and the transfection efficiency.Enzyme-linked Immunosorbent Assay of ICAM-3 Fc
Proteins
Immulon-3 96-well plates were precoated with 1
µg/well goat-anti human-Fc Ig overnight at 4 °C, blocked with
PBS, 0.4% bovine serum albumin (Fraction V, Sigma) for 2 h at room
temperature, and then coated with chimeric proteins (5 µg/ml) in
PBS for at least 2 h at room temperature. Antibody was added in
saturating amounts as either neat supernatant or appropriately diluted
ascitic fluid, followed by peroxidase-conjugated goat anti-mouse Ig
(1:500 dilution, Dako, Glostrup, Denmark). Each layer was incubated for
30 min at room temperature and followed by six washes. The assay was
visualized with O-phenylenediamine dihydrochloride (Sigma) and
absorbance read at 450 nm.
Regulation of LFA-1 Activation State in
Nonhematopoietic Cells
In order to dissect the interaction of
ICAM-3 with the leukocyte integrin LFA-1, the experimental system
involved quantitative binding assays of LFA-1 cells to
soluble recombinant fusion proteins. Previous work has established that
LFA-1
leukocytes bind to an ICAM-3-Fc chimeric protein
containing only the N-terminal two domains of ICAM-3 (Fawcett et
al., 1992). Recent work has shown that ligand itself may influence
the binding avidity of T cell LFA-1 (Cabanas and Hogg, 1993). As
leukocytes express LFA-1, ICAM-1, and ICAM-3, potential problems might
occur when trying to dissect the nature of LFA-1 binding to ICAM-3, due
to the influence of intercellular LFA-1/ICAM-1 or ICAM-3 interaction.
In order to avoid these possible complications, COS cells were used as
the recipient cells for LFA-1. DEAE-dextran transfection of COS cells
with pCD18 and pCD11a results in transient expression of LFA-1 on
15-40% of cells as detected by flow cytometry with mAbs MHM24
(CD11a) or MHM23 (CD18). In contrast, no surface expression of CD11a or
CD18 was detected when either pCD11a or pCD18 were transfected alone
(data not shown).
COS were allowed to adhere to soluble
recombinant fusion proteins containing the two N-terminal domains of
ICAM-1 or ICAM-3 (Fawcett et al., 1992). The activation state
of COS cell LFA-1 could be regulated both by the protein kinase C
agonist PMA and by an ``activating'' anti-LFA-1 monoclonal
antibody (Fig. 1). The anti-CD18 mAb KIM185 is known to induce a
change in the conformation of CD18 and promote LFA-1-dependent adhesion
(Andrew et al., 1993). Adhesion of LFA-1
COS
to ICAM-1 and ICAM-3 is also enhanced by the addition of KIM185 (Fig. 1).
) were allowed to adhere to ICAM-1(D1-D2)-Fc or
ICAM-3(D1-D2)-Fc. Where indicated, cell binding occurred in the
presence of monoclonal antibodies: mAb38 is an anti-CD11a mAb which
blocks LFA-1/ICAM interactions and KIM185 is an anti-CD18 mAb which
induces LFA-1-dependent adhesion. Where indicated, COS cells were
treated with 80 nM PMA. Results are expressed as means
± 1 S.D. unit (n =
5).
ICAM-3 Domains 1 and 2 Are Necessary and Sufficient for
LFA-1 Binding
Previous work has established that ICAM-3 domains
1 and 2 contain the LFA-1-binding site (Fawcett et al., 1992).
However, the possibility that there might be additional contributions
to this binding from domains 3, 4, and 5 has not been addressed. To
investigate this a series of ICAM-3-Fc chimeric proteins, comprising a
nested set of ICAM-3 domain deletion mutants, was generated (Fig. 2). Fusion protein ICAM-3(D1-D5)-Fc is the wild-type
molecule containing all five immunoglobulin domains whereas proteins
ICAM-3(D1-D4)-Fc, ICAM-3(D1-D3)-Fc, and ICAM-3(D1-D2)-Fc have
sequential, C-terminal, domain deletions. Cell binding assays were
performed with chimeric proteins immobilized onto plastic via anti-Fc
polyclonal antibodies which allowed normalization of the amount of
wild-type and mutant proteins presented to the cells.
COS that had been activated
with PMA. Binding was not significantly increased by the presence of
domains 3-5 of ICAM-3 (Fig. 3). This binding is LFA-1
specific as it is blocked by the anti-CD11a mAb 38.
