From the Joseph J. Jacobs Center for Thrombosis and
Vascular Biology and the Department of Molecular Cardiology, Lerner
Research Institute, Cleveland, Ohio 44195 and the § J.
Holland Laboratory, American Red Cross, Rockville, Maryland 20855
Received for publication, November 8, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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The leukocyte integrin
Integrin In previous studies, Altieri et al. (10, 11) demonstrated
that a peptide (designated P1), corresponding to residues 190-202 of
the Within the heterodimeric Proteins, Peptides, and Monoclonal Antibodies--
Human kidney
293 cells expressing wild-type and the mutant forms of the
Expression of Recombinant
To express the
Site-directed mutagenesis of the Generation of the Flow Cytometry--
FACS analyses were performed to assess the
expression of Adhesion Assays--
The wells of tissue culture plates (Costar,
Cambridge, MA) were coated with 6.1, 12.5, 25, 50, and 100 µg/ml P2-C
peptide or 1, 5, and 25 µg/ml of D100 fragment for 3 h at 37 °C. The amount of peptides immobilized onto the wells was
measured by utilizing radiolabeled peptides (12). The coated wells were
postcoated with 0.5% polyvinylpyrrolidone for 1 h at 22 °C.
The 293 cells expressing wild-type or mutated forms of
Solid Phase Binding Assays--
To test the interaction of the
wild-type
To examine the interaction of the D fragment with the I-domain
peptides, 96-well plates (Immulon 2 HB, Dynex Technologies Inc.) were
coated with peptides Pro147-Arg152,
Pro201-Lys217,
Lys245-Tyr252, and
Glu253-Arg261 at 100 µM (0.1 ml/well) overnight at 4 °C and postcoated with 3% BSA for 1 h
at 22 °C. 10 µg/ml biotinylated D98 in 50 mM Tris-HCl buffer, pH 7.5, and 0.05% Tween 20 were added
to the wells and incubated for 2.5 h at 37 °C. In parallel, the
same amount of the D98 was mixed with different
concentrations of P1, P2 peptides, or NIF and added to the wells with
immobilized
To demonstrate the direct binding of the P2-C region ( Statistical Analyses--
Values of adherent cells bound to
substrates are given as means ± S.E. and are based on two to six
independent experiments with each Binding of Wild-type Recombinant Binding of P2-C to Mutant Cell Lines--
As the first step to
define the binding site for P2-C within the
The adhesion of each mutant was measured with increasing concentrations
of immobilized P2-C and D100 fragment to determine the
maximal level of adhesion. In all cases where a cell line bearing a
mutant receptor did adhere to either substrate, the adhesion was
dependent upon the concentration of the immobilized ligand and reached
a plateau. This maximal adhesion was compared with that of the cells
expressing wild-type
The results of adhesion of 16 mutants to P2-C and the
D100 fragment are summarized in Fig.
3, A and B. The
data are expressed as the percent adhesion of the wild-type
The following mutants supported the same or higher levels of
adhesion to P2-C compared with the cells expressing the wild-type Interaction of P2-C and D Fragment with the
The interaction of the D98 fragment with
Lys245-Tyr252 and
Glu253-Arg261 was characterized further. The
binding of D98 to immobilized Lys245-Tyr252 and
Glu253-Arg261 was effectively inhibited by P2-C
and P2 (
In addition, we were able to detect direct binding of P2-C to the
immobilized Grafting of the
Identification of Residues within the
In this study, we have identified key elements of the binding site
for a small amino acid sequence of Fg, M
2 (Mac-1, CD11b/CD18) is a cell
surface adhesion receptor for fibrinogen. The interaction between fibrinogen and
M
2 mediates a range of
adhesive reactions during the immune-inflammatory response. The
sequence
383TMKIIPFNRLTIG395, P2-C, within
the
-module of the D-domain of fibrinogen, is a recognition site for
M
2 and
X
2.
We have now identified the complementary sequences within the
MI-domain of the receptor responsible for recognition of
P2-C. The strategy to localize the binding site for P2-C was based on
distinct P2-C binding properties of the three structurally similar
I-domains of
M
2,
X
2, and
L
2,
i.e. the
MI- and
XI-domains
bind P2-C, and the
LI-domain did not bind this ligand.
The Lys245-Arg261 sequence, which forms
a loop
D-
5 and an adjacent helix
5 in the three-dimensional
structure of the
MI-domain, was identified as the
binding site for P2-C. This conclusion is supported by the following
data: 1) mutant cell lines in which the
MI-domain segments 245KFG and
Glu253-Arg261 were switched to the homologous
LI-domain segments failed to support adhesion to P2-C;
2) synthetic peptides duplicating the Lys245-Tyr252 and
Glu253-Arg261 sequences directly bound the D
fragment and P2-C derivative,
384-402, and this interaction was
blocked efficiently by the P2-C peptide; 3) mutation of three amino
acid residues within the Lys245-Arg261 segment,
Phe246, Asp254, and Pro257,
resulted in the loss of the binding function of the recombinant
MI-domains; and 4) grafting the
M(Lys245-Arg261) segment
into the
LI-domain converted it to a P2-C-binding
protein. These results demonstrate that the
M(Lys245-Arg261) segment, a site
of the major sequence and structure difference among
MI-,
XI-, and
LI-domains,
is responsible for recognition of a small segment of fibrinogen,
Thr383-Gly395, by serving as ligand binding site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M
2 participates in the
attachment of leukocytes to the endothelial lining of blood vessels and
the subsequent transmigration of adherent cells during
immune-inflammatory responses (1-3). The engagement of fibrinogen
(Fg)1 by
M
2 on the surface of leukocytes and by
intercellular adhesion molecule-1 (ICAM-1) on the endothelium may play
a role in mediating the adhesion of leukocytes to the vessel wall (4,
5) and in facilitating their subsequent extravasation across the
endothelial monolayer (6). In addition, the binding of deposited
fibrinogen or fibrin to
M
2 may mediate
leukocyte adhesion at extravascular sites of inflammation (7-9).
-chain of the D-domain of Fg, was recognized by
M
2. However, when residues key to the
recognition of P1 by
M
2-bearing cells
were mutated in the
-module,
148-411, this recombinant fragment
was as active as its wild-type counterpart in supporting
M
2-mediated adhesion (12). This
observation led to the search for additional
M
2 recognition sites within the
-chain, and ultimately the P2 peptide, corresponding to
377-395,
was identified (12). Indeed, in comparative analyses, P2 was
10-15-fold more potent than P1 in inhibiting adhesion of the
M
2-expressing cells to the D fragment of
Fg. Further analyses of the adhesion-promoting activity of overlapping
peptides showed that its COOH-terminal part,
383TMKIIPFNRLTIG395, designated P2-C, was
the primary site of its biological activity (12). Recently, a second
leukocyte integrin,
X
2, which is highly
homologous to
M
2, was demonstrated to
bind to the
-module and P2-C peptide (13), and soluble P2-C peptide
efficiently blocked the
X
2-mediated adhesion.
