(Received for publication, July 25, 1994; and in revised form, November 7, 1994)
From the
The epitopes recognized by eight independently isolated
monoclonal antibodies to the chain of human and murine leukocyte
function-associated antigen 1 (LFA-1), all able to inhibit receptor
function, were identified. Initial localization of epitopes was
accomplished using chimeric proteins constructed by splicing fragments
of cDNAs encoding the
subunit of LFA-1 (CD11a) and the
subunit of the closely related leukocyte integrin, Mac-1 (CD11b).
Antibody binding to CD11a/CD11b chimeras, expressed in the 293 human
kidney cell line, demonstrated that the epitopes recognized by six
monoclonal antibodies to human CD11a were located in a
200-amino
acid sequence found in all
-integrin
subunits,
termed the inserted (I) domain. Three distinct epitopes within the I
domain (IdeA, IdeB, and IdeC) were identified using a series of mutants
in which sequences from murine CD11a were substituted into human CD11a.
A series of mutants incorporating single amino acid substitutions was
used to identify individual amino acids essential for antibody binding.
The location of these residues accounts for the binding specificity of
LFA-1-blocking antibodies and identifies particular conserved sequences
(residues 126-150) in the I domain of CD11a and homologous
sequences in other
-integrin
subunits that may
be important for ligand binding.
Integrins are a large family of homologous, heterodimeric cell
surface receptors that mediate cell to cell and cell to substratum
adhesion(1, 2) . All integrins possess
,
subunit structure where the
chains and the
chains associate through noncovalent interactions.
Integrin families are classified on the basis of their
subunits (3) , which typically form heterodimers with any of several
different
subunits. In the immune system, integrins serve as
accessory molecules that facilitate intercellular interactions required
for antigen recognition, cellular activation, and leukocyte
trafficking, as well as effector functions such as cytotoxic killing,
phagocytosis, and antibody-dependent
cytotoxicity(4, 5, 6) . The
family of leukocyte integrins consists of three homologous
receptors, LFA-1 (
)(CD11a/CD18), Mac-1 (CD11b/CD18), and
p150,95 (CD11c/CD18), each consisting of a unique
chain (CD11a,
CD11b, and CD11c, respectively) binding to a common
chain (CD18).
The distribution of the
-integrins is tissue-specific,
with LFA-1 present on all leukocytes and Mac-1 and p150,95 occurring
primarily on macrophages, neutrophils, and other myeloid cells. Like
other integrins, the members of the
family are
promiscuous and interact with several different ligands or
contrareceptors. LFA-1 is known to bind to the three structurally
related immunoglobulin superfamily members termed intercellular
adhesion molecule 1
(ICAM-1)(7, 8, 9, 10, 11) ,
intercellular adhesion molecule 2 (ICAM-2)(12, 13) ,
and intercellular adhesion molecule 3 (ICAM-3)(14) . Mac-1 is
known to bind several diverse and unrelated ligands including the C3Bi
component of complement(15) , ICAM-1(16, 17) ,
fibrinogen (18) , factor X(19) , and an unidentified
ligand on neutrophils that mediates homotypic aggregation(20) .
The functional activity of
-integrins has been
investigated through the use of monoclonal antibodies (mAbs) to LFA-1,
Mac-1, and p150,95 (21) that are able to block adhesive
interactions that mediate leukocyte function. These studies have shown
that mAbs to either the
or
chains can be potent inhibitors
of leukocyte
function(15, 22, 23, 24, 25) .
Additional insight into the function of
-integrins has
come through the analysis of a rare genetic disease, leukocyte adhesion
deficiency, which results from the inability to synthesize functional
CD18(26) . Individuals with this disease are unable to express
LFA-1, Mac-1, or p150,95 and, as a consequence, are severely
immunocompromised and exhibit multiple defects in lymphocyte and
granulocyte function (27, 28) .