COS cells were assayed for
binding to the domain deletion series of ICAM-3-Fc chimeric proteins
coated onto plastic via anti-Fc polyclonal antibodies. LFA-1
COS (15% COS cells LFA-1
) were activated with 80
nM PMA. NCAM-Fc is included as a negative control having 5
C2-set IgSF domains. Results are expressed as means ± 1 S.D.
unit (n = 10).
Further Definition of the LFA-1-binding Site on ICAM-3 by
Directed Mutagenesis
Having established that the LFA-1-binding
site is contained within domain 1 and 2, a mutagenesis screen of these
domains was undertaken. We aligned the primary amino acid sequences of
ICAM-1 and ICAM-3, and, based on a molecular model for ICAM-1 (Berendt et al., 1992), predicted which ICAM-3 residues contributed to
the strands of the immunoglobulin structure (Fig. 4). The
key residues for ICAM-1 binding to LFA-1 (Glu and
Gln
) are present on the CC`FG face of domain 1. To
determine the importance of ICAM-3 domain 1 for interaction with LFA-1,
six specific mutations were targeted onto the CC`FG face and three onto
the opposite ABE face. The CC`FG face mutations included substitution
of residues homologous to ICAM-1 Glu
and Gln
.
In addition, five mutations were targeted onto domain 2, particularly
in connecting loops between
strands, to assess its contribution
toward the LFA-1-binding site. We selected residues predicted to be
solvent accessible and exposed at the surface of ICAM-3. For the most
part charged amino acids were substituted with alanine (Cunningham and
Wells, 1989) by site-directed mutagenesis of pICAM-3(D1-D2)-Fc, and the
location of the mutations is indicated in Fig. 4. Mutant
ICAM-3(D1-D2)-Fc fusion proteins produced by expression of
ICAM-3(D1-D2)Fc plasmids in COS cells were checked by
SDS-polyacrylamide gel electrophoresis analysis and found to migrate at
the predicted size of 80 kDa (Bossy et al., 1994).
sheets of the immunoglobulin domains is
indicated below the alignment. Asterisks denote those residues
that were targeted for site-directed mutagenesis. Potential N-linked glycosylation sites are numbered (-1-, etc.)
Effect of ICAM-3 Mutations on Interaction with
LFA-1
To address the effect of the mutations on LFA-1 binding,
adhesion assays were performed onto a concentration range of mutant and
wild-type ICAM-3 protein using LFA-1 COS cells as the
ligand bearing cells (Fig. 5). When compared to an equivalent
concentration of wild-type ICAM-3, no binding was exhibited to mutants
E37A, L66K, S68K, Q75H, and Q75A. These residues all map onto the CC`FG
face of domain 1 and are critical for interaction with LFA-1.
Glu
is also located in this region of ICAM-3, and LFA-1
binding to E43A is reduced to 30-50% of wild-type. In contrast,
substitution of Asp
to alanine does not affect interaction
with LFA-1. Compared with those residues critical for LFA-1 binding,
Asp
is located on the opposite face of domain 1. The
domain 1 mutants F21A and P12A are also located on this ABE face, and
binding of LFA-1 to these proteins is reduced to 50% of wild-type.
) were treated with PMA and then allowed to
adhere to a concentration range (1, 5, 10, and 20 µg/ml) of
ICAM-3(D1-D2)-Fc mutant and wild-type chimeric proteins. The
concentration range was achieved by precoating with 1 µg/well
anti-human-Fc Ig and then coating with chimeric protein at the
indicated concentration. Results are expressed as means ± 1 S.D.
unit (n = 6).
2 activating mAb
KIM185 and allowed to bind to mutant and wild-type ICAM-3 chimeric
proteins (Fig. 6). Under these conditions of enhanced LFA-1
activation, there is still no binding to mutants E37A, L66K, S68K,
Q75H, and Q75A. This emphasizes the critical nature of these residues
for interaction with ligand. In contrast, under these optimal
conditions binding to mutants on the ABE face of domain 1 and within
domain 2 is largely restored and so may be less important for
interaction with LFA-1.
) were treated with PMA plus mAb KIM185.
Cells were allowed to adhere to ICAM-3(D1-D2)-Fc mutant and wild-type
proteins or NCAM-Fc as a negative control. Results are expressed as
means ± 1 S.D. (n =
6).