M
2 receptor, the
I-domain, a region of ~200 amino acid residues, inserted in the
M subunit, contributes broadly to the recognition of
ligands by
M
2 (14) and specifically to
the binding of Fg to this integrin (14, 15). In addition to Fg, this
region also was implicated in the binding of ICAM-1(15), iC3b (16), and
neutrophil inhibitory factor, NIF (17, 18). We have shown previously
that P2 interacts with the recombinant
MI-domain and
that NIF partially blocked this interaction (12). Previous studies
suggested that overlapping but not identical sites are involved in the
recognition of iC3b, NIF, and Fg (19). However, although the binding
sites for iC3b and NIF in the
MI-domain were mapped
extensively (20-23), the recognition site for Fg has not been studied.
Recently, sequences key to the binding of NIF and iC3b to the
MI-domain were mapped using a homolog scanning mutagenesis strategy (22, 24). This approach is based upon the
structural similarity of the I-domains of
M and
L and the differences in their ligand recognition;
i.e. the crystal structures of the I-domains of
M and
L are very similar (25-28), but
only the I-domain of
M binds NIF with high affinity (17,
18). Fg, together with NIF and iC3b, does not bind to
L
2, suggesting that differences in the
structure of the
MI- and
LI-domains may
be responsible for their distinction in ligand binding specificity. In
this study, we have sought to localize the binding site for the P2-C
sequence of Fg within the
MI-domain. The strategy
developed was based on the differences in the binding of P2-C to the
MI-,
XI-, and
LI-domains
and involved several independent approaches, including screening of
mutant cells, synthetic peptides, site-directed mutagenesis, and the
gain-in-function analyses. The binding site for P2-C was localized
within the segment
M(Lys245-Arg261), a site of the
major structure divergence between
MI-,
XI-, and
LI-domains. The grafting of this
segment into the
LI-domain converted it to the
P2-C-binding protein. Thus, a small amino acid sequence, P2-C, with a
defined structure within a crystallized domain of fibrinogen (29) is
shown to interact with a small segment that also has a defined
structure within the
MI-domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M
2 receptor were described and
characterized in detail previously (19, 22). These cells were grown as
adherent monolayers in Dulbecco's modified Eagle's medium/F-12 medium
(BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine
serum, 25 mM HEPES, and antibiotics. Human Fg was purified
from fresh human blood by differential ethanol precipitation (30) or
obtained from Enzyme Research Laboratories (South Bend, IN). The
D100 (Mr 100,000) fragment was
prepared by digestion of human Fg with plasmin (Enzyme Research
Laboratories, South Bend, IN) and purified as described (31). The
D98 fragment (Mr 98,000) was
produced by digestion of the D100 with plasmin. This
fragment lacks 5-15 amino acid residues from the COOH terminus of the
-chain and will be described
elsewhere.2 D98
was biotinylated with EZ-link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's instructions. P1 (
190-202), P2 (
377-395), P2-C (
383-395), H19 (
340-357), H2O
(
350-374), L10 (
402-411), and H12 (
400-411) peptides were
synthesized and purified as described (12). In addition, an analog of
P2-C, P2-Ce, a peptide with the extended COOH-terminal end,
384-402, was also synthesized to test for direct binding to
MI-domain peptides. The following peptides duplicating
selected sequences within the
MI-domain were
synthesized: 147PHDFRR152
(Pro147- Arg152),
201PITQLLGRTHTATGIRK217
(Pro201-Lys217),
245KFGDPLGY252
(Lys245-Tyr252),
253EDVIPEADR261
(Glu253-Arg261),
245KFGDPLGYEDVIPEADREG263
(Lys245-Gly263), and
223FITNGARKN232
(Phe223-Asn232). The numbers indicate the
positions of the residues within the
M subunit (numbered
according to Arnaout et al. (32)). NIF (a gift from Corvas
International, San Diego) was labeled with EZ-link Sulfo-NHS-LC-Biotin
according to the manufacturer's protocol. mAbs OKM1, 44a, and 904 were
obtained from ATCC (Rockville, MD). mAb 4-2 (33) was a generous gift
from Dr. B. Kudryk, the New York Blood Center, and mAb 4A5 (34) was
obtained from Dr. G. Matsueda (Bristol-Meyers Squibb).
MI-,
XI-,
and
LI-Domains and Site-directed Mutagenesis--
The
I-domains were expressed as fusion proteins with glutathione
S-transferase (GST) and purified from soluble fractions
of Escherichia coli lysates by affinity
chromatography. The coding regions for the
MI-domain
(residues Asp132-Ala318) and
LI-domain (Gly153-Ile333) were
amplified by polymerase chain reactions using as template plasmids
pCIS2M-
M (19) and
pCIS2M-
L, which contain the cDNA fragments coding for the full-length of
M and
L, respectively. The primers used for the
MI-domain were
5'-TGTCCTGGATCCGATAGTGACATTGCCTTCTTGA (forward) and
5'-TGAGTACCCGCGGCCGCCGCAAAGATCTTCTCCC (reverse). The
primers used for the
LI-domain were
5'-CAGGAAGGATCCAAGGGCAACGTAGACCTGGTATT (forward) and
5'-GTCCTGTTTGCGGCCGCCCTCAATGACATAGA (reverse). The underlined nucleotides are BamHI and NotI
recognition sequences that were introduced in the primers. The
fragments were digested with BamHI and NotI and
cloned in the expression vector pGEX-4T-1 (Amersham Pharmacia Biotech).
The accuracy of the DNA sequence was verified by sequencing. The
plasmid was transformed in E. coli strain BL-21(DE3)pLysS
competent cells, and expression was induced by adding 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3-5 h at
37 °C.
XI-domain, the following primers were
used to amplify a cDNA fragment encoding the
XI-domain (residues
Glu148-Ala335) from a randomly primed cDNA
library of U937 monocytoid cell line:
5'-AGGCTACCGGGATCCAGACAGGAGTGCCCAAGA (forward) and
5'-CATCTCCCAATTTGGCGGCCGCACTGCTTGTGGTC (reverse). The
product was digested with BamHI and NotI and
cloned into pGEX-4T-1. The plasmid was transformed in E. coli BL-21(DE3)pLysS cells, and the correctness of the
XI-domain insertion was confirmed by sequencing. The
XI-domain was expressed and purified from the cell
lysates as a fusion protein with GST under the conditions used for the
MI- and
LI-domains.
