(Received for publication, April 28, 1995)
From the
Previous studies have shown that lymphocyte function-associated
antigen-1 (LFA-1) molecules containing the human (CD11a) and
human
(CD18) subunits but not the murine
and human
subunits can bind to human intercellular adhesion molecule 1 (ICAM-1).
Using human/mouse LFA-1
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
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.
The lymphocyte function-associated antigen-1 (LFA-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
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
2 integrin subunit and have homologous
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 subunit,
L, 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
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
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 -sheet surrounded by amphipathic
-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
-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
1 and
2 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 subunits in intact
heterodimers expressed on the cell surface to define in detail
structural regions of the LFA-1
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
subunit
(Johnston etal., 1990). The human and
murine LFA-1
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.
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.
Chimeric 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 h
L,
residues 154-359 are from m
L, and residues 360 to the C
terminus are from h
L. 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 h
L or m
L, respectively, and 2.1-kb fragments
from complete MscI and HindIII digestion of m
L
or h
L cDNA, respectively. Chimera h359m was constructed by
ligation of a 8.2-kb fragment from complete HindIII and
partial Sse8387I digestion of h
L cDNA and a 1.2-kb
fragment from complete HindIII and Sse8387I digestion
of m
L cDNA. m153h359m was constructed with a 0.6-kb fragment from
complete HindIII and BspHI digestion of m
L 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 h
L with HindIII and BspHI was ligated to a 8.3-kb fragment
from complete HindIII and partial BspHI digestion of
m
L 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 h
L to make h153m359h.
Figure 1:
Structure-function
studies with subunit chimeras. A, three restriction
sites were used to generate
subunit chimeras (openbars, human sequence; hatchedbars,
mouse sequence). Sites and the aa residue at which they occur are
indicated. B, human
L (h), mouse
L (m), and human/mouse
L subunit chimeras or vector alone
were coexpressed with the human
subunit in transfected COS cells.
2 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;
,
2 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
2 subunit or bound to
ICAM-1.
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 2 subunit expression of COS cells
cotransfected with wild-type or scanning mutant
subunits and the
human
2 subunit was measured as described in Fig.1.
, binding to ICAM-1;
,
2 subunit expression. Data
are mean and S.D. of at least three
experiments.
Figure 5:
Mapping of mAb epitopes on the
subunit. A, chimeras. B, scanning mutants. COS cells
cotransfected with wild type, chimera, or scanning mutant
subunits and the human
subunit- or mock-transfected cells were
stained with mAb to the
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 hL. 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 h
L and a
neighboring fragment from m
L. 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
L 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 h
L cDNA fragment in Ap
M8
produced by digestion with the same enzymes. Scanning mutants m57h,
h57m74h, h74m93h, and h93m117h (Fig.2A) were
transferred into
L 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
L 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.
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.
COS cells were cotransfected with the chimeric
subunits and the wild-type human
2 subunit. To test for
association of the
subunit chimeras with the
2 subunit and
expression on the cell surface, the transfected COS cells were stained
with anti-
L mAb and anti-
2 mAb TS1/18 and subjected to flow
cytometry. Association between
and
subunits is required for
efficient surface expression of the LFA-1
and
subunits
(Hibbs etal., 1990; Larson etal., 1990); as shown by stimulation of
subunit expression, all five
subunit chimeras associated with the
2 subunit and were expressed on the COS cell surface comparably to
wild type human and murine
subunits (Fig.1B).
The overall conformation of the chimeric
subunits on the cell
surface was intact, because they reacted with a variety of antibodies
to different epitopes on the
subunits (see below, Fig.5A). The molecular size of the chimeric
subunits was the same as human or mouse
as shown by
I labeling of COS cell transfectants and
immunoprecipitation with both anti-
and anti-
mAbs (data not
shown).
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 L, 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.
Figure 3:
Alignments of the human and mouse LFA-1 I
domains, and the human Mac-1 I domain. The strands and
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
2 subunit expression of COS cells cotransfected with wild-type or
scanning mutant
subunits and the human
2 subunit was
measured as described in Fig.1. Data are mean and S.E. of at
least three experiments.
, binding to ICAM-1;
,
2
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.
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).
We have used two approaches to define segments of the LFA-1
subunit important for binding to ICAMs. The first approach relied
on the observation that the human but not mouse
subunit enabled
binding of the LFA-1
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 L 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 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 5
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
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 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
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
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
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 subunit. Amino acid residue
numbers are for the human LFA-1
subunit. The boldlines over the
subunit indicate the regions that
restrict species-specific binding to ICAM-1. The thinlines below the
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 1 helix and the long loop between
the
3 and
4 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
subunits expressed in the absence of
subunit, whereas we used
immunofluorescent labeling of
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
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
subunit
(Sanchez-Madrid etal., 1983) and mutation
of a MIDAS-like site in the conserved region of the
subunit (Bajt
and Loftus, 1994; Lee etal., 1995) also
inhibit ligand binding. How both the
and
subunit contribute
to ligand binding and work together to regulate integrin adhesiveness
remains to be established.