Effect of ICAM-3 Mutations on Interaction with
Anti-ICAM-3 mAbs
Eight anti-ICAM-3 mAbs were mapped to domain 1
by enzyme-linked immunosorbent assay onto ICAM-3(D1-D2)-Fc and a
mouse-human chimeric protein composed of D1 of mouse ICAM-1 linked to
domains 2-5 of human ICAM-3. MAbs CH3.1, CH3.2, CH3.3, BY44,
CG106, CAL3.10, CAL3.38, and CAL3.41 recognized ICAM-3(D1-D2)-Fc but
not the mouse-human ICAM-1/ICAM-3 chimera, indicating that these mAbs
have epitopes in domain 1 or possibly at the interface between domains
1 and 2.
, Glu
,
Arg
, and Asp
are in close association, a
prediction that is supported by modeling data (see below).
Molecular Model for ICAM-3
In order to map the
relative position of the mutations, a model of the two N-terminal
domains of ICAM-3 was constructed (Fig. 7). The protocol for
replacement of side chains and protein fragments (usually loop regions)
was initiated by a critical comparison of the primary protein sequence
alignment of ICAM-1 and ICAM-3 (Fig. 4) and then followed by
methodologies previously described for the ICAM-1 model (Berendt et
al., 1992). An important feature of the molecular model is the
relative position of the two domains. For both ICAM-1 and ICAM-3, a
close association between the domains is predicted with domain 2
rotated at approximately 160 ° with respect to domain 1. Key
conserved hydrophobic patches at the base of domain 1 and at the top of
domain 2 support this arrangement (data not shown) as does the epitope
mapping in this present study.
strands are labeled in the
normal convention for immunoglobulin folds, A to G, with the two
sheets CC`FG and ABE, indicated by shading differences. Disulfide bonds
are denoted by dark zig-zag lines. The locations of
site-directed mutations are indicated by residue number and side chain,
and potential N-linked glycosylation sites are numbered (1-7) and shown as 
. This diagram was
produced with the aid of the display program MOLSCRIPT (Kraulis,
1991).
,
Leu
, Ser
, and Gln
) are aligned
on the CC`FG face of domain 1. The residue Asp
, within
the F-G loop of domain 2, also contributes to the binding site. The
model shows the CC`FG face of domain 1 and the F-G loop of domain 2 to
be associated along the same side of ICAM-3 and this supports the
participation of these two regions in the binding site for LFA-1.
/COS cells.
All ICAM-3 site-directed mutants were produced as recombinant D1-D2-Fc
fusion proteins, and this allowed even small differences in the binding
properties between the mutants to be quantitatively assessed. This
system overcomes the problems of expressing equivalent amounts of
mutant proteins in COS cells and eliminates the possibility of
accessory cell-cell interactions which may contribute to background
binding in a two-cell system.
cells in the COS
cell population) and higher expression of LFA-1/COS cell. These factors
might affect avidity by altering the distribution of LFA-1 on the COS
cell surface.
which is
also critical for association with LFA-1 ligand (Staunton et al., 1990). To extend the study of ICAM-3 E37, we made substitutions of
the amino acids to which Glu
is predicted to hydrogen
bond. Modeling data associates Glu
in an interaction triad
with Leu
and Ser
. By generating mutants L66K
and S68K, binding of ICAM-3 to LFA-1 was prevented, presumably by
disrupting the inter-residue contacts within this triad.
. The
homologous residue is Gln
in ICAM-1 and is essential for
LFA-1 binding. Staunton et al. (1990) found that mutating
ICAM-1 Gln
to histidine decreased LFA-1 binding 10-fold,
and substitution to threonine decreases binding 2-fold. In contrast, we
find that mutation of ICAM-3 Q75 to either a histidine or an alanine
eliminates LFA-1 binding. Therefore, ICAM-3 residue Gln
distinguishes the binding site for LFA-1 on ICAM-3 from that on
ICAM-1.
and Ser
on the F strand in the middle of
the CC`FG face indicates that those residues important for interaction
with LFA-1 are aligned along one face of the molecule. This region of
ICAM-3 is predicted to be free from oligosaccharide cover and therefore
accessible for ligand binding. Indeed, a recent study (Landis et
al., 1994) shows that the glycosylation of ICAM-3 domain 1 does
not have a role in ligand binding. Although our assignment of
oligosaccharide chains to the periphery of the CC`FG face is
speculative, a recent study of the glycan structures of human CD2
suggests a similar situation with regard to the ligand-binding site
(Withka et al., 1993). The three-dimensional structure of the
fully glycosylated form of domain 1 of human CD2, determined by nuclear
magnetic resonance spectroscopy, places the oligosaccharide at the top
of the connecting loops between the
strands, at the perimeter of
the CD58 ligand-binding site. and Phe
, did perturb ligand
binding. This raises the possibility that there are regions of ICAM-3
D1, in addition to the CC`FG face, that have a role in ligand binding.