MI-domain was performed
by using QuickChangeTM mutagenesis kit (Stratagene, San
Diego). The pGEX-4T-1 construct containing DNA encoding the
MI-domain was modified by site-directed mutagenesis
using two mutagenic primers containing the desired mutation. The
mutations introduced in the
MI-domain and the primers used are listed in Table I. The
oligonucleotide primers, each complementary to opposite strands of the
vector, were extended during temperature cycling by using
PfuTurboTM DNA polymerase. Following temperature
cycling, the product was treated with DpnI endonuclease to
digest the parental DNA template. The nicked vector DNA incorporating
the desired mutations was then transformed into the Epicurian
Coli® XL1-Blue supercompetent cells, and cDNA from
individual bacterial clones was analyzed by sequencing. The E. coli BL-21(DE3)pLysS host cells were then transformed with the
mutant plasmids, and the mutant
MI-domains were prepared
as described above for the recombinant wild-type
MI-domain. The immunoreactivity of wild-type and mutant
recombinant I-domains was analyzed by enzyme-linked immunosorbent assay
with mAbs 44a and 904 using our standard protocol (35).
Mutations in the Lys245-Arg261 sequence of the
recombinant MI-domain and nucleotide sequences used for
their constructing
L(
M
(Lys245-Arg261))I-Domain Chimera--
The
segment corresponding to the sequence
Ala267-Asp278 within the
LI was exchanged to the homologous segment of the
MI-domain Lys245-Arg261. The
segment switch was created by oligonucleotide-directed mutagenesis using polymerase chain reaction. The construction of the chimera was
based on the observation that oligonucleotide sequence corresponding to
M(Lys245-Arg261) contains a
unique restriction site for Eco81I (SauI),
whereas the second site for this enzyme is present between 4760 and
4761 of the pGEX-4T-1 sequence. To switch the
Lys245-Arg261 from
M to
L, the mutagenic primers were designed to contain the
Eco81I site within the
M(Lys245-Arg261) segment and
additional unchanged
L bases:
5'-CATCCCTGAGGCAGACAGAATCATCCGCTACATCATCG (forward), 5'-GCTGCCTC
AGGGGATCACATCTTCATAACCCAATGGATCTCCAAACTTCTCCCCATCCGTGATGATGATAAG (reverse) (the
M sequences are in bold, and the
restriction site for Eco81I is underlined). The pGEX-4T-1
vector containing DNA encoding the
LI-domain was
modified by polymerase chain reaction using PfuTurbo DNA
polymerase with the following cycling parameters: 95 °C for 30 s, 55 °C for 1 min, 68 °C for 11.5 min. Following temperature
cycling, the product was treated with DpnI to digest the
parental DNA template. The linear product was purified and digested
with Eco81I to produce the two cDNA fragments with
cohesive ends. These fragments were ligated and transformed into
Epicurian Coli XL1-Blue supercompetent cells. The accuracy of the DNA
sequence and the correctness of the I-domain direction were verified by sequencing. The E. coli BL-21(DE3)pLysS cells were
transformed with the mutated plasmid, and the chimeric molecule was
prepared following the procedure described above for the wild-type and mutant I-domains.
M
2 on the surface of the
cells transfected with wild-type and mutant forms of the receptor. The
cells were harvested, and 106 cells were incubated with
M-specific mAbs OKM1 or 44a at 15 µg/200 µl of cell
suspension for 30 min at 4 °C. The cells were then washed and
incubated with fluorescein isothiocyanate-goat anti-mouse IgG (at a
1:1,000 dilution) for additional 30 min at 4 °C. Finally, the cells
were washed and analyzed in a FACS Star (Beckton Dickinson, Mountain
View, CA). Populations of cells expressing a similar amount of the
receptor were selected by FACS. A clonal population of each mutant was
isolated by limiting dilution. After propagation in culture, the amount
of the
M
2 was again evaluated by FACS
analysis with mAb OKM1.
M
2 were harvested with cell-dissociating
buffer (Life Technologies, Inc.) for 1 min at 22 °C and washed twice
in HBSS/HEPES solution containing 10 mg/ml BSA, and resuspended at
5 × 105/ml in HBSS (without phenol red)/HEPES
supplemented with 1 mM Ca2+ and 1 mM Mg2+ and 10 mg/ml BSA. 100-µl aliquots of
the cells were added to each well and incubated on the adhesive
substrates for 25 min at 37 °C in a 5% CO2 humidified
atmosphere. The nonadherent cells were removed by three washes with
colorless HBSS. The adherent cells were frozen overnight at
20 °C,
then thawed and lysed by the addition of a buffer containing the
CyQuant dye (Molecular Probes, Eugene, OR). The fluorescence was
measured in a Cytofluorimeter (PerSeptive Biosystems, Framington, MA),
and the number of adherent cells was determined from a reference
standard curve prepared according to the manufacturer's protocol.
Additionally, several experiments were performed using our established
protocol with 51Cr-labeled cells (12), and the results were
found to be identical with those obtained using the CyQuant reagent
(see "Results").
M,
X,
L, mutant
M, and chimeric I-domains, 96-well plates (Immulon 4BX,
Dynex Technologies Inc., Chantilly, VA) were coated with P2-C or P2-Ce at 50 µg/ml overnight at 4 °C and postcoated with 3% BSA or 0.5% polyvinyl alcohol for 2 h. The GST-I-domains in 20 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, 0.05% Tween 20, and 5% glycerol were added to the wells and incubated for 3 h at
22 °C. After washing, bound I-domains were detected with an anti-GST mAb (Upstate Biotechnology, Lake Placid, NY) at a 1:5,000 dilution. After washing, goat anti-mouse IgG conjugated to alkaline phosphatase was added for 1 h, and the binding of the I-domains was measured by reaction with p-nitrophenyl phosphate. As a control, the
binding of GST to immobilized P2-C was typically ~5-10% that of
wild-type
MI-domain, and background binding to BSA or
polyvinyl alcohol was subtracted.
MI-domain peptides. After washing,
streptavidin conjugated to alkaline phosphatase (Pierce) was added and
incubated for 45 min at 37 °C. D98 binding was detected
by reaction with p-nitrophenyl phosphate, measuring the
absorbance at 405 nm.
383-395) to
the
MI-domain peptides, experiments were performed as follows. Different concentrations of the P2-Ce peptide,
384-402, in
50 mM Tris-HCl and 0.05% Tween 20 were added to wells with immobilized
MI-domain peptides and incubated for 3 h at 37 °C. After washing, the binding of the
384-402 was
detected with mAb 4-2. The mAb 4-2 recognizes an epitope within this
peptide, although it reacts poorly with authentic
P2-C.3 The binding of this
mAb to
384-402 was measured by reaction with goat anti-mouse IgG,
conjugated to alkaline-phosphatase (Pierce) and using
p-nitrophenyl phosphate for detection.
