©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Binding Interface on the I Domain of Lymphocyte Function-associated Antigen-1 (LFA-1) Required for Specific Interaction with Intercellular Adhesion Molecule 1 (ICAM-1) (*)

(Received for publication, April 28, 1995)

Chichi Huang Timothy A. Springer (§)

From theCenter for Blood Research, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that lymphocyte function-associated antigen-1 (LFA-1) molecules containing the human alpha (CD11a) and human beta (CD18) subunits but not the murine alpha and human beta subunits can bind to human intercellular adhesion molecule 1 (ICAM-1). Using human/mouse LFA-1 alpha subunit chimeras, we mapped regions required for binding to ICAM-1 N-terminal to amino acid (aa) residue 359. Ligand binding sites were mapped in greater detail by scanning this region with murine sequences from 56 down to 17 aa in length and finally by introducing single or few murine aa residue replacements into the human sequence. Replacement of two non-contiguous regions of aa residues 119-153 and 218-248 in the I domain with the corresponding mouse sequences abolished most binding to human ICAM-1, without affecting alphabeta subunit association or expression on the surface of transfected COS cells. Specific residues within the I domain found to be important were Met-140, Glu-146, Thr-243, and Ser-245. Using the recently solved structure of the Mac-1 (CD11b) I domain as a model (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R.(1995) Cell 80, 631-638), these residues are shown to be located on the surface of the I domain surrounding the site to which Mg is chelated, and define a ligand binding interface. Mapping of the epitopes of a panel of mouse anti-human and rat anti-mouse monoclonal antibodies gave concordant results. Epitopes were mapped to two different regions in the N-terminal domain, four regions within the I domain, and two regions between the I domain and the EF hand-like repeats. Monoclonal antibodies to epitopes within the mid- to C-terminal portion of the I domain and the N-terminal portion of the region between the I domain and the EF hand-like repeats gave good inhibition of LFA-1-dependent homotypic aggregation with cells that express either ICAM-1 or ICAM-3 as the major LFA-1 ligand.


INTRODUCTION

The lymphocyte function-associated antigen-1 (LFA-1, (^1)CD11a/CD18) was initially identified with mAb because of its importance in antigen-specific T lymphocyte interactions (Larson etal., 1989). Subsequently, LFA-1 was shown to be important in a wide variety of lymphocyte functions and to be required for interactions with vascular endothelium in immigration of lymphocytes, monocytes, and neutrophils in inflammation and homing and recirculation of lymphocytes (Springer, 1990, 1994, 1995). LFA-1 is an integrin alphabeta heterodimer. Two other integrins restricted in expression to leukocytes, Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18) (Sanchez-Madrid etal., 1983), share the same CD18 or beta2 integrin subunit and have homologous alpha subunits. LFA-1 binds to three counter-receptors, ICAM-1, ICAM-2, and ICAM-3, that are members of the Ig superfamily (IgSF) and have distinctive patterns of surface expression and in the aggregate are constitutively or inducibly expressed on almost all cells in the body (Springer, 1995). Binding of LFA-1 to ICAMs is Mg-dependent (Marlin and Springer, 1987). Although LFA-1 is constitutively expressed on most leukocytes, binding to its counter-receptors requires cellular activation, which is thought to induce conformational changes in LFA-1 that affect its affinity for ligand and interaction with the cytoskeleton (Diamond and Springer, 1994).

Structure-function studies on LFA-1 are crucial for mapping ligand binding sites and determining the molecular basis for the interaction with ICAMs and the activation of adhesiveness of LFA-1. The LFA-1 alpha subunit, alphaL, has two prominent structural features, an inserted or I domain of about 200 aa residues present in some but not most integrins, and three EF hand-like putative divalent cation binding repeats that are shared with all other integrin alpha subunits (Hynes, 1992). The I domain is homologous to motifs in other proteins including the three A domains of von Willebrand factor, which have been implicated in ligand binding, multiple repeats in cartilage matrix protein and collagen type VI, and single repeats in complement component C2 and factor B (Colombatti and Bonaldo, 1991). Mac-1 and p150,95 alpha subunit chimeras were previously used to map epitopes of function-blocking mAb to Mac-1 and thus to map a site important for binding of the ligands iC3b, fibrinogen, and ICAM-1 to the I domain of Mac-1 (Diamond etal., 1993). Subsequently, multiple studies on Mac-1 (Lee etal., 1995; Michishita etal., 1993; Muchowski etal., 1994; Rieu etal., 1994; Zhou etal., 1994), LFA-1 (Champe etal., 1995; Landis etal., 1993, 1994; Randi and Hogg, 1994), VLA-1 (Kern etal., 1994), and VLA-2 (Kamata etal., 1994; Kamata and Takada, 1994), have implicated the I domain in ligand binding.

