From the Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
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ABSTRACT |
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Using a solid phase assay, we show that isolated LFA-1 I domain binds ICAM-1 in a Mg2+-dependent manner and is blocked by anti-I domain monoclonal antibodies. This activity mirrors that of the intact receptor (Dransfield, I., Cabañas, C., Craig, A., and Hogg, N. (1992) J. Cell Biol. 116, 219-226) and suggests that the I domain controls divalent cation-dependent receptor function. In ICAM-1, domain 1 residues Glu-34 and Gln-73 have been identified as critical for binding of LFA-1 as an intact receptor (Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254). For the first time, we show that isolated I domain binds to domain 1 of ICAM-1 and that this interaction is inhibited partially by mutation of Glu-34 but not by Gln-73. The anti-ICAM-1 monoclonal antibody RR1/1, which maps to Gln-73 (Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254), enhances I domain binding, suggesting potential allosteric control or coordinate binding by this region. Finally, I domain binding inhibited by Glu-34 ICAM-1 mutation correlates with divalent cation dependence, indicating that this residue might be in direct contact with the metal ion-dependent adhesion site. Thus, we describe the interaction between the LFA-1 I domain and ICAM-1, an event that controls the function of the intact receptor but includes only part of the complete ligand binding site.
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INTRODUCTION |
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Cell-cell interactions have an essential role in maintaining an
immune response and are a requirement for effective functioning of
leukocytes (3, 4). For example, adhesion of leukocytes to endothelium
is critical for their transmigration from the circulation to sites of
inflammation, and firm contact with antigen-presenting cells is
required for T lymphocyte stimulation and proliferation. The leukocyte
integrin LFA-11
(L
2; CD11a/CD18) is an important receptor
for these adhesive interactions and has three major ligands: ICAM-1,
ICAM-2, and ICAM-3 (CD54, CD102, CD50), which are members of the
immunoglobulin superfamily (IgSF). ICAM-1 is the principle ligand for
LFA-1 in many of its interactions. Mutagenesis studies of ICAM-1 have
shown the residues Glu-34 and Gln-73 in domain 1 to be essential for binding of purified LFA-1 to ICAM-1 (2, 5, 6). The importance of these
residues in adhesion has also been established for other IgSF adhesion
ligands, where residues in homologous positions are both conserved and
essential for integrin function; for example, ICAM-3 contains Gln-75
and Glu-37 (7-9), and VCAM-1, the ligand for
4
1, contains Asp-40 (10-12). However, it
has not been shown which region of integrin interacts with these
residues.
Eight integrins have an I domain in the subunit to which ligand
binding sites have been mapped (13). In LFA-1, the I domain contains a
major ICAM-1 binding site (14). The I domains of the two
2 integrins LFA-1 and Mac-1 have recently been
crystallized and an important part of the structure shown to be a
Mg2+/Mn2+ binding site termed the MIDAS motif
(15-17). Since divalent cations are required by integrins for activity
this suggests an important role for the I domain in activation as well
as ligand binding. Other regions of LFA-1 that have been implicated in
binding to ICAM-1 and also potentially bind to divalent cation are
domains V/VI of the
subunit (18) and the I domain-like region of
the
2 subunit (19). Whether these different domains form
one large ligand binding pocket or bind ICAM-1 separately remains to be determined (13). In this study, we use an isolated LFA-1 I domain to
locate residues of ICAM-1 that interact with this domain.
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EXPERIMENTAL PROCEDURES |
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Monoclonal Antibodies-- The following monoclonal antibodies (mAbs) were used in this study. Anti-LFA-1 (CD11a) mAbs mapping to the I domain (14) were mAb 38 (this laboratory), MEM 25, MEM 30, MEM 83 (gifts from Dr. V. Horesji, Institute of Molecular Genetics, Prague), and F110.22 (gift from Dr. T. Plesner, Herlev Hospital, Denmark). Anti-ICAM-1 (CD54) mAbs were 15.2 (this laboratory), RR1/1 (gift from Dr. R. Rothlein, Boehringer Ingleheim, Inc., Ridgefield), 8.4A6 (gift from Dr. D. Haskard, RPMS, Hammersmith Hospital, London) and WEHI-CAM-1 (gift from Dr. A. Boyd, Queensland Institute for Medical Research, Australia). The anti-CD64 mAb 10.1 (this laboratory) was used most frequently as a control.