Little is known of
the molecular basis of ligand recognition for the family of integrins. Like other integrins, the
chains in
the
family possess multiple divalent cation binding
sites (29, 30, 31) and require
Ca
, Mg
, or Mn
to
function(32, 33) . The ligands for
-integrins are unique in that none are known to
possess the tripeptide receptor recognition sequence, RGD, that is
common in many of the ligands for other integrin families (e.g.
and
family)(1, 2, 3, 34) . Besides
the difference in ligand specificity, the
family of
integrins is unusual in that the
subunits are larger (molecular
size, 150-170 kDa) than most other integrins by virtue of a
unique insertion of approximately 200 amino acids, termed the inserted
domain (I domain). The I domain contains sequences homologous with the
type A domains of von Willebrand factor, the repeats of cartilage
matrix-binding protein, and complement factor B and is located
approximately 150 amino acids from the NH
terminus(29, 31, 35) . Recent studies (20) have used antibodies to localize the binding sites for
C3Bi, ICAM-1, fibrinogen, and an as yet unidentified ligand responsible
for homotypic aggregation of neutrophils to the I domain of the
CD11b/CD18 heterodimer (Mac-1). However, the regions within the I
domain responsible for ligand binding have not yet been described.
In the present study, we have localized the epitopes recognized by eight mAbs to human and mouse CD11a that are all known to inhibit LFA-1 function. Mutagenesis to replace I domain sequences of human CD11a with those of murine CD11a permitted the identification of three discrete sequences in the I domain of CD11a that are recognized by antibodies able to block the binding of LFA-1 to ICAM-1.
Figure 1:
Immunoprecipitation of recombinant
human CD11a, CD11b, and CD11a/CD11b chimeras with monoclonal antibodies
to human CD11a. The human kidney adenocarcinoma cell line 293 was
transfected with expression plasmids containing genes encoding
full-length human CD11a, full-length human CD11b, or chimeric genes
produced from the ligation of complementary regions of human CD11a and
CD11b genes (LM2, LMN, LM3, and ML9). The chimeric genes, but not the native genes, were
mutated so as to delete sequences encoding the transmembrane domains
and cytoplasmic tails. A schematic diagram of these constructions is
shown in Fig. 2. Murine monoclonal antibodies to human CD11a
CLB-LFA-1/2, MHM24, and 50G1, (A) and 32E6, 3D6, 25.3, and
TS-1/22 (B) or a rabbit polyconal antisera to human CD11b (A) were incubated with detergent lysates of metabolically
labeled [S]methionine-transfected 293 cells and
immunoprecipitated as described previously(43) . The
immunoprecipitated proteins were resolved by SDS-PAGE and visualized by
autoradiography as described
previously(43) .
Figure 2: Diagram of CD11a/CD11b chimeric proteins. Intermolecular chimeras between CD11a and CD11b were produced by ligation of complementary sequences of cDNAs encoding CD11a and CD11b. Regions selected for chimerization exhibited a high degree of sequence homology. For all but one of the chimeras, site-directed mutagenesis was used to introduce common restriction sites at the chimeric junctions without changing the predicted amino acid sequence. To construct the LM2 and ML9 plasmids, SacII sites were introduced to join the fragments of CD11a and CD11b. In the LMN plasmid, NheI sites were introduced into CD11a and CD11b to join the complementary fragments. To construct the LM3 plasmid, naturally occurring Sse8387I sites were used to splice the complementary CD11a and CD11b fragments. Sequences from CD11a are shown as open bars; sequences from CD11b are shown as dark bars. The locations of the I domain, the transmembrane domain, and the intracellular domain are also indicated. Amino acid numbering for CD11a and CD11b and the resulting chimeras is indicated.