Very recently, the three-dimensional structure of the Mac-1 I domain has been determined (Lee etal., 1995). It has a double-twisted fold, with a central hydrophobic beta-sheet surrounded by amphipathic alpha-helices. A single Mg ion is bound to residues in three connecting loops at what will be referred to as the top of the I domain, above one end of the central beta-sheet. The Mg is coordinated by the hydroxyls of Thr-209, Ser-142, and Ser-144, and by 2 water molecules. Asp-140 and Asp-242 are in an outer sphere of coordination and coordinate with a water molecule and Ser-142 and Ser-144 that directly coordinate Mg. Asp-140 and Asp-242 are buried beneath the Mg, and unavailable for contact with ligand. This structural motif has been called a metal ion-dependent adhesion site (MIDAS). In the crystal structure, the Mg forms an adventitious coordination with a Glu residue from a neighboring I domain. In ICAM-1, Glu-34 in IgSF domain 1 is the most important residue for binding to LFA-1 (Staunton etal., 1990) and might form an analogous coordination with Mg. Mutation of residues that form the primary or secondary coordination shell with the Mg ion, including Asp-140 and Asp-242 in the I domain of Mac-1 (Lee etal., 1995; Michishita etal., 1993), their homologues in the I domain of VLA-2 (Kamata etal., 1994) and VLA-1 (Kern etal., 1994), and the homologue of Thr-209 in the I domain of VLA-2 (Kamata and Takada, 1994), disrupt ligand binding. Such mutations abolish divalent cation binding in Mac-1 (Michishita etal., 1993) and are predicted to do the same in the alpha1 and alpha2 subunits, suggesting that the divalent cation may indirectly stabilize a ligand binding site, or that the cation may directly coordinate with ligand. Other residues required for ligand binding and that are likely to form direct contacts with ligand have yet to be identified in any integrin I domain.

In this study, we have utilized chimeric LFA-1 alpha subunits in intact heterodimers expressed on the cell surface to define in detail structural regions of the LFA-1 alpha subunit that are important for binding to ICAM-1. Our studies were made possible by a previous observation that human but not mouse LFA-1 would bind to human ICAM-1, and that this species specificity mapped to the LFA-1 alpha subunit (Johnston etal., 1990). The human and murine LFA-1 alpha subunits have 72% amino acid sequence identity (Kaufman etal., 1991), allowing interspecies chimeric subunits to be constructed with little disruption of conformation. The chimeras have been used to map residues required for binding of LFA-1 to ICAM-1, and also to map epitopes for a panel of 20 mAb and to correlate epitope location with inhibition of binding to ICAMs. We demonstrate that four specific amino acid residues in two noncontiguous regions of the I domain, aa residues 119-153 and 218-248, are crucial for binding to ICAM-1. These residues surround the Mg ion on the top of the I domain, and define a ligand-binding interface. mAbs that block binding to ICAM-1 and ICAM-3 map to epitopes within or adjacent to these regions.


MATERIALS AND METHODS

Cell Lines and Monoclonal Antibodies

COS-7 cells, Epstein-Barr virus-transformed B-lymphoblastoid cell line, JY, and the T cell line, SKW3, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.

The mouse anti-human CD11a mAbs TS1/11, TS1/12, TS2/4, TS2/14, TS1/22, and TS2/6 and CD18 mAb TS1/18 (Sanchez-Madrid etal., 1982b) and the rat anti-mouse CD11a mAbs M17/7 (Sanchez-Madrid etal., 1982a) and M7/14 (Davignon etal., 1981) have been described previously. The rat anti-mouse CD11a mAb FD441.8 (Sarmiento etal., 1982) was obtained from ATCC. The mAb S6F1 (Morimoto etal., 1987), BL5, F8.8, MAY.035 (Ohashi etal., 1992), 25-3-1 (Fischer etal., 1986), YTA-1 (Nakamura etal., 1989), G-25.2, NKI-L16 (Keizer etal., 1988), CBR LFA-1/10, CBR LFA-1/9, CBR LFA-1/3, and CBR LFA-1/1, were obtained through the 5th International Leukocyte Workshop.

Wild-type and Chimeric Integrin Subunit Constructs

The human LFA-1alpha cDNA in pSP65 (Larson etal., 1990) was excised with HindIII and SphI and cloned into the polylinker of pUC19. It was excised with XbaI and cloned into the XbaI site of Ap^rM8, a derivative of CDM8 containing the beta-lactamase gene from pBluescript. (^2)The murine LFA-1alpha cDNA (Kaufman etal., 1991) and human beta cDNA (Kishimoto etal., 1987) were in Ap^rM8.

Chimeric alpha subunits with exchanges at BspHI (aa residue 153), Sse8387I (aa residue 359), and MscI (aa residue 654) sites occurring in both the human and murine cDNA were prepared by restriction enzyme digestion, purification of fragments by agarose gel electrophoresis, and ligation (see Fig.1A). Chimeras or scanning mutants were named according to the species origin of their segments. For example, h153m359h indicates that residues 1-153 are from halphaL, residues 154-359 are from malphaL, and residues 360 to the C terminus are from halphaL. Amino acid sequence numbering was according to the human sequence (Larson etal., 1990). Chimeras h654m and m654h were constructed by ligation of 7.3- or 6.8-kb fragments from partial MscI and complete HindIII digestion of halphaL or malphaL, respectively, and 2.1-kb fragments from complete MscI and HindIII digestion of malphaL or halphaL cDNA, respectively. Chimera h359m was constructed by ligation of a 8.2-kb fragment from complete HindIII and partial Sse8387I digestion of halphaL cDNA and a 1.2-kb fragment from complete HindIII and Sse8387I digestion of malphaL cDNA. m153h359m was constructed with a 0.6-kb fragment from complete HindIII and BspHI digestion of malphaL and a 8.3-kb fragment from complete HindIII and partial digestion with BspHI of h359m. h153m359h was constructed in two steps. A 0.6-kb fragment from complete digestion of halphaL with HindIII and BspHI was ligated to a 8.3-kb fragment from complete HindIII and partial BspHI digestion of malphaL to generate h153m. Then a 1.2-kb fragment from complete HindIII and Sse8387I digestion of h153m was ligated with a 8.2-kb fragment from complete HindIII and partial Sse8387I digestion of halphaL to make h153m359h.