M
2 cell
line, performing triplicates at each experimental point.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MI-,
XI-, and
LI-Domains to the P2-C
Peptide of Fg--
In previous studies, we have demonstrated that the
P2-C peptide binds directly to the recombinant
MI-domain
(12).
X
2 also recognizes P2-C (13); but
the role of the
XI-domain in this interaction was not
evaluated, and the recognition of P2-C by the
LI-domain
had not been tested. Therefore, the three I-domains were expressed as
GST fusion proteins and tested for their ability to interact with the
immobilized P2-C peptide. As shown in Fig. 1, the recombinant
XI-domain exhibited a dose-dependent and
saturable binding to the P2-C peptide similar to that of the
recombinant
MI-domain. In contrast, the recombinant
LI-domain did not interact with P2-C even at the highest
concentration of the
LI-domain added (200 µg/ml
maximal testable concentration). The binding characteristics of the
isolated I-domains parallel the binding properties of the corresponding
intact receptors on cell surfaces, i.e. the
M
2- and
X
2-expressing cells adhere to Fg and
P2-C, whereas the
L
2-bearing cells do not
(see Figs. 2 and 3). Thus, these data confirm that binding of P2-C to
two highly homologous integrins,
M
2 and
X
2, is mediated by
MI- and
XI-domains, respectively, and further suggest that the
lack of binding of P2-C to
L
2 may be the
result of sequence and/or structural differences.
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Fig. 1.
Binding of the recombinant wild-type
MI- (
),
XI- (
), and
LI- (
) domains to the P2-C peptide
of Fg. Different concentrations of recombinant I-domain-GST
proteins in Tris buffer, pH 7.4, containing 100 mM NaCl, 1 mM Mg2+, 1 mM Ca2+,
0.05% Tween 20, and 5% glycerol were added to the microtiter plates
coated with 50 µg/ml P2-C peptide and postcoated with 0.5% polyvinyl
alcohol and incubated for 3 h at 22 °C. After washing, anti-GST
mAb (1:5,000) was added to the wells and incubated for an additional
1.5 h. The binding of the I-domains then was detected with a
secondary goat anti-mouse IgG conjugated to alkaline phosphatase with
subsequent development of the reaction with p-nitrophenyl
phosphate.
MI-domain,
mutant cell lines, each expressing a mutant
M
2 in which a short
MI-domain sequence was replaced for the corresponding region of the
LI-domain, were tested for their adhesion
to immobilized P2-C or D100 fragment. These mutant cell
lines have been used previously to examine the binding of NIF and iC3b
to
M
2 (22, 24). For such experiments to
be readily interpretable, cell lines expressing high and similar levels
of wild-type and mutant receptors were selected by cell sorting using
mAbs OKM1 and 44a followed by cloning by limiting dilution. The
expression of the receptors as assessed by the mean fluorescence
intensity in FACS analyses differed by less than 2-fold from the
internal wild-type control.
M
2 receptor and mock-transfected cells in the same experiment to allow normalization of
results. The cell lines exhibiting adhesion similar to or greater than
the wild-type
M
2 cells were identified as
"positive" mutants. The pattern of cell adhesion to P2-C obtained
for wild-type
M
2 and a representative
mutant,
M(K231NAF), is shown in Fig.
2. Adhesion of
M(K231NAF) to P2-C was
dose-dependent and saturable with ~70% of added cells
adherent to P2-C at the plateau. Adhesion of wild-type and mutant cells
to a control peptide, H19, was tested and was found to be less than 5%
of added cells. Also, as the essential control, the
L
2-expressing cells adhered poorly to
either adhesive substrates, P2-C and D100, (~10-15% of
added cells) consistent with lack of interaction of
L
2 with Fg. All negative mutants did not
adhere to P2-C; adhesion of these cells was similar to that of
mock-transfected cells.
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Fig. 2.
Adhesion of the
M(K231NAF) mutant and the
cells expressing wild-type
M
2
and
L
2
to different adhesive substrates. 0.1-ml aliquots of 5 × 104 cells expressing
M(K231NAF)
(
), wild-type
M
2 (
), and
L
2 (
) in HBSS/HEPES, supplemented with
1 mM Ca2+ and 1 mM
Mg2+, were incubated in the wells of 48-well plates coated
with different concentrations of P2-C. Adhesion of
M(K231NAF)-expressing cells to H19 is shown
for comparison (dashed line). After 25 min at 37 °C in a
humidified atmosphere containing 5% CO2, the nonadherent
cells were removed by three washes with HBSS, and the amount of
adherent cells was determined using the fluorescent dye CyQuant as
described under "Experimental Procedures." Data are expressed as a
percentage of added cells and are the mean ± S.E. of four
individual experiments. The actual amounts of P2-C and H19 immobilized
onto the plastic wells were determined as described (12) and are shown
on the abscissa.
M
2-expressing cells to each substrate.
Substitutions for the following regions of
MI-domain
abrogated adhesion:
M(Pro147-Arg152),
M(Pro201-Gly207),
M(Arg208-Lys217),
M(K245FG), and
M(Glu253-Arg261). These cell
lines form a group of negative mutants. The lack of adhesion of these
mutants was not the result of decreased surface expression of the
receptor because there was no correlation between adhesion and
expression. Specifically, although surface expression of
M(R208-K217) was 2.0 lower than
that of the wild-type
M
2 cells, adhesion was decreased 6-fold. On the other hand, surface expression of the
M(K245FG) was 1.2-fold higher than that of
the cells expressing the wild-type
M
2
cells, but adhesion was abrogated completely. Surface expression of two
other negative mutants,
M(P147-R152) and
M(E253-R261), was very similar
to that of the wild-type receptor.
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Fig. 3.
Adhesion of the wild-type and the
MI-domain mutants to P2-C (panel
A) and D100 (panel B).
0.1-ml aliquots of 5 × 104 cells were added to the
wells coated with 5, 25, 50, and 100 pmol of each peptide and 1, 5, and
25 µg/ml D100 fragment. Adhesion was performed as
described in Fig. 2. Adhesion of each mutant reached the maximal level
at 25 pmol of P2-C and at 5 µg/ml of the D100 fragment.
The results are presented as a percentage of maximal adhesion attained
with the wild-type expressing cells (WT) at 25 pmol of the
P2-C and 5 µg/ml D100. Dashed lines on each
graph are drawn at the maximal level of adhesion achieved with the
wild-type cells, and dotted lines show the maximal level of
adhesion with mock-transfected cells (Mock). Adhesion of the
cells expressing
L
2 and
X
2 is also shown. Data for each mutant
are from two to six adhesion assays, performed in triplicate at each
experimental point.