The sequences of CD11a and CD11b were examined, and regions of high homology were selected as locations for chimerization. A schematic diagram of the four chimeras that were constructed is shown in Fig. 2. The chimeric genes, mutagenized to remove the transmembrane domains and cytoplasmic tails, were all cloned into the pRK expression vector and transiently expressed in 293 cells as described previously(43) . It was found that all of the chimeric proteins, but not CD11a, could be immunoprecipitated from lysates of transfected cells with polyclonal rabbit sera to recombinant soluble Mac-1 (Fig. 1A). When the binding of the CD11a mAbs to the panel of CD11a/CD11b chimeras was evaluated, it was found that none of the antibodies known to block LFA-1 function bound to the ML9 construction containing residues 1-340 of CD11b fused to amino acids 332-1063 of CD11a (Fig. 2). However, the 3D6 mAb, known to bind to CD11a without blocking function, was able to immunoprecipitate this protein (Fig. 1, A and B, ML9). These results suggested that the epitopes recognized by the LFA-1-blocking mAbs were located within the first 340 residues of CD11a. The mAbs were then tested for binding to the LM3 protein that contained the first 25 amino acids of CD11a fused to residues 62-1092 of CD11b (Fig. 2). In contrast to the ML9 protein, neither 3D6 nor any of the LFA-1-blocking mAbs were able to immunoprecipitate this protein (Fig. 1, A and B, LM3). Thus the amino-terminal sequence of CD11a did not appear to contain epitopes recognized by any of the mAbs. When mAb binding to the LM2 protein was examined, all six of the CD11a-blocking antibodies were able to immunoprecipitate the protein (Fig. 1, A and B, LM2) that consisted of amino acids 1-331 of CD11a fused to amino acids 341-1092 of CD11b (Fig. 2). Similarly, all six of the CD11a-blocking mAbs bound to the LMN protein (Fig. 1, A and B, LMN) that consisted of residues 1-278 of CD11a fused to residues 289-1092 of CD11b (Fig. 2). These studies localized the epitopes recognized by the six LFA-1-blocking mAbs to an amino-terminal fragment of CD11a spanning residues 26-278 that contains the I domain but excludes the divalent cation-binding sites (Fig. 2).
Figure 3: Alignment of I domain sequences from human and murine CD11a and description of human to mouse mutants. The specific amino acid changes incorporated into each member of the H/M mutant series are indicated. The boxes above the alignment identify the three epitopes defined by the H/M mutants.
Figure 4:
Immunoprecipitation of human CD11a
variants incorporating selected sequences from murine CD11a. Human
kidney adenocarcinoma cell line 293 was transfected with expression
plasmids containing genes encoding full-length human CD11a or
full-length human CD11a mutated to contain short stretches of murine
CD11a sequence. Murine monoclonal antibodies to human CD11a 3D6, 32E6,
CLB-LFA-1/2, and 50G1 (A) and 25.3, TS-1/22, and MHM24 (B) or rat monoclonal antibodies to murine CD11a M17 and I27/1 (B) were incubated with detergent lysates of metabolically
labeled [S]methionine-transfected 293 cells and
immunoprecipitated as described previously(43) . The
immunoprecipitated proteins were resolved by SDS-PAGE and visualized by
autoradiography as described
previously(43) .
Figure 5:
Localization of residues in I domain
epitopes B and C using human CD11a variants incorporating single amino
acid substitutions. Human kidney adenocarcinoma cell line 293 was
transfected with expression plasmids containing genes encoding
full-length human CD11a or full-length human CD11a with single amino
acid substitutions as indicated. Murine monoclonal antibodies to human
CD11a 3D6, 32E6, 50Gl, CLB-LFA-1/2, and MHM24 were incubated with
detergent lysates of metabolically labeled
[S]methionine-transfected 293 cells and
immunoprecipitated as described previously(43) . The
immunoprecipitated proteins were resolved by SDS-PAGE and visualized by
autoradiography as described
previously(43) .