Figure 1: Structure-function studies with alpha subunit chimeras. A, three restriction sites were used to generate alpha subunit chimeras (openbars, human sequence; hatchedbars, mouse sequence). Sites and the aa residue at which they occur are indicated. B, human alphaL (h), mouse alphaL (m), and human/mouse alphaL subunit chimeras or vector alone were coexpressed with the human beta subunit in transfected COS cells. beta2 subunit expression was detected with anti-CD18 monoclonal antibody TS1/18 and flow cytometry. Binding to ICAM-1 was measured with carboxyfluorescein-labeled transfected COS cells in microtiter wells coated with purified ICAM-1. , binding to ICAM-1; black square, beta2 subunit expression. Data are mean and S.D. of at least three experiments and are normalized to the percent of human wild-type transfectants that expressed the beta2 subunit or bound to ICAM-1.



Scanning Mutant Constructs

The region from the N terminus to the N-terminal end of the EF hand-like repeats was scanned by insertion of short segments of mouse sequence into the human alphaL subunit (Fig.2A). To make scanning mutants h217m248h, h249m303h, and h300m442h, we took advantage of unique NruI, ClaI, and SalI restriction sites and two adjacent BglII sites at residues 300 and 303 in halphaL. None of these sites were present at the same position in malphaL; therefore, oligonucleotides containing these sites were used to amplify with PCR the corresponding mouse sequences. PCR fragments were restriction digested and NruI-ClaI, ClaI-BglII, or BglII-SalI fragments were ligated to halphaL cut with the same enzymes to construct h217m248h, h249m303h, and h300m442h, respectively. A PCR fragment amplified from m359h was digested with BglII-SalI and ligated into halphaL cut with the same enzymes to construct h300m359h (Fig.2A).


Figure 2: Structure-function studies with scanning mutants. A, schematic structure. Restriction sites with asterisks were introduced with silent mutations. h300m359h was used only in epitope mapping (see Fig.5below). B, binding to ICAM-1 and beta2 subunit expression of COS cells cotransfected with wild-type or scanning mutant alpha subunits and the human beta2 subunit was measured as described in Fig.1. , binding to ICAM-1; black square, beta2 subunit expression. Data are mean and S.D. of at least three experiments.




Figure 5: Mapping of mAb epitopes on the alpha subunit. A, chimeras. B, scanning mutants. COS cells cotransfected with wild type, chimera, or scanning mutant alpha subunits and the human beta subunit- or mock-transfected cells were stained with mAb to the alpha subunits and subjected to immunofluorescence flow cytometry. +, percent of positive cells was comparable to COS cells transfected with wild-type LFA-1. -, mAb staining was not significantly different from staining with the negative control X63 IgG1 myeloma. nd, not done.



Other scanning mutants were constructed with the PCR overlap extension technique (Ho etal., 1989). Two successive PCR were used to generate a chimeric fragment, which was then restriction digested and inserted in halphaL. A silent substitution in the sequence of the overlap oligonucleotide was frequently used to introduce a restriction site at or nearby the mouse-human junction. These sites were used diagnostically and in some cases for construction of subsequent scanning mutants. In the first PCR, two separate reactions were performed to generate one fragment from halphaL and a neighboring fragment from malphaL. The two oligonucleotide primers at the overlap region were complementary for at least 18 bases (Table1). The two PCR reactions used 5` upstream and 3` complementary primers, and 5` complementary and 3` downstream primers, respectively. Restriction sites that were unique in alphaL or the vector were included in the 5` upstream and 3` downstream primers. The products of each PCR reaction were separated on an agarose gel, and bands of correct size were purified using Promega's PCR DNA preparation system (Promega). One tenth of the purified DNA samples were mixed and served as templates for the second PCR reaction, with the 5` upstream and 3` downstream primers of the previous two PCR reactions. The PCR products were digested with the proper restriction enzymes and ligated into the halphaL cDNA fragment in Ap^rM8 produced by digestion with the same enzymes. Scanning mutants m57h, h57m74h, h74m93h, and h93m117h (Fig.2A) were transferred into alphaL using HindIII and KasI sites. The h57m117h construct was used as an intermediate in construction of h93m117h and h57m93h. The h57m93h construct was used as an intermediate in construction of h57m74h and h74m93h. The sequence of all these mutants was confirmed from the HindIII to KasI site by dideoxysequencing using Sequenase (U. S. Biochemical Corp.) according to manufacturer's instructions. Scanning mutants h118m153h, h153m183h, and h185m215h (Fig.2A) were transferred into alphaL using unique KasI and ClaI sites. The h118m215h construct was used as an intermediate in construction of h153m215h, which in turn was used in construction of h153m183h and h184m215h. The sequence of all these mutants was confirmed from the KasI to ClaI sites.