M
2 receptor (positive mutants):
M(M153-T159),
M(E162-L170),
M(E178-T185),
M(Q190-S197),
M(K231NAF234), deletion
mutant
M(D248PLGY252),
M(R281-I287), and
M(Q309-E314). Adhesion of
dE262G and
M(F297-T307)
was partially affected; the maximal level of adhesion to P2-C reached
64 ± 5% and 83 ± 7, respectively, of wild-type
M
2 cells. These two receptors were
classified as "intermediate" mutants.
M(D273-K279) was the only mutant
to exhibit differential recognition of P2-C and the D fragment; it
supported adhesion to P2-C effectively (65%) but mediated adhesion to
D100 poorly (15%).
MI-DomainPeptides--
In subsequent analyses,
we focused on the five negative mutants. Peptides corresponding to the
wild-type
MI-domain sequences were synthesized and
tested for their ability to interact with the D fragment and P2-C.
Because the sequences within two of the negative mutants,
M(Pro201-Gly207) and
M(Arg208-Lys217), were
contiguous, one linear peptide spanning
Pro201-Lys217 was prepared. Also, the peptide,
Lys245-Tyr252, containing the sequence of the
negative mutant
M(K245FG) was extended at
its COOH terminus to include the D248PLGY252
sequence. Thus, four peptides were synthesized:
Pro147-Arg152,
Pro201-Lys217,
Lys245-Tyr252, and
Glu253-Arg261. In these experiments, we used a
derivative of the D100 fragment, D98, which has
a higher affinity for
M
2.2
D98 contains the entire P2-C but differs from
D100 in the length of the constituent
-chain, which
terminates at
397/405 in D98 compared with the intact
411 in D100. These
MI-domain peptides were immobilized onto microtiter plates, and the binding of
biotinylated D98 fragment to them was assessed. As shown in
Fig. 4A, two peptides, Lys245-Tyr252 and
Glu253-Arg261, bound D98
effectively. The immobilized Pro201-Lys217
peptide exhibited low binding of D98, and
Pro147-Arg152 and control peptide
Phe223-Asn232 did not bind the fragment.
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Fig. 4.
Binding of the D98 fragment to
the peptides from within the
MI-domain. Panel A, 10 µg/ml biotinylated D98 fragment in 50 mM
Tris-HCl, pH 7.5, and 0.05% Tween 20 was added to the wells coated
with 0.1 ml/well 100 µM each
Pro147-Arg152,
Pro201-Lys217,
Lys245-Tyr252,
Glu253-Arg261, and
Phe223-Phe232 and incubated for 2.5 h at
37 °C. After washing, the bound D98 fragment was
detected using streptavidin conjugated to alkaline phosphatase and
p-nitrophenyl phosphate for disclosure. Panel B,
10 µg/ml biotinylated D98 in 50 mM Tris-HCl
buffer, pH 7.5, with 0.05% Tween 20 was mixed with different
concentrations of P2 (
377-395) (
), P1 (
190-202) (
), H19
(
340-354) (
), and H2O (
350-374) (
) and added to the wells
coated with 100 µM Glu253-Arg261
peptide for 2 h at 37 °C. The binding of the D98
was determined as above. Data are expressed as a percentage of the
D98 binding in the absence of peptides. Shown in
panels A and B are the representative experiments
of three to five independent determinations.
377-395), which contains the P2-C sequence (Fig.
4B; inhibition of D98 binding to
Glu253-Arg261 by P2 is shown). In addition, P1
(
190-202) inhibited the binding of D98 to
Lys245-Tyr252 and
Glu253-Arg261 (Fig. 4B; the effect
of P1 on the binding to Glu253-Arg261 is
shown). Two control peptides duplicating fibrinogen sequences
340-357 (H19) and
350-374 (H2O) did not affect the interaction. P1 inhibited the interaction as efficiently as P2, consistent with
ability of this peptide to compete with P2 for binding to
M
2 (12). The interaction of the
D98 with immobilized
MI-domain peptides was
cation-independent; in fact, the higher level of binding was
observed in the absence than in the presence of 1 mM
Ca2+ or Mg2+ (Table
II). Interestingly, NIF did not inhibit
binding of the D98 fragment to the immobilized
Lys245-Tyr252 and
Glu253-Arg261 peptides at concentrations as
high as 100 µg/ml (Table II), although it efficiently inhibited
adhesion of the
M
2-expressing cells to
the D fragment and P2-C peptide at a concentration as low as 0.1 µg/ml (not shown). These data suggest that NIF does not interact with
the I-domain peptides, which are capable of binding P2-C and the
D98 fragment. Indeed, when direct binding of biotinylated NIF to the immobilized
MI-domain peptides was tested,
NIF did not bind to Lys245-Tyr252 and
Glu253-Arg261 peptides (not shown).
Effect of cations and NIF on the binding of D98 fragment and
the P2-C derivative, 384-402, to immobilized
Glu253-Arg261
384-402 was detected by using the mAb 4-2 as described in
Fig. 5. The data shown are the mean values (±S.D.) of the absorbance
at 405 nm of a representative experiment done in triplicate.
MI-domain peptides. Using a derivative
peptide P2-Ce,
384-402, which contains an epitope for the reporting
mAb 4-2, we demonstrated that P2-Ce efficiently bound to immobilized Lys245-Tyr252 and
Glu253-Arg261 in a dose-dependent
and saturable manner (Fig. 5). The
binding of P2-Ce to Pro201-Lys217 was low,
whereas Pro147-Arg152 and control peptide
Phe223-Asn232 did not bind P2-Ce. Similar to
the interaction of the whole D98 fragment, the binding of
P2-Ce to Lys245-Tyr252 and
Glu253-Arg261 was cation-independent (Table
II). To exclude further the possibility that the binding of P2-Ce to
immobilized Lys245-Tyr252 and
Glu253-Arg261 was nonspecific, we tested two
control fibrinogen peptides,
402-411 (L10) and
400-411 (H12).
These peptides duplicate sequences that reside in close proximity to
P2-Ce (
384-402) and interact with platelet integrin
IIb
3. These peptides contain an epitope
at
405-411 recognized by the mAb 4A5 (34). L10 and H12 did not bind
to the immobilized
MI-domain peptides as judged by the
lack of the mAb 4A5 immunoreactivity, providing further evidence for the specificity of the interaction between P2-Ce and two
MI-domain peptides,
Lys245-Tyr252 and
Glu253-Arg261. Thus, the D98 and
P2-C-derivative bound strongly to the same peptides from within the
MI-domain, Lys245-Tyr252 and
Glu253-Arg261, and the interaction of both
ligands with these peptides followed the same pattern. This is
consistent with the binding of the D98 fragment being
mediated by P2-C.