We next explored the
importance of residues 198-200 in IdeC, which were found to be
important for the binding of mAbs 32E6, 50G1, CLB-LFA-1/2, and MHM.24 (Fig. 4). It was found that replacement of Lys with Asp completely inhibited the binding of all four mAbs but
did not affect the binding of 3D6 (Fig. 5, K197D).
Replacement of the adjacent His
with Ala, had no effect
on the binding of the four CD11a-blocking mAbs or 3D6 (Fig. 5, H198A). Replacement of the neighboring residue,
Lys
, with Asp (Fig. 5, K200D) diminished
the binding of MHM.24 without affecting the binding of any of the other
mAbs. Replacement of His
with Ala (Fig. 5, H201A) completely inhibited the binding of 32E6, diminished
the binding of MHM.24, and had little effect on the binding of the 50G1
and CLB-LFA-1/2 mAbs.
Finally, the mutations identified in IdeA that
prevented the binding of 25.3 and TS1/22 and conferred binding of rat
mAbs to murine CD11a (M17 and 121/7 mAbs) were investigated.
Replacement of Ile with Met or Asp (Fig. 6, I126M and I126D) failed to prevent the binding of
mAbs 25.3 and TS1/22. Similarly, replacement of Asn
with
Lys also failed to affect the binding of these antibodies. Thus,
whereas the H/M 53 double mutant that incorporated both of these
substitutions blocked the binding of both antibodies (Fig. 4B, H/M 53), replacement of either
residue alone had no effect on antibody binding. To determine if other
nearby residues might affect antibody binding, Lys
was
replaced with Ala (Fig. 6, K127A) and Gly
was replaced with Ala (Fig. 6, G128A). It was
found that mutagenesis of position 127 reduced the binding of both mAbs
TS1/22 and 25.3, whereas mutagenesis at position 128 had no effect.
When the rat mAbs to murine CD11a (M17 and I21/7) were tested against
this series of variants, no binding could be detected (Fig. 6).
Thus, like the mAbs to human CD11a, the mAbs to murine CD11a required
the double mutation at residues 126 and 129 for binding (Fig. 4B, H/M 53).
Figure 6:
Localization of residues in I domain
epitope A using human CD11a variants incorporating single amino acid
substitutions. Human kidney adenocarcinoma cell line 293 was
transfected with expression plasmids containing genes encoding
full-length human CD11a or full-length human CD11a with single amino
acid substitutions as indicated. Murine monoclonal antibodies 3D6,
25.3, and TS 1/22 were incubated with detergent lysates of
metabolically labeled [S]methionine-transfected
293 cells and immunoprecipitated as described previously. The
immunoprecipitated proteins were resolved by SDS-PAGE and visualized by
autoradiography as described previously.
The location and structure of ligand binding sites on
integrin and
subunits have been the subject of intense
speculation. The discovery that all three
-integrin
chains possess an I domain suggested that this family of
integrins evolved a unique strategy for ligand binding. Data supporting
the role of the I domain in ligand binding by the closely related
integrins, Mac-1 and p150,95, were provided in experiments (20, 21) in which the binding properties of
intermolecular chimeras between CD11b and CD11c were analyzed. Direct
data demonstrating that the I domain of CD11a contains the ligand
binding was provided in studies utilizing a bivalent chimeric protein
consisting of the CD11a I domain fused to the Fc fragment of
immunoglobulin G(52) . In these studies, binding of CD11a I
domain/Fc chimeras to purified recombinant soluble ICAM-1 could be
detected by enzyme-linked immunosorbent assay. Neither of these
studies, however, provided any indication as to which part of the
200-amino acid I domain might actually be involved in ligand binding.
Data suggesting that the ICAM-1 binding site of CD11a might involve
residues outside the I domain was provided in other studies (53) in which fragments of CD11a lacking the I domain expressed
in an in vitro translation system could be precipitated using
a chimeric ICAM-1 variant that contained the first three domains of
ICAM-1 fused to the Fc fragment of immunoglobulin G
.