Construction of Multiple and Single Point Mutants

PCR amplification with primers that encompassed the KasI or ClaI sites and encoded mutations near these sites was used to generate the F122Y, I126M, L224S, T243S, and S245K mutants (Table1). The PCR products were digested with KasI and ClaI and ligated into halphaL digested with the same enzymes. PCR overlap extension was used to produce mutants N129K, M140Q, Q143D, P144R, D145K, E146K, Q148E, D152E, I231V, E218H/R221K/L224S, and I235V/T243S/S245K. The 5` upstream and 3` downstream primers encompassed the KasI and ClaI sites, respectively, and these enzymes were used to move the PCR fragments into halphaL as described above for scanning mutants. The entire KasI-ClaI fragment was sequenced for each mutant.

COS Cell Transfection and Flow Cytometry

Plasmids were purified with a Wizard Maxi preparation kit (Promega) and ethanol precipitation. COS cells were transiently cotransfected with wild type, mutant, or chimeric LFA-1 alpha subunits and human beta2 subunit constructs using DEAE-Dextran (Aruffo and Seed, 1987). Transfected cells were treated with trypsin-EDTA on day 2 and replated. On day 3, cells were harvested in 5 mM EDTA/phosphate-buffered saline and washed with L15 medium (Sigma) supplemented with 2.5% fetal bovine serum.

Immunofluorescence flow cytometry was as described previously (Hibbs etal., 1990), with a first incubation of 10 µg/ml purified mAb or 1:200 dilution of ascites and staining with 1:20 dilution of fluorescein isothiocyanate-conjugated second antibody.

Adhesion Assay

A soluble form of human ICAM-1 (sICAM-1) truncated before the transmembrane domain (Y452E/F*) was expressed in SF9 insect cells and purified by immunoaffinity chromatography (Casasnovas etal., 1994). Transfected COS cells were labeled with 2`,7`-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester and assayed for binding to purified ICAM-1 absorbed to 96-well plates with a fluorimeter as described (Bilsland and Springer, 1994), except binding was for 25 min at 37 °C and washing was with a 26-gauge 5/8-inch needle. Binding of transfected cells to ICAM-1 was expressed as a percent of wild type = 100 (mutant - mock binding)/(wild type - mock binding). Triplicates in each experiment were averaged and considered a single data point for calculation of S.D. among different experiments.

Aggregation Assay

JY or SKW3 cells were harvested near confluence (about 6 10^5 cells/ml). Cells (2 10^6/ml) preincubated with 1:200 ascites or 10 µg/ml purified protein for 20 min were stimulated with phorbol 12-myristate 13-acetate at a final concentration of 50 ng/ml in 100 µl in microtiter plates gently shaken for 30 min for JY cells or 2 h for SKW3 cells at 37 °C, as described previously (Rothlein etal., 1986), except with 5% fetal bovine serum.


RESULTS

Construction and Expression of Human/Mouse CD11a Chimeras

To localize sites on the human LFA-1 alpha subunit cognate for human ICAM-1, a set of five human/mouse alpha subunit chimeras were constructed in the expression vector Ap^rM8 (Fig.1A). These chimeras swapped the N-terminal region, I domain, metal binding domain, and the remaining C-terminal portion between the human and mouse alpha chains. Chimeras and scanning mutants (see below) were named according to the species origin of their segments, e.g. h153m359h is an alpha subunit with human aa residues 1-152, mouse residues 153-358, and human residues 359 to C terminus.

COS cells were cotransfected with the chimeric alpha subunits and the wild-type human beta2 subunit. To test for association of the alpha subunit chimeras with the beta2 subunit and expression on the cell surface, the transfected COS cells were stained with anti-alphaL mAb and anti-beta2 mAb TS1/18 and subjected to flow cytometry. Association between alpha and beta subunits is required for efficient surface expression of the LFA-1 alpha and beta subunits (Hibbs etal., 1990; Larson etal., 1990); as shown by stimulation of beta subunit expression, all five alpha subunit chimeras associated with the beta2 subunit and were expressed on the COS cell surface comparably to wild type human and murine alpha subunits (Fig.1B). The overall conformation of the chimeric alpha subunits on the cell surface was intact, because they reacted with a variety of antibodies to different epitopes on the alpha subunits (see below, Fig.5A). The molecular size of the chimeric alpha subunits was the same as human or mouse alpha as shown by I labeling of COS cell transfectants and immunoprecipitation with both anti-alpha and anti-beta mAbs (data not shown).