Synthesis of ICAM-1Fc Proteins-- Chimeric wild type (wt) ICAM-1Fc protein consists of the five extracellular domains of ICAM-1 plus the hinge, CH2, and CH3 domains of IgG1 (20). The cDNA construct was ligated into the vector pEE14 and stably transfected into CHO-K1 cells by calcium phosphate precipitation (21). Protein was isolated from high expressing transfectants after 10 days in serum-free medium. ICAM-1Fc mutants E34A and Q73N were made by polymerase chain reaction using site-directed mutagenesis by standard protocols and were fully sequenced by dideoxy sequencing. Proteins were synthesized by electroporated COS cells and collected in serum-free medium for up to 4 days. All proteins were purified on a Protein A-Sepharose column and eluted with 0.1 M citrate, pH 3.0, using standard methods.
The VCAM-1Fc chimeric protein contains VCAM-1 domains 1 and 2 and the same Fc domain as ICAM-1Fc (22) (a kind gift from Dr. R. Lobb, Biogen). Both ICAM-1Fc and VCAM-1Fc were equally able to bind stimulated T cells at equivalent molarity.2Synthesis of I-GST Protein--
The LFA-1 I domain (Leu-111 to
Ser-327) including the predicted sequence and a 17-amino acid
N-terminal extension, was made by polymerase chain reaction from an
LFA-1 subunit cDNA by a standard protocol and ligated into the
vector pGEX2T (Pharmacia Biotech) to produce an I-GST coding sequence.
The sequenced DNA was transformed into Escherichia coli Topp
2 (Stratagene) and grown to log phase before inducing with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside for
4 h. I-GST was extracted from bacterial pellets using the GST
purification module kit (Pharmacia) essentially following the
manufacturer's instructions. Isolated I domain was made by cleaving
directly from glutathione-Sepharose with thrombin. The I-GST and I
domain proteins ran as single bands of ~50 and 25 kDa,
respectively, on SDS-polyacrylamide gel electrophoresis. The proteins
were tested by ELISA with 10 I domain mAbs (14) and shown to express
all epitopes. The I-GST protein was stable for 4-6 weeks, but the
cleaved I domain frequently lost activity in less than 1 week.
Solid Phase ELISA Assay-- The wells of Maxisorp 96-well plates (Nunc) were coated for 16 h with 500-750 ng/well ICAM-1Fc in phosphate-buffered saline at 4 °C, then blocked with 3% BSA (Calbiochem) in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS) for 1 h at room temperature.
The wells were washed three times with TBS, and 50 µl/well I-GST diluted in TBS, 1 mM MgCl2, 1 mg/ml BSA was added and incubated at 37 °C for 3 h. Bound I-GST and GST were detected with 50 µl/well rabbit anti-GST antibody at 5 µg/ml (Sigma) in TBS, 1 mM MgCl2, 0.5 mg/ml BSA for 30 min at room temperature followed by 50 µl of peroxidase-conjugated goat anti-rabbit IgG (Dako) diluted 1:1000 in TBS, 1 mM MgCl2, 0.5 mg/ml BSA added for 30 min at room temperature. The wells were washed three times with TBS between each layer. Bound I-GST was detected with 75 µl/well 0.5 mg/ml o-phenylenediamine dihydrochloride (Sigma, made up as recommended), added for 10 min, stopped by addition of 50 µl of 3 M H2SO4 and plates read in an ELISA reader at 492 nm. In experiments where isolated I domain was used, it was detected using 5 µg/ml mAb F110.22 followed by a 1:1000 dilution of peroxidase-conjugated goat anti-mouse IgG (Dako). When divalent cations or blocking antibodies were added, these and the I-GST were made up to 2-fold concentrations and 30 µl of each added to the wells. I-GST or GST was always added last. Comparison of binding between VCAM-1Fc and ICAM-1Fc or I-GST and GST used equimolar concentrations as determined by protein estimation and SDS-polyacrylamide gel electrophoresis.T Cell Binding to ICAM-1Fc-- These assays were carried out as described previously (1). Briefly, 5 × 107 T cells were labeled with 25 µCi of [3H]thymidine for 16 h at 37 °C, washed three times in HEPES buffer, and 2 × 105 cells/well in HEPES buffer containing 2 mM MgCl2 and 1 mM EGTA were added to 96-well plates coated with ICAM-1Fc. Blocking control mAbs were added at 10 µg/ml. Plates were spun down and incubated at 37 °C for 35 min. Wells were washed three times, harvested, and read in a Betaplate counter (Pharmacia).