The
present study provides evidence that the I domain of CD11a, like that
of CD11b(20) , contains sequences that are important for ligand
binding. Using reciprocal intermolecular chimeras of CD11a and CD11b,
it was possible to localize the epitopes recognized by six
receptor-blocking mAbs to human LFA-1 and two blocking mAbs to murine
CD11a to sequences within the NH-terminal region of the
CD11a I domain. A series of novel CD11a mutants constructed by
replacement of human CD11a sequences with sequences from murine CD11a
further localized the epitopes recognized by all eight blocking mAbs to
three distinct epitopes within the I domain. Proteins incorporating
amino acid substitutions at residues 126-129 (H/M 53) completely
inhibited the binding of the TS-1/22 and 25.3 mAbs without affecting
the binding of the other CD11a mAbs. This epitope (IdeA) falls
immediately NH
-terminal to the sequence that previously was
considered the start of the I domain(30) . In other studies, we
found that the same mutations that blocked the binding of the TS-1/22
and 25.3 mAbs to human CD11a allowed for the binding of the rat mAbs to
murine CD11a (M17 and I21/7). The possibility that these antibodies
actually bind to the ICAM-1 binding site on LFA-1 is supported by the
experiments of Lollo et al.(54) , who have shown that
M17 competes with soluble recombinant murine ICAM-1 for binding to
LFA-1.
A second epitope (IdeB) important for the binding of
CD11a-blocking mAbs was identified by mutations in the region of
143-149 (H/M 52). This epitope is located 12 amino acids away
from the H/M 53 site and does not appear to contain residues important
for the binding of TS-1/22 or 25.3. Interestingly, the sequence located
between these two epitopes (i.e. residues 130-143) is
highly conserved among the -integrins and among the
domains of the other proteins (i.e. cartilage matrix protein,
von Willebrand factor, and factor B) that share homology with the
-integrin I domains (Fig. 7). In contrast, the
sequences of the two mAb recognition sites themselves (126-129
and 143-149) differ between the three
-integrin
chains (CD11a, CD11b, and CD11c) and exhibit no homology with the
domains of the other proteins (i.e. von Willebrand factor,
cartilage matrix protein, and factor B) that share homology with the I
domain. Thus, these results suggest that these two classes of
antibodies may inhibit LFA-1 binding by disrupting the function of
highly conserved structural elements important for ligand binding. The
fact that the residues important for mAb binding are located in
sequences that vary among the
-integrin
subunits
accounts for their LFA-1 binding specificity.
Figure 7:
Alignment of I domain sequence homologies.
The amino acid sequences of the NH-terminal portion of the
I domains of human CD11a, mouse CD11a, human CD11b, and human CD11c
aligned with each other and with three regions of human von Willebrand
factor A domains (VWFA) and human cartilage matrix protein (CMP). Boxed regions represent regions of sequence
homology. Shaded regions represent nonhomologous regions
corresponding to the epitopes identified in this study. The locations
of I domain epitopes IdeA, IdeB, and IdeC are indicated above the alignment.
The third epitope
(IdeC) recognized by CD11a-blocking mAbs was localized to residues
197-203 (H/M 54). Of the four mAbs that bound to this site, two
distinct specificities could be identified: mAbs that depended on
epitopes at residues 143-149 and 197-203 (CLB-LFA-1/2,
32E6, and 50G1) and mAbs (e.g. MHM.24) whose binding depended
only on residues 193-207. The sequence at 193-207, like the
other two epitopes, is unique to CD11a but lies between two conserved
sequences (PXXLL and TXTXXA(I/L)) that are
found in all the -integrin
chains and are
homologous to sequences in von Willebrand factor A repeats, cartilage
matrix protein, and factor B (Fig. 7). These results suggest
that the mAb MHM.24 might inhibit LFA-1 function by interacting with a
sequence that is distinct from the epitopes recognized by the other
LFA-1-blocking mAbs.