Binding of LFA-1 with Human/Mouse Chimeric alpha Subunits to ICAM-1

The functional activity of LFA-1 was tested by measuring binding of transfected COS cells to soluble ICAM-1 absorbed on plastic microtiter wells (Fig.1B). COS cells cotransfected with halphaL and beta2 subunit cDNAs bound to human ICAM-1, whereas COS cells that were mock-transfected or transfected with the beta2 subunit alone did not bind. By contrast, COS cells cotransfected with the mouse alphaL and human beta2 subunit cDNAs did not bind human ICAM-1, confirming previous results (Johnston etal., 1990; Kaufman etal., 1991). The h654m chimera bound to ICAM-1 as well as human LFA-1 (Fig.1B), whereas the reciprocal chimera m654h did not bind to ICAM-1, suggesting that the region controlling the species specificity is located N-terminal to the MscI site. The SseI site at residue 359 is intermediate between the I domain and the EF hand-like repeats (Fig.1A). The chimera h359m bound to ICAM-1 as well as human LFA-1, further mapping the species specificity of binding N-terminal to the SseI site. The reciprocal chimera m359h was not expressed on the cell surface (data not shown). The BspHI site at residue 153 is in the I domain (Fig.1A). Chimeras in which the segment between aa residues 153 and 359 was exchanged, h153m359h and m153h359m, showed binding to ICAM-1 that was less than for human alphaL, but greater than for mouse alphaL or m654h. Taken together with the finding that h359m had full activity, these results suggested that both the aa residue 1-153 and 154-359 segments contributed to binding to ICAM-1.

Two Separate Regions, Residues 119-153 and 218-248, Crucial for ICAM-1/LFA-1 Recognition

To map binding sites in more detail, the region from residues 1 to 442 was scanned by replacing short segments of human sequence with the corresponding mouse sequence (Fig.2A). The expression of each mutant in COS cells was determined by flow cytometry using mAb to the beta2 subunit (Fig.2B) and to the alphaL subunit (see Fig.5B below), and compared to binding of each mutant to purified ICAM-1 (Fig.2B).

The region from the N terminus to the beginning of the I domain (residues 1-117) was scanned with four different mutants. Replacements with larger segments of mouse sequence in this region (1-117, 57-117, 57-93), which is 71% identical with human, resulted in chimeras that were not expressed (not shown). Replacements with the four smaller segments resulted in chimeras that were expressed, but not always as well as wild-type. Scanning mutant m57h was consistently expressed at about 60% the level of human alphaL, and bound about 35% as well, consistent either with a contribution to binding or an altered conformation. Scanning mutants h57m74h and h74m93h gave expression and binding to ICAM-1 that were concordant and were slightly depressed relative to wild-type. Binding and expression of h93m117h was not significantly different from human LFA-1.

The I domain was scanned with five different mutants, from residues 118-303. All five scanning mutants were expressed in COS cells as well as wild-type mouse and human LFA-1 (Fig.2B). Scanning mutant h118m153h bound only 30% as well as human LFA-1 to ICAM-1, despite a difference of only 11 amino acids between mouse and human over the 28-aa residue segment that was exchanged. Mutants h153m183h and h184m215h showed only a slight and nonsignificant reduction in binding activity. By contrast, scanning mutant h217m248h was dramatically deficient in binding to ICAM-1. The 30 amino acid residues exchanged in this region contained only six differences between mouse and human. Exchange of the final segment of the I domain in chimera h249m303h had no effect on binding to ICAM-1.

The region between the I domain and the first EF hand-like putative divalent cation binding repeat was scanned in h300m442h. Substitution of the 142 residues in this region with murine sequence had no effect on surface expression or binding of LFA-1 to human ICAM-1.

Amino Acid Substitutions in Two Subregions of the I Domain

The two subregions of the I domain important in species-specific recognition of ICAM-1 (Fig.3) contained 11 and 6 amino acid differences, respectively. To identify the critical residues in these regions, we replaced single or multiple aa residues of the human alpha chain with corresponding mouse alpha chain residues. The differences in residues 119 to 153 were scanned with 10 single substitutions (Fig.4A). All mutants were well expressed. Two mutants, M140Q and E146D, retained only 35% and 50% of ICAM-1 binding activity, respectively. Binding by these mutants was almost as depressed as h118m153h, suggesting that residues Met-140 and Glu-146 make the major contributions to species-specific binding to ICAM-1 in this region. The other mutants in this region retained most or all binding activity.


Figure 3: Alignments of the human and mouse LFA-1 I domains, and the human Mac-1 I domain. The beta strands and alpha helices of the Mac-1 I domain are underlined, and residues in the primary or secondary Mg coordination shell are indicated by asterisks. Species-specific differences studied by mutation of individual or several amino acids in regions 119-153 and 218-248 are shown in reversetype. Residues are numbered that were found to be important for binding of human ICAM-1 (Met-140, Glu-146, Thr-243, Ser-245), or binding of mAb CBR LFA-1/9 and F8.8 (Pro-144).




Figure 4: Mapping of species-specific recognition of ICAM-1 to specific aa residues. A, mutation of individual human residues to mouse residues in region 119-153. B, mutation of residues in region 218-248. Binding to ICAM-1 and beta2 subunit expression of COS cells cotransfected with wild-type or scanning mutant alpha subunits and the human beta2 subunit was measured as described in Fig.1. Data are mean and S.E. of at least three experiments. , binding to ICAM-1; black square, beta2 subunit expression.



The six species-specific residues from 218 to 248 were divided in two groups by constructing mutant E218H/R221K/L224S and mutant I235V/T254S/S245K. Point mutants in this region were also constructed. All mutants were well expressed (Fig.4B). Mutant E218H/R221K/L224S and a point mutant with its least conservative substitution, L224S, bound to ICAM-1 with nearly wild-type activity. By contrast, binding to ICAM-1 of I235V/T245S/S245K was almost as depressed as for h217m248h. The point mutants T243S and S245K bound to ICAM-1 only 40-30% as well as human LFA-1, suggesting T243S and S245K contribute to species-specific binding.

