(Received for publication, September 18, 1995; and in revised form, November 20, 1995)
From the
The VLA-4 (4
1) integrin is involved in the adhesion of
cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In
order to study
4 structure-function relationships, we have
expressed mutated
4 subunit by transfection into VLA-4-negative
K562 cells. Substitutions at
4 residues
Arg
-Asp
, which show the highest surface
probability indexes inside the N-terminal
fragment, resulted in a reduction in the reactivity of all
anti-
4 epitope A monoclonal antibodies (mAbs) tested, compared
with the reactivity with anti-
4 epitopes B1, B2, and C mAb, both
by transfectant flow cytometry, and by immunoprecipitation and
SDS-polyacrylamide gel electrophoresis analysis of transfectant
surface-iodinated proteins