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RESULTS |
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ICAM-1 Mutants E34A and Q73N Are Not Recognized by T Cell LFA-1-- The location of the LFA-1 I domain binding site on ICAM-1 has not been identified, but residues Glu-34 and Gln-73 in domain 1 of ICAM-1 have been demonstrated to be critical for binding of the intact integrin LFA-1 (2, 5, 6). Previous studies have showed the sensitivity of LFA-1 to these mutated ICAM-1 proteins expressed as cell transfectants. Here, we confirm this finding by mutating these residues in an ICAM-1Fc chimeric protein and showing that neither mutant E34A nor Q73N ICAM-1Fc immobilized on plastic support any T cell adhesion compared with wt ICAM-1Fc (Fig. 1). Nine mAbs specific for epitopes in ICAM-1 domains 1 and 2 (20) bound wt ICAM-1Fc and the two mutants to an equivalent extent, showing that the mutant proteins are correctly folded (data not shown). The epitope recognized by mAb RR1/1 is eliminated by Q73N mutation as shown previously (2).
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Characterization of Isolated LFA-1 I Domain Binding to ICAM-1 Using an ELISA Assay-- To investigate the specificity of LFA-1 I domain binding to ligand, we tested the ability of isolated recombinant I domain to bind to ICAM-1Fc in a solid phase assay. The specificity of adhesion is demonstrated by the ability of LFA-1 I domain, as a GST fusion or cleaved protein, to bind immobilized ICAM-1Fc in a saturable fashion compared with a lack of binding to either BSA (Fig. 2A) or a second IgSF, protein VCAM-1Fc (Fig. 2B). The GST fusion partner protein expressed alone did not interact with ICAM-1Fc or VCAM-1Fc (Fig. 2B), showing that adhesion to ICAM-1Fc is mediated solely by the I domain in the fusion protein.
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Identifying the ICAM-1 Binding Site for LFA-1 I Domain-- To locate the I domain binding site on ICAM-1, we first tested a panel of anti-ICAM-1 (CD54) mAbs that interfere with LFA-1 binding and map to distinct epitopes on the CFG face of domain 1 and to the top of domain 2 (20). Four of the five mAbs tested had no effect on I domain/ICAM-1Fc binding compared with control isotype-matched mAbs (Fig. 4B), which suggests that the I domain does not bind to ICAM-1 domain 1 at the sites recognized by them. The fifth mAb, RR1/1, which maps to the Gln-73 sequence on the G strand at the top of domain 1 (2), enhanced binding of I domain to ICAM-1Fc (Fig. 4B). This was unexpected, as mAb RR1/1 is a good blocker of adhesion between ICAM-1 and cell surface LFA-1. A capture ELISA assay showed that there is no direct binding of I-GST to any ICAM-1 mAb tested (data not shown).
As a further approach to identifying the I domain binding site on ICAM-1, the two ICAM-1Fc mutants E34A and Q73N were tested in the ELISA assay. The E34A mutant substantially blocked I domain binding (Fig. 5), showing for the first time that the I domain interacts with domain 1 of ICAM-1 and involves residue Glu-34. Unlike the E34A mutant, binding of I domain to the Q73N mutant resembled wt ICAM-1Fc (Fig. 5). This result, together with the enhanced binding shown with mAb RR1/1 (Fig. 4B), which maps to Gln-73 on the G strand, suggests that this region of ICAM-1 influences binding of the I domain but does not directly interact with it.
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A Role for the Glu-34 Residue of ICAM-1 in Divalent Cation-dependent Binding-- The presence or absence of divalent cation has been shown to result in little alteration to the structure of the LFA-1 I domain (17). It has therefore been suggested that, during activation, Mg2+ may be involved either in conformational change between integrin domains or binds directly to ligand. As demonstrated in this study, Mg2+ produces a marked increase in I domain binding, showing that it has a direct role in the function of the isolated I domain and suggesting involvement in ligand binding. If, as has been predicted (26, 27), the Glu-34 residue is involved directly in cation-dependent ligand binding, then the limited binding of I domain to the Glu-34 mutant ICAM-1Fc should be insensitive to divalent cation. We tested the effect of Mg2+ and EDTA on the level of I domain binding to the E34A mutant and found that it was equivalent (Fig. 6). In addition, EDTA-treated wt ICAM-1Fc binding was comparable to the level of G34A ICAM-1Fc binding in Mg2+, suggesting that the Glu-34 residue has an essential role in divalent cation-dependent binding. Addition of anti-LFA-1 blocking mAb 38 results in binding equivalent to the GST control, showing that the binding not blocked by EDTA is specific. This result is consistent with the interpretation that residue Glu-34 does interact with Mg2+ at the MIDAS site. The mutant Q73N ICAM-1Fc showed equivalent binding to wt ICAM-1Fc in Mg2+ (Fig. 6) and was blocked similarly by EDTA (data not shown).