Epitope Mapping of CD11a Antibodies

An alternative approach to map the binding site for ICAMs is to use chimeras and mutants to map the epitopes of mAbs, and test the mAbs for inhibition of LFA-1 function, to establish an epitope and function relationship map. Eighteen mouse mAb to human LFA-1 alpha, and three rat mAb to mouse LFA-1 alpha, were tested for immunofluorescent staining of alpha subunit chimeras, scanning mutants, and point mutants cotransfected with the human beta subunit in COS cells. All but two groups of mAb could be assigned to one of three segments of the LFA-1 alpha subunit defined by the chimeras: residues 1-153, 154-359, and 360-654 (Fig.5A). The mAbs CBR LFA-1/9, BL5, and F8.8 reacted with h359m, but not with h153m359h or m153h359m, suggesting that they recognize residues both in regions 1-153 and 154-359. The mAb G-25.2, NKI-L16, and CBR LFA-1/3 reacted with an epitope C-terminal to aa residue 359 as shown by staining of h153m359h and not m153h359m or h359m. However, the three mAb were negative on both h654m and m654h, suggesting the epitope(s) included residues in regions on both sides of residue 654.

The epitopes were more precisely mapped with the scanning mutants (Fig.5B). The S6F1 and TS2/4 mAb were negative on m57h and positive on all the other scanning mutants and thus mapped to residues 1-57. mAb CBR LFA-1/10 was positive on all mutants except for h74m93h and h93m117h and thus appears to recognize an epitope with contributions from both residues 75-93 and 93-117, and thus maps to residues 75-117. Lack of reaction with two different scanning mutants mapped mAb CBR LFA-1/9, BL5, and F8.8 to two separate regions, residues 119-153 and 185-215, confirming results with chimeras. Lack of reaction with individual scanning mutants mapped mAb TS2/6 to residues 154-183; mAb MAY.035, TS1/11, and TS1/12 to residues 185-215; mAb TS1/22, TS2/14, and 25-3-1 to residues 250-303; and mAb CBR LFA-1/1 to residues 301-359. mAb YTA-1 lost reactivity with h300m442h but reacted with h300m359h; thus, at least a portion of its epitope localizes to residues 360-442. mAb G-25.2, NKI-L16, and CBR LFA-1/3 reacted with h300m442 and could be localized C-terminal to residue 442.

Three rat mAb to mouse LFA-1, all of which block function, were localized. mAb M17/7 and M7/14 were localized to residues 154-359 with the chimeras (Fig.5A). M17/7, M7/14, and FD441.8 reacted with h249m303h and were negative on all other scanning mutants on which they were tested (Fig.5B). Thus, murine residues 250-303 in a completely human background are sufficient for expression of the epitope(s) recognized by these mAb.

The point mutants in the segment from residues 119-153 were tested for reactivity with mAb to this region, CBR LFA-1/9, BL5, and F8.8. The P144R substitution, but none of the other point mutations, abolished binding of CBR LFA-1/9 and F8.8 (data not shown).

Inhibition of Lymphoid Cell Homotypic Aggregation with mAb to LFA-1

mAb were tested for inhibition of homotypic aggregation by two cell lines that utilize different ICAMs. JY cells express ICAM-1, less ICAM-2, and little ICAM-3 (de Fougerolles and Springer, 1992). Phorbol 12-myristate 13-acetate-stimulated JY cell homotypic aggregation is largely blocked by mAb to ICAM-1, unaffected by mAb to ICAM-2, and completely blocked by a combination of mAb to ICAM-1 and ICAM-2 (de Fougerolles etal., 1991). SKW3 cells express ICAM-3, less ICAM-2, and no ICAM-1 (de Fougerolles and Springer, 1992). Phorbol 12-myristate 13-acetate-stimulated SKW3 cell homotypic aggregation or binding to purified LFA-1 is partially inhibited by mAb to ICAM-3 and completely inhibited by a combination of mAb to ICAM-2 and ICAM-3 (de Fougerolles etal., 1994; de Fougerolles and Springer, 1992). Inhibition of SKW3 and JY cell aggregation by LFA-1 mAb was concordant (Table2). Inhibition by mAb of aggregation was a more stringent assay than inhibition of binding of lymphoid cells to purified ICAM-1 on a substrate, i.e. the same trends were seen, but mAb that were partial blockers of aggregation were more complete blockers of binding to ICAM-1 (data not shown). Of 18 mAb, six completely inhibited homotypic aggregation of JY and SKW3 cells. These mAb mapped to residues 154-183, 185-215, and 250-303 within the I domain, and residues 301-359 just C-terminal to the I domain. mAb to adjacent residues 119-153 gave partial inhibition, whereas mAb to residues 1-117 and 360-1063 gave little or no inhibition.




DISCUSSION

We have used two approaches to define segments of the LFA-1 alpha subunit important for binding to ICAMs. The first approach relied on the observation that the human but not mouse alpha subunit enabled binding of the LFA-1 alphabeta complex to human ICAM-1 (Johnston etal., 1990). The human and murine ICAM-1 molecules are 55% identical in overall amino acid sequence and 52% identical in the first IgSF domain, where the binding site for LFA-1 has been mapped. The inability of murine LFA-1 to bind human ICAM-1 no doubt reflects the divergence of at least some of the ICAM-1 residues that are present at the binding interface with LFA-1.