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Fig. 5.
Binding of the P2-Ce peptide to the
MI-domain peptides. Different
concentrations of P2-Ce (
384-402) in 50 mM Tris-HCl, pH
7.5, with 100 mM NaCl and 0.05% Tween 20 were added to the
well coated with 100 µM solutions of
Pro147-Arg152 (
),
Pro201-Lys217 (
),
Lys245-Tyr252 (
),
Glu253-Arg261 (
), and
Phe223-Asn232 (
) peptides and incubated for
3 h at 37 °C. After washing, the mAb 4-2 at 1:1,000 dilution
was added for 1 h at 37 °C. To detect the binding of the mAb
4-2, the second goat anti-mouse IgG was added, and its binding was
determined by reaction with p-nitrophenyl phosphate,
measuring absorbance at 405 nm.
M(Lys245-Arg261) Segment into
the
LI-Domain Converts It to a P2-C-binding
Protein--
We have sought to provide direct evidence that the two
identified
MI-domain segments,
Lys245-Tyr252 and
Glu253-Arg261, contain a binding site for P2-C.
Guided by the crystal structure of the
MI- and
LI-domains, the whole contiguous
Lys245-Arg261 segment was switched from the
MI-domain into the counterpart region of the
LI-domain. The appropriate DNA sequence of the entire
mutated I-domain was confirmed, and the chimeric I-domain was expressed
as a GST fusion protein. The functional consequence of this switch is
shown in Fig. 6A. The P2-C
peptide was immobilized onto microtiter plates, and the binding of the
chimeric
L(
M(Lys245-Arg261))I-domain
molecule and the parental
LI-domain was tested. Whereas the parental
LI-domain does not bind to P2-C, the
grafting of the
M segment imparted the P2-C binding
function to the chimeric I-domain. Although the affinity of the
interaction could not be assessed accurately from the experimental
format used, the concentration of the immobilized P2-C required to
obtain 50% of the chimeric I-domain binding was similar to that for
wild-type
MI-domain, i.e. ~12 µg/ml for
the chimera compared with ~20 µg/ml for the wild-type
MI-domain. The interaction of the chimeric molecule with
P2-C was blocked by soluble P2-C, thus confirming specificity (Fig.
6B).
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Fig. 6.
Binding of wild-type
LI-domain and
L(
M
(Lys245-Arg261)) chimeric I-domain to
P2-C. Panel A, different concentrations of wild-type
LI- (
) and
L(
M(Lys245-Arg261))I-
(
) domains as fusions with GST in 20 mM Tris-HCl, pH
7.4, 100 mM NaCl, 1 mM Mg2+, 1 mM Ca2+, 0.05% Tween 20, and 5% glycerol were
added to the wells coated with 50 µg/ml P2-C and incubated for 3 h at 22 °C. After washing, bound I-domains were detected by anti-GST
mAb (1:5,000 dilution). After washing, goat anti-mouse IgG conjugated
to alkaline phosphatase was added for 1 h, and the binding of the
I-domains was measured by reaction with the p-nitrophenyl
phosphate. Background binding to BSA or polyvinyl alcohol was
subtracted. Panel B, inhibition of the chimeric I-domain
binding by P2-C. 50 µg/ml chimera was mixed with different
concentrations of P2-C (
) or H19 (
) and then added to the wells
coated with P2-C. The binding of the I-domain was determined as in
panel A. Results are presented as a percentage of maximal
chimera binding in the absence of P2-C.
M(Lys245-Arg261) Segment
Critical for P2-C Binding--
The residues within the
M(Lys245-Arg261) segment,
responsible for the P2-C binding, were identified by site-directed
mutagenesis. The selection of residues for mutational analyses was
based on the following considerations: 1) the side chains of residues
that participate in direct docking of P2-C should be exposed on the hydrated surface of the
MI-domain; 2) given the
similarities in the binding function between
M and
X I-domains, it is likely that the residues that
interact with P2-C should be identical or conserved between two
I-domains; 3) because the switch of the 248DPLGY252 segment did not affect adhesion of
mutant cell line, this sequence was not included in mutational
analyses. Nine residues, Lys245, Phe246,
Gly247, Glu253, Asp254,
Pro257, Glu258, Asp260, and
Arg261 are exposed on the surface of the
MI-domain in the Lys245-Arg261
stretch, and eight residues are identical in the
M(Lys245-Arg261) and
X(Lys262-Ala277) sequences (see
Fig. 7A). Thus, only 5 of the
17 residues in Lys245-Arg261 segment meet both
criteria, being exposed and identical between
M and
X: Lys245, Gly247,
Asp254, Pro257, and Asp260 (Fig.
7A). To examine the contribution of these residues in the ligand binding function, single or multiple point mutations to alanine
were introduced into the
MI-domain (Fig. 7A),
and the capability of mutant proteins to interact with the immobilized P2-C was tested (Fig. 7B). The binding of mutants containing
single or double mutations of Asp254 and Pro257
(mutants 3, 4, 5, and 6) was significantly impaired compared with
wild-type
MI-domain. In contrast, I-domains with
substitutions of Lys245, Gly247, and
Asp260 bound similarly to the wild-type
MI-domain. Because the switch of 245KFG
impaired adhesion of the
M
2-expressing
cells (see Fig. 3, A and B), Phe246
might be critically involved in P2-C recognition. Accordingly, Phe246 was mutated to Arg because previous data had
indicated that substitution of Phe246 to the positively
charged residue abrogated binding of the
MI-domain to
iC3b, whereas mutation to Ala was without effect (23). As shown in Fig.
7B, the binding of the
MI-domain with
mutation F246R was reduced significantly. The reduced binding of the
three mutants, F246R, D254A, and P257A, to P2-C was apparently not
caused by perturbation in conformation based upon the following
observations: 1) The reactivity of the mutant I-domains with
anti-
MI-domain mAbs 44a and 904 was similar to that the
wild-type
MI-domain (not shown), consistent with what
was reported previously for other mutations in this region (20). 2)
Inspection of the three-dimensional structure of the
MI-domain suggested that introduction of these substitutions would not produce conformational changes because side
chains of the three mutated residues do not interact with neighboring
residues and thus do not contribute into stabilization the overall
structure; instead, alanine substitution of Pro257 should
improve the slightly imperfect
5 helix. 3) Typical proteins can
tolerate major mutations within a single loop and remain properly folded (36). Thus, these data suggest that the three residues, Phe246, Asp254, and Pro257, are
directly involved in the P2-C binding.
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Fig. 7.