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DISCUSSION |
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An ELISA assay showing isolated LFA-1 I domain binding to ICAM-1 was characterized with the aim of identifying the residues of ICAM-1 that are critical for this interaction. The main findings of the study are that: 1) I domain binding to ICAM-1 is Mg2+-dependent and mirrors the cation-dependent behavior of cell surface LFA-1; 2) intact LFA-1 receptor binding is inhibited by ICAM-1 domain 1 mutations at Glu-34 and Gln-73, in confirmation of previous studies; 3) the I domain is sensitive to mutation of residue Glu-34 but not Gln-73; 4) anti-ICAM-1 mAb RR1/1, which maps to Gln-73, enhances binding of the I domain to ICAM-1; and 5) the limited binding of I domain to the Glu-34 mutant is cation-independent, which adds evidence to previous suggestions that this residue may form the point of ligand contact with the MIDAS motif in the I domain.
The ELISA assay detecting LFA-1 I domain binding to ICAM-1 reflected several characteristics of the interaction of intact receptor with ligand. The binding was specific for ICAM-1, with no recognition of the closely related IgSF adhesion protein VCAM-1. Additionally, monoclonal antibodies previously mapped to the I domain (14) and shown to block the interaction between intact LFA-1 and ICAM-1 by varying amounts (25) had a comparable blocking pattern in the isolated I domain assay.
The divalent cation binding characteristics of the isolated I domain in
our assay mirror the behavior of intact LFA-1 (1), with
Mg2+ promoting maximal binding to ICAM-1 and
Ca2+ failing to support binding. This suggests that the I
domain controls the divalent cation-dependent function of
the native receptor. Although the majority of LFA-1 I domain binding is
dependent upon the presence of Mg2+, there is a low level
of binding in the assay, which occurs in the absence of divalent cation
and is specific as it is blocked by anti-LFA-1 mAb. The specific
binding of LFA-1 I domain with and without divalent cation supports
both previous conflicting reports about divalent cation requirement of
2
1 I domain (23, 24). The LFA-1 I domain
structure solved in the presence of Mg2+ or absence of
cation showed little difference in tertiary structure (17) and
therefore does not exclude the possibility of low level cation-independent binding as observed here. In the intact LFA-1 receptor, this may not be possible due to insufficient binding strength
or masking of the binding face between
and
subunits, which are
believed to undergo conformational change during activation (13,
26).
A main objective of this study was to determine the regions of ligand ICAM-1 that are important for recognition by the LFA-1 I domain to understand how integrin and ligand interact. As the anti-I domain mAbs blocked similarly in ELISA and whole cell assays, anti-ICAM-1 mAbs were also tested. Monoclonal antibodies that interfere with ICAM-1 binding of cell surface-expressed LFA-1 (20) were unable to block the ELISA assay. The explanation for this may be that, in intact LFA-1, the anti-ICAM-1 mAbs block function by interfering with a binding site on ICAM-1 not involving the I domain or that they block sterically rather than directly.
The domain 1 residues Glu-34 and Gln-73 of ICAM-1 have been shown to be
essential for binding of intact LFA-1 receptor to ICAM-1 (2, 5, 6), as
we have confirmed. However, the demonstration here that LFA-1 I domain
binding is sensitive to Glu-34 mutated ICAM-1 is the first evidence
that the LFA-1 I domain interacts with this region of ICAM-1 domain 1. Mutation of equivalent residues Glu-37 in ICAM-3 (7-9) and Asp-40 in
VCAM-1 (10-12) are also inhibitory in whole cell assays. The I domain
may be expected to interact with Glu-37 of ICAM-3, but VCAM-1 is the
ligand of 4
1, an I domain-negative
integrin. If residue Asp-40 in VCAM-1 has a similar function to Glu-34
in ICAM-1, then it must be recognized by a different integrin domain,
potentially the
subunit I domain.