By dividing the alphaL subunit into four portions with chimeras, we mapped a region important for species-specific interaction with ICAM-1 to aa residues 1-359, which contains the I domain. The chimeras also provided evidence for multiple subregions within the I domain required for specific interactions, since substitution of human LFA-1 with either of two different murine segments (aa residues 1-153 or residues 154-359) greatly reduced binding to human ICAM-1. Working with scanning mutants provided further evidence for two subregions. The major differences between human and mouse ICAM-1 that restrict recognition of ICAM-1 were localized to residues 119-153 and 218-248. These subregions correspond to the first and third segments, respectively, of five I domain segments that we studied.

The human LFA-1 alpha subunit was substituted with single or several murine aa residues for fine localization of residues required for specific binding to ICAM-1. Two of 10 point mutants in region 119-153, M140Q and E146D, lost a large portion of ICAM-1 binding activity. In the region of 218-248, the multiple substitution mutant I235V/T243S/S245K lost most of the ICAM-1 binding activity, and the individual point mutations T243S and S245K lost a large portion of the binding activity.

After this work was completed, the three-dimensional structure of the I domain of Mac-1 was reported (Lee etal., 1995). We have used this structure as a model for the I domain of LFA-1; the only significant difference between the structures is predicted to be a shortening of the alpha5 helix of LFA-1 (Fig.3). The four residues in LFA-1 important in species-specific binding to ICAM-1 are superimposed on the position of the homologous residues in Mac-1 in Fig.6. Residue Met-140 is in between Ser-139 and Ser-141, which coordinate with the Mg ion; the Met-140 side chain is a prominent feature of the I domain surface. Glu-146 forms the base of a depression near the Mg. The Thr-243 and Ser-245 residues are located on the other side of the Mg from Met-140 and Glu-146. Previous mutagenesis studies have identified residues in integrin I domains that form the primary or secondary coordination shell with Mg and are required for ligand recognition (Kamata etal., 1994; Kamata and Takada, 1994; Kern etal., 1994; Lee etal., 1995; Michishita etal., 1993). These mutations are known (Michishita etal., 1993) or predicted (Lee etal., 1995) to prevent divalent cation binding. The Mg in the MIDAS motif is predicted to form one coordination with ligand (Lee etal., 1995); divalent cations have for some time been predicted to bridge integrins and their ligands (Corbi etal., 1987). Thus, these mutations suggest the importance of the Mg, rather than particular amino acid residues, in ligand binding. By contrast, none of the four residues we have identified are predicted to coordinate Mg. Rather, the striking feature of the residues we have identified is that they surround the Mg binding site, and for the first time define a ligand binding face for an integrin I domain. We hypothesize that the Mg in the MIDAS motif coordinates with Glu-34 in ICAM-1, by far the most important residue yet identified for binding to LFA-1 (Staunton etal., 1988). Glu-34 is completely conserved in ICAM-1 in the mouse, human, and three other species, and in murine and human ICAM-2 and in human ICAM-3 (Vonderheide etal., 1994). We hypothesize that residues surrounding Glu-34 in the binding interface on ICAM-1 that differ between mouse and human are responsible for the species-specific differences we have mapped on the I domain of LFA-1. A residue that may play an analogous role to Glu-34 in ICAM-1 is Asp-40 in VCAM-1, although the integrin to which VCAM-1 binds, VLA-4, lacks an I domain (Osborn etal., 1994; Renz etal., 1994; Vonderheide etal., 1994). Asp-40 in VCAM-1 is in a prominent loop between the C and D beta strands of the first IgSF domain (Jones etal., 1995), as predicted previously for Glu-34 in ICAM-1 (Staunton etal., 1988).


Figure 6: The Mac-1 I domain, with residues in LFA-1 shown to be important in specific interaction with ICAM-1 superimposed on the position of the homologous residues in Mac-1. Alignment was as shown in Fig.3.



Our second approach to define segments of the LFA-1 alpha subunit important for binding ICAM-1 was to construct a structure-function map with mAb. We mapped epitopes of a panel of eighteen mouse anti-human CD11a and three rat anti-mouse CD11a antibodies. These mAbs were mapped to nine different segments of the alpha subunit (Fig.7). Furthermore, we examined the ability of these mAbs to block LFA-1-dependent homotypic aggregation that was primarily dependent on ICAM-1 for JY cells and ICAM-3 for SKW3 cells. These results extended previous studies that have mapped mAb to one to four different segments of the LFA-1 alpha subunit (Champe etal., 1995; Landis etal., 1993, 1994; Randi and Hogg, 1994). Our epitope-function map (Fig.7) showed that function-blocking mAb localized to all four subregions to which epitopes were mapped in the I domain and to a segment C-terminal to the I domain, but not to two N-terminal or two C-terminal subregions. Strongest blocking was obtained with mAb that bound to epitopes in segments from aa residues 154-359. However, a group of three mAb that recognized epitopes in both segments 119-153 and 185-215 gave intermediate inhibition; the epitopes of two of these mAb, CBR LFA-1/9 and F8.8, included Pro-144, which is located just prior to the beginning of alpha helix 1. The studies with mAb suggest that multiple segments of the I domain contribute to the interaction with ICAM-1 and further suggest that the segment between the I domain and EF hand-like repeats is important for binding to ICAM-1. Our epitope mapping results are in agreement with previous cross-blocking and functional studies on the TS (Ware etal., 1983) and M series (Sanchez-Madrid etal., 1982a) of mAb. Thus TS1/11 and TS1/12 that map to residues 183-215 cross-blocked one another and were distinct from all other TS series mAb; TS1/22 and TS2/14 that map to residues 248-303 also showed cross-blocking. Studies on T cell-mediated killing showed that TS mAb that map to the I domain were inhibitors, whereas TS2/4 that maps to the N terminus was not. The mAb M7/4 and M17/7, which block T cell-mediated killing by 80%, cross-blocked one another and all other tested function-blocking rat anti-mouse mAb (Sanchez-Madrid etal., 1982a); less sequence divergence between mouse and rat than between mouse and human may have led to a more focused response to epitopes in the residue 249-303 segment, where all three studied rat anti-mouse mAb map.