Binding of the recombinant wild-type
I-domains and MI-domain mutants to
P2-C. Panel A, alignment of the
MI-,
XI-, and
LI-domain sequences. The
M(Lys245-Arg261) sequence was
aligned with human
X(Lys262-Ala277) and
L(Ala269-Asp280) sequences using
the NCBI Blast program. The solvent-exposed residues in
M(Lys245-Arg261) are in
bold, and the residues identical between
M
and
X are underlined. The residues mutated in
the
MI-domain are illustrated in the lower
part of panel A. Panel B, different
concentrations of wild-type (WT)
MI-,
XI-, and
LI-domains and mutant
MI-domains as fusions with GST were added to microtiter
wells coated with 50 µg/ml P2-C and incubated for 3 h at
22 °C. The binding of the recombinant I-domains was detected as in
Fig. 6. The binding of each mutant reached the maximal level at 100 µg/ml of the added I-domain, and results are presented as a
percentage of the binding attained with wild-type
MI-domain. The dashed line indicates the
maximal level of binding achieved with wild-type
MI-domain, and the dotted line is drawn at
the level of binding attained with the recombinant
LI-domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
383-395 (P2-C), within the
MI-domain of
M
2. The
strategy to define the ligand binding site was based on the difference
in the P2-C binding properties of the
MI-,
XI-, and
LI-domains and entailed four
complementary approaches. In the first approach, a series of
homolog-scanning mutants, used previously to map the binding regions
for NIF, iC3b, and Candida albicans (22, 24, 37), were
screened for adhesion to P2-C and D100 fragment of Fg. In
these mutants, 16 segments from the
MI-domain were
replaced with the corresponding segments from the homologous
LI-domain, which does not bind Fg. Because all of these
swapped segments are located at the hydrated surface of the
MI-domain, they should identify candidate sequences for interaction with the ligand. Five mutants lacked the ability to support
adhesion to P2-C and D100:
M(P147-R152),
M(P201-G207),
M(R208-K217),
M(K245FG), and
M(E253-R261). Therefore, these
MI-domain segments may be critical for binding of these
ligands. Alteration of three other regions, dE262G,
Asp273-Lys279, and
Phe297-Thr307, resulted in the partial loss of
adhesive function. These segments may play an accessory role in ligand
binding. Thus, the initial insight provided by these mutant receptors
indicated that the P2-C binding interface within the
MI-domain was composed of several nonlinear sequences.
Based on the crystal structure of the
MI-domain (25),
the segments critical for recognition encompass a portion of helix 1, the loop between helix 3 and helix 4, the small
245KFG segment in the loop between helix 5 and
-strand
D, and the entire helix 5 (Fig. 8,
left). These sequences form an almost continuous stretch on
the upper face of the
MI-domain (colored in different
shades of green in Fig. 8, left).
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Fig. 8.
Binding site for P2-C in the
MI-domain. Left, ribbon
model of the
MI-domain based upon its crystal structure
(25), code 1JLM. The 245KFG in the
D-
5 loop
and Glu253-Arg261 in the helix
5 are
bright green. The segments
Pro147-Arg152 and
Pro201-Lys217 are light green. The
numbers indicate the positions of segments. The side chains
of residue mutations that were found to affect the P2-C binding,
Phe246, Asp254, and Pro257, are
shown in purple.
-Strands and helix assignments are
shown. Right, ribbon model of the
LI-domain,
based upon its crystal structure, code 1LFA (27), shown for comparison.
The region homologous to
M(Lys245-Arg261) is light
blue. The models were drawn using the computer program Molscript,
Bobscript and Raster (44-46).
The second approach entailed the use of synthetic peptides duplicating
the sequences of the critical segments in the M
I-domain. These analyses showed that two of four critical segments,
M(245KFG) and
M(Glu253-Arg261), may contain
amino acid residues that participate directly in binding the P2-C
sequence of Fg because the peptides that duplicated Lys245-Tyr252 and
Glu253-Arg261, bound P2-C. Although the peptide
Pro147-Arg152 did not interact with the Fg
derivatives, and Pro201-Lys217 interacted
weakly, the negative results do not exclude a role for these peptides
in binding function; the immobilized peptide may simply not assume the
appropriate conformation for recognition by the ligand.
The Lys245-Tyr252 and
Glu253-Arg261 sequences are contiguous, and the
entire M(Lys245-Arg261) sequence
might serve as the primary binding site for P2-C. Of note, this segment
is the most divergent between the
MI- and
LI-domains in terms of sequence homology and folding
(Fig. 8, right). In fact,
L lacks most of
helix 5, which is formed by residues
Tyr252-Arg261 of
M (27, 28). In
addition, the loop
D-
5 is longer in
M than in
L and assumes a different conformation. Therefore, this
difference between
M and
L could account
for inability of
L to bind Fg. To obtain direct evidence
that the
D-
5 loop-
5 helix in the
MI-domain
constitutes the functional binding site for the Fg ligand, the third
approach entailed grafting of the entire
Lys245-Arg261 segment into the corresponding
region of the
LI-domain. As demonstrated in Fig. 6, this
manipulation imparted P2-C binding capacity to the chimeric molecule,
and the binding affinity of the chimeric receptor for P2-C was very
similar to that of wild-type
MI-domain. Thus, the role
of the
D-
5 loop-
5 helix in P2-C binding, which initially was
inferred from the loss-in-function experiments, was verified by the
gain-in-function approach.
Finally, the implementation of the fourth method, site-directed
mutagenesis, served the dual purpose. First, it provided the independent confirmation that the Lys245-Arg261
segment is important for P2-C binding because mutations of the three
residues resulted in the significant loss of P2-C binding. Second, it
implicated Phe246, Asp254, and
Pro257 as contact residues. Taken together, the four
approaches substantiated independently the role of the D-
5
loop-
5 helix in the P2-C binding and provided evidence that three
residues participate in ligand docking.
It is unclear why switches of the
M(Pro147-Arg152) and
M(Pro201-Lys217) resulted in the
loss of function given that two other unsubstituted segments,
M(245KFG) and
M(Glu253- Arg 261) could
potentially support adhesion. The effect of the switches of
M(Pro147-Arg 152) and
M(Pro201-Lys217) on P2-C
recognition could conceivably arise from changes of conformation of the
ligand binding region residing in the
M(Lys245-Arg261). In this
regard, a subtle perturbation of the structure of
M(P201-G207) and
M(P147-R152) mutants was
suggested previously by an altered reactivity with the
conformation-dependent mAb 24 (22). In addition to the
MIDAS motif (25), which is known to be required for the normal binding function (21, 38), single point mutations in the regions outside MIDAS
also may abrogate ligand binding by altering conformation (21, 38, 39).
For example, alanine substitution of Asp248 or
Tyr252, the residues that are not exposed on the surface of
the
MI-domain, eliminated the binding of mutants to iC3b
(21), suggesting that a structural alteration might have been involved
in the loss-of-binding function. Thus, even relatively small
perturbations in the folding of the I-domain can lead to gross
alterations in binding affinities.