In contrast, binding of I domain to the Gln-73 ICAM-1 mutant resembled
wt ICAM-1. Since there is a definite requirement for Gln-73 not only
for cell-surface LFA-1 but also for LFA-1 in cell-free systems (2, 5,
6), this result implies that Gln-73 interacts with a region of LFA-1
other than the I domain either on the or
subunit. However, the
enhanced binding caused by anti-ICAM-1 mAb RR1/1, which maps to Gln-73
on the G strand, suggests that this region of ICAM-1 influences binding
of the I domain. The mAb might mimic an allosteric alteration of ICAM-1
following LFA-1 binding or replace the cooperative binding of another
LFA-1 domain by altering the orientation of the G strand to facilitate
I domain binding. In ICAM-3, the comparable Gln-75 residue is also
critical for intact LFA-1 binding and may have a similar role in I
domain interaction. This residue is not conserved in VCAM-1.
In the solved Mac-1 I domain crystal structure, Mg2+ bound to the MIDAS motif coordinated a glutamic acid (Glu) residue in an adjacent I domain (15). This was suggested to mimic the ligand bound state and led to the prediction that the function of critical acidic residues found in ligands might be to coordinate Mg2+ (26, 27). As demonstrated in this study, Mg2+ produces a marked increase in I domain binding, showing that it has a direct role in the function of the isolated I domain consistent with a critical role in ligand binding. However, Glu-34 mutated ICAM-1 shows limited binding, which is not enhanced by addition of Mg2+. The equivalence in level of cation-independent I domain binding to wt ICAM-1 in the presence of EDTA and the binding of Glu-34 mutant supports the suggestion that the Glu-34 residue has an essential role in cation-dependent binding. This result is consistent with the interpretation that residue Glu-34 interacts with Mg2+, but this issue will be finally solved only by co-crystallization of I domain with ICAM-1. The mutant Q73N ICAM-1 showed equivalent binding to wt ICAM-1 in Mg2+ (Fig. 4) and was blocked similarly by EDTA (data not shown). Recently, ICAM-1 domain 1 residues Lys-39, Met-64, Tyr-66, and Asn-68 have also been highlighted as important for binding of purified LFA-1 to ICAM-1 (5). However, none of these residues has the appropriate characteristics for direct MIDAS coordination.
Although there has been speculation that the I domain associates with
domain 1 (27, 28), this report is the first direct demonstration of the
interaction. The binding site includes the Glu-34 residue on the C
strand, which appears to influence
Mg2+-dependent binding. In the modeled
structure of ICAM-1 (20) and the solved ICAM-2 structure (29), Glu-34
is central to a flat area, which may serve as the ligand binding face.
The region surrounding the MIDAS motif, which is also flat, contains
residues required for binding of ligand (30), suggesting the I domain and ICAM-1 could interact with one another at several contact points.
The enhancing activity of mAb RR1/1, and lack of effect of the Gln-73
mutant on isolated I domain binding, implies that the G strand region
cooperatively or allosterically contributes to I domain binding
possibly at the Glu-34 site. The LFA-1 binding interface with ICAM-1 is
likely to be complex. Other regions of LFA-1 that might interact with
Gln-73 as well as other areas of ICAM-1 are the I domain-like region of
the subunit (19) and sequences within domain V/VI of the
subunit (18). In summary, by identifying the Glu-34 but not the Gln-73
region of ICAM-1 in specifically binding the LFA-1 I domain, the
initial steps have been taken toward understanding how integrin and
ligand recognize each other.
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ACKNOWLEDGEMENTS |
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We thank our colleagues Paul Bates, Alison McDowall, Joanna Porter, and Matthew Robinson for many helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Imperial Cancer Research Fund.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. Tel.: 44-171-269-3255;
Fax: 44-171-269-3093; E-mail: hogg{at}europa.lif.icnet.uk.
1 The abbreviations used are: LFA-1, leukocyte function-associated antigen-1; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; MIDAS, metal ion-dependent adhesion site; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; IgSF, Ig superfamily; mAb, monoclonal antibody; TBS, Tris-buffered saline; wt, wild type; BSA, bovine serum albumin.
2 J. Porter, personal communication.
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REFERENCES |
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