Figure 7: Schematic map of functionally important regions and epitopes of the LFA-1 alpha subunit. Amino acid residue numbers are for the human LFA-1 alpha subunit. The boldlines over the alpha subunit indicate the regions that restrict species-specific binding to ICAM-1. The thinlines below the alpha subunit indicate mAb epitope localization. Inhibition refers to potency of mouse anti-human mAb in inhibition of LFA-1-dependent homotypic aggregation. All three rat anti-mouse mAb were previously selected for strong inhibition of LFA-1-dependent T cell-mediated killing.



There are agreements and also contradictions between our study and a recent study in which eight mAb shown in the literature to inhibit LFA-1 function were mapped to three regions within the I domain (Champe etal., 1995). Three mAb were mapped to segments 143-148 and 197-203, and one was mapped to 197-203 only. The three mAb thus recognize an epitope similar to CBR LFA-1/9, BL5, and F8.8, which were shown here to recognize regions 119-153 and 185-215. Both Champe et al.(1995) and our group found that reactivity of these mAb was abolished by the P144R human to mouse substitution. These residues are located on the first turn of the alpha1 helix and the long loop between the alpha3 and alpha4 helices, and are on the same face of the I domain as the residues involved in specific interactions. This localization is consistent with inhibition of function that we found with the mAb to this epitope. Champe et al. (Champe etal., 1995) found that the anti-human mAb TS1/22 and 25-3-1, as well as the anti-mouse mAb M17/4 and I21/7, mapped to residues Ile-126 and Asn-129. In contrast, we mapped TS1/22, 25-3-1, and three anti-mouse mAb, including M17/7 and M7/14 (which are known to cross-block M17/4; Sanchez-Madrid etal., 1982a), to residues 250-303. Each study grouped the same antibodies as reacting with a common epitope, but mapped them to completely different sites. Champe et al. used immunoprecipitation of biosynthetically-labeled transfected alpha subunits expressed in the absence of beta subunit, whereas we used immunofluorescent labeling of alphabeta complexes on the cell surface, but this cannot explain the discrepancy. The mapping to Ile-126 and Asn-129 was supported by a single mutant construct, and no effect of individual mutations at residues 126 and 129 was found, including an I126D substitution more radical than the I126M human-mouse substitution. Champe et al.(1995) made no substitutions C-terminal to residue 218, and thus did not study the C-terminal half of the I domain where we localized this group of mAb. Our localization to 250-303 as opposed to 126-129 was supported by four constructs: h153m359h, m153h359m, h118m153h, and h249m303h. The group of mAb we localized to residues 250-303 all gave maximal inhibition of LFA-1 function, implying the epitope is localized close to the MIDAS motif. By contrast, residues 126 and 129 are at the very beginning of the I domain, prior to beta strand A, and are on the opposite side of the I domain from the MIDAS motif.

Our study has identified residues that surround the Mg-binding site on the LFA-1 I domain that are required for specific binding to ICAM-1. Other studies on multiple integrin I domains have shown that residues that directly or indirectly coordinate Mg in the MIDAS motif are required for ligand binding. Together, this work defines a ligand binding interface on integrin I domains. The epitope structure-function mapping experiments confirm the importance of multiple subregions of the I domain in ligand binding, and also suggest that the region C-terminal to the I domain residues 301-359, is important. Monoclonal antibodies that bind to the beta subunit (Sanchez-Madrid etal., 1983) and mutation of a MIDAS-like site in the conserved region of the beta subunit (Bajt and Loftus, 1994; Lee etal., 1995) also inhibit ligand binding. How both the alpha and beta subunit contribute to ligand binding and work together to regulate integrin adhesiveness remains to be established.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 31798. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Blood Research, Rm. 251, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3232.

^1
The abbreviations used are: LFA, lymphocyte function-associated antigen-1; ICAM, intercellular adhesion molecule; IgSF, Ig superfamily; MIDAS, metal ion-dependent adhesion site; VLA, very late antigen; mAb, monoclonal antibody; kb, kilobase pair(s); PCR, polymerase chain reaction.

^2
L. B. Klickstein, unpublished data.


ACKNOWLEDGEMENTS

We thank Jie-oh Lee and Robert Liddington for discussions of I domain structure and for Fig.6.


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