Residue Pro
is located on the A strand of ICAM-3 D1.
Interestingly, mutation of the A strand of ICAM-1 D1 does not affect
binding to LFA-1 (Staunton et al., 1990). Since LFA-1
binds selectively to ICAM-1 and ICAM-3 (Landis et al., 1994),
the binding interfaces are likely to be distinctive. For both ICAM-1
and ICAM-3, the CC`FG face of domain 1 appears to have a dominant role
in interaction with ligand, but the binding sites may be defined by
other areas within domain 1.
and Asp
reduced binding to
LFA-1 but may also destabilize the Ig structure. Thus, the second
domain of ICAM-3 may contribute to the LFA-1-binding site, but the
extent of the interaction face has yet to be determined. There may be
regions within domain 2 that are critical for LFA-1 binding, or it is
possible that this domain has only a supportive role in association
with ligand LFA-1.
strands of the Ig-related domains may be as
important for ligand interaction as the connecting loop regions.
Indeed, the binding site for LFA-3 (CD58) is located along one face (in
fact the CC`FG face) of CD2 (Somoza et al., 1993; Arulanandam et al., 1993). A molecular model of the first two domains of
ICAM-3 indicates that the domains are closely associated, with the
second domain rotated at approximately 160 °. Although molecular
modeling cannot predict domain packing with certainty, solved
structures for CD4 and CD2 support this orientation (Wang et al., 1990; Ryu et al., 1990: Jones et al., 1992;
Brady et al., 1992). Thus, the LFA-1 binding surface on ICAM-3
encompasses the CC`FG face of domain 1 and may extend into domain 2.
Although it is possible that, within domains 1 and 2, there are other
important binding areas to be defined. and Gln
within ICAM-3 domain 1 may represent an
essential, common motif in the ligand-binding site of all ICAMs.
Homologous residues are found in all members of the ICAM family, ICAM-1
(Simmons et al., 1988; Staunton et al., 1988),
ICAM-2 (Staunton et al., 1989), and ICAM-3 (Fawcett et
al., 1992; Vazeux et al., 1992; de Fougerolles et
al., 1993), and are critical for binding of LFA-1 to ICAM-1 and
ICAM-3. These key residues are also conserved across species and are
present in chimpanzee ICAM-1 (Hammond and McClelland 1993), rat ICAM-1
(Kita et al., 1992), mouse ICAM-1 (Horley et al., 1989), and mouse ICAM-2 (Xu et al., 1992). This motif may
be generally important for the interaction of integrins with members of
the immunoglobulin superfamily (IgSF). To date, there are three
examples of IgSF/integrin interactions: ICAMs with LFA-1 and/or Mac-1
(Simmons et al., 1988; Staunton et al., 1989; Diamond et al., 1990), VCAM with VLA-4 (
41) and 47
integrins (Elices et al., 1990; Ruegg et al., 1992),
and MadCAM-1 with the 47 integrin (Berlin et al., 1993). Acidic residues at equivalent locations on VCAM-1 domains 1
and 4 (i.e. within the C-C` region) are also important for
binding to its cognate integrin VLA-4 (41) (Osborn et
al., 1994; Vonderheide et al., 1994; Renz et
al., 1994).
)
We thank Martyn Robinson (Celltech) for the generous
gift of mAb KIM185, Alister Craig (Institute of Molecular Medicine,
Oxford) for ICAM-1(D1-D5)-Fc; Paul Crocker (IMM, Oxford) for NCAM-Fc
protein; David Mason (Nuffield Department of Pathology) for gifts of
ICAM-3 and CD11a/CD18 mAbs; Michael J. E. Sternberg, Alister Craig,
Andy Gearing, and John Clements (British Bio-technology, Oxford) for
discussions; and Paul Crocker and Anthony Berendt for comments on the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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