Although all of the mAbs used in this study
have been shown to inhibit functional responses in vitro (e.g. CTL killing and mixed lymphocyte reactions), the
possibility exists that the mechanism of action may differ between
antibodies to different epitopes. Several different mechanisms have
been discussed in accounting for the activities of integrin-blocking
mAbs. In principle, the simplest mechanism whereby antibodies can block
adhesive interactions is by competitive binding to the ligand binding
site. Studies by Lollo et al.(54) suggest that this
is the case for the M17 mAb. Because mAbs 25.3 and TS-1/22 appear to
bind to the same epitope as M17, they would also be expected to inhibit
LFA-1 function competitively binding to the ligand (ICAM-1) binding
site. Another mechanism by which antibodies are thought to modulate
LFA-1 function is stabilization of ``activated'' or
``unactivated'' conformations. Dransfield et al.(32) have described an antibody that is thought to inhibit
LFA-1 function by preventing ``de-adhesion,'' thus locking
LFA-1 and Mac-1 into a permanently activated state. Finally, antibody
binding could inhibit function by altering interactions with divalent
cations mediating the transition between activated and unactivated
conformations. Several studies have described cation-dependent epitopes
recognized by antibodies that can activate or inhibit LFA-1
function(32, 33) . This type of mechanism might
account for the activity of the CLB-LFA-1/2 mAb that binds to the IdeB
site and is known to activate T cells in T cell receptor co-stimulation
studies and to inhibit adhesion required for cytotoxic T cell-mediated
lympholysis in studies with human T cell
clones(37, 38) . Michishita et al.(55) have described a novel divalent cation binding site
in the I domain of CD11b that is essential for ligand binding.
Mutagenesis experiments identified two sites of CD11b
(Asp/Ser
and Asp
) that were
critical for cation binding and receptor function but did not affect
the binding of several mAbs known to block CD11b function. In addition,
a third residue, Pro
, was identified that appeared to
shift the optimal cation concentration required for ligand binding but
did not inhibit the binding of CD11b mAbs known to inhibit receptor
function. These residues are conserved among all three
-integrin
chains and correspond to residues
Asp
, Ser
, Asp
, and
Pro
of CD11a. The observations that Pro
was
located just 5 residues away from Lys
(critical for the
binding of mAb MHM.24) and that Asp
and Ser
were located in the conserved region between the epitopes IdeA
and IdeB (critical for the binding of the 25.3, TS-1/22, M17, I21/7,
32E6, 50G1, and CLB-LFA-1/2 mAbs) support our hypothesis that the
conserved regions between residues between IdeA and IdeB, as well as
the regions flanking IdeC, are important for CD11a function. These data
are consistent with the possibility that the CD11a-blocking mAbs that
we have studied may inhibit LFA-1 by affecting the conformation or the
activity of this novel cation binding site. This model is also
consistent with a recent report (53) suggesting that ICAM-1 can
bind to CD11a variants that lack the I domain.
Whether or not the I domain sequences that we have identified contain the LFA-1 ligand (ICAM-1) binding site remains to be established. Because murine LFA-1 does not bind human ICAM-1(56) , analysis of the H/M mutants in functional assays provides a means to test the role of these sequences in ICAM-1 binding. Studies utilizing the mutants described in this paper in cell-based adhesion assays are in progress to determine which of the mutations that inhibit antibody binding also inhibit ligand binding. In this regard it will be interesting to test the inhibitory activity of synthetic peptides corresponding to the I domain sequences identified by the epitope-mapping studies described herein. Studies of this type should help to determine whether the I domain participates in receptor function by providing contact residues that interact with ligand or by providing structural elements necessary to maintain the conformation of a ligand binding site in a remote part of the molecule. An interesting possibility is that the I domain represents an ion-sensitive regulatory domain that has been incorporated into the prototypic integrin structure to govern the transition between activated and unactivated conformations.