Although M(Lys245-Arg261)
resides in close proximity to the cation binding MIDAS motif, none of
the residues in this sequence is directly involved in coordination of
the divalent cation (25). Our results indicate that the binding of Fg
derivatives, the D fragment and P2-C, to immobilized peptides
duplicating Lys245-Arg261 region was
cation-independent. This finding is consistent with previous data
showing that EDTA or mutation of Asp242, a residue which
coordinates to the bound metal, only partially impairs the binding of
Fg to the
MI-domain although it abolished the binding of
other ligands, including NIF and iC3b to the recombinant fragment (25).
Another report also suggests that ligands can bind to I-domains
independent of cations. Peptides duplicating
D-
5 loop in the
MI-domain or immediately preceding it bound to iC3b in a
cation-independent manner (16). Thus, at least in the case of P2-C, its
binding to the
MI-domain does not occur through direct
interaction with the metal ion as was proposed (25). This conclusion is
further supported by the fact that P2-C sequence in Fg does not contain
a candidate acidic residue to provide a missing coordination to the
metal. At the same time, P2-C does contain an arginine residue,
Arg391, which could displace cation, a model suggested from
the crystal structure of the
1I-domain (40).
The sequence M(Lys245-Tyr252),
which overlaps with the identified Fg-binding region
M(Lys245-Arg 261), was
implicated previously in the binding of iC3b. Deletion of
Phe246-Tyr252 abolished rosetting of
iC3b-coated erythrocytes with
M
2 (21). In
addition, mutation of Lys245 to Ala (24) and
Phe246 to Lys (24) also significantly reduced iC3b binding.
However, although the binding site for iC3b overlaps with the Fg
binding site, the contribution of this region in recognition of two
ligands appears to be distinct. For example, deletion of
248DPLGY did not affect adhesion to P2-C or
D100 fragment in our experiments, but it reduced to some
extent iC3b binding (19).
The overlapping nature of the NIF and Fg binding sites within the
MI-domain was suggested previously based on the ability of NIF to inhibit interaction of the
M
2-bearing cells with Fg (18, 19). The
same segments which were identified as critical for Fg binding,
M(Pro147-Arg152),
M(Pro201-Gly207),
M(Arg208-Lys217), and
M(Glu253-Arg261), also have been
shown to participate in NIF binding (22). The significant differences
in the binding of
MI-domain mutants to these two ligands
were: 1) switch of 245KFG, which completely abrogated
adhesion to Fg peptides, was not critical for NIF binding; and 2)
deletion of 248DPLGY, which affected NIF binding, was not
detrimental for adhesion to Fg derivatives. The binding site for NIF
was verified previously by grafting the identified segments into the
XI-domain because these swaps imparted NIF binding
capacity to the chimeric receptor (22). However, because the identified
segments were grafted simultaneously, it is uncertain whether all of
these sequences contain NIF contact sites or if some may provide
structural elements necessary to maintain a permissive conformation for
NIF binding. That NIF did not inhibit the binding of the D fragment or
P2-C to immobilized Lys245-Tyr252 and
Glu253-Arg261 suggests that these segments of
the
MI-domain may not contain contact sites for NIF.
Therefore, one possibility is that the primary binding site for NIF may
reside in the two other identified segments,
M(Pro147-Arg152) and
M(Pro201-Lys217). Indeed,
switching
M(Pro147-Arg152),
M(Pro201-Gly207), and
M(Arg208-Lys217) decreased
affinity for NIF 33-, 305- and 206-fold, respectively (22). At the same
time, affinity of the
M(E253-R261) swapping mutant or
M(D248PLGY252) deletion mutant
was reduced 8- and 13-fold, respectively (22). In addition, Rieu
et al. (20) demonstrated the importance of Gly143, Asp149, and Arg208, which
reside close or within
M(Pro147-Arg152) and
M(Pro201-Lys217), for NIF
binding. The extension of this hypothesis is that NIF and P2-C do not
compete for the same binding site on the
MI-domain but
rather that NIF blocks adhesion to Fg by steric or allosteric effects.
Also, the epitope for the mAb CBRM1/5 was recently mapped to a region
that includes Pro147, His148,
Arg151 within
M(Pro147-Arg152) and
Lys200, Thr203, and Leu206 within
the
M(Pro201-Arg208) segment
(41). This mAb blocks the
M
2-mediated
adhesion to several ligands including Fg (42). Furthermore, the epitope for the second inhibitory mAb, 7E3, which recognizes the active I-domain conformation and interferes with binding of Fg to stimulated leukocytes resides in the
M sequence
Gly127-Phe150 (43). Therefore, this region in
the
MI-domain may contain a unique regulatory element
that controls ligand recognition to other portions of the
MI-domain.
In summary, we have determined the binding site for the P2-C sequence
of Fg, 383-395, within the
MI-domain of
M
2. The binding site was localized in the
17-mer linear sequence, Lys245-Arg261, thus
mapping the site to a very narrow region on the MIDAS face of the
MI-domain. The interaction between P2-C and the
MI-domain Lys245-Arg261 may
represent a minimal functional recognition unit and, thus, defines
complementary binding sites within the
MI-domain and ligand. The
M(Lys245-Arg261)
sequence is a region of the major difference between the I-domains of
two related integrins,
M
2 and
L
2, in terms of sequence and structure.
Thus, the regions of structural divergence may provide the potential
mechanism whereby integrins specify ligand recognition.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. C. Forsyth for valuable technical advice regarding the mutant cells propagation, Dr. B. Kudryk for providing mAb 4-2, Dr. G. Matsueda for mAb 4A5, and Corvas International Inc. for NIF. We thank Jane Rein for secretarial assistance. FACS analyses were performed at the Institutional core facility established with a gift from the W. M. Keck Foundation.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL-63199 (to T. P. U.) and HL-54924 (to E. F. P).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, NB50, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8209; Fax: 216-445-8204; E-mail: ugarovt@ccf.org.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010174200
2 T. P. Ugarova, V. P. Yakubenko, B. Kudzyk, E. F. Plow, and V. C. Yee, manuscript in preparation.
3 V. P. Yakubenko and T. P. Ugarova, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
Fg, human
fibrinogen;
ICAM-1, intercellular adhesion molecule-1;
I-domain, region
of ~200 residues "inserted" in the -subunit of
M
2,
X
2, and
L
2;
NIF, neutrophil inhibitory factor;
mAb, monoclonal antibody;
GST, glutathione S-transferase;
FACS, fluorescence activated cell sorting;
HBSS, Hanks' balanced salt
solution;
BSA, bovine serum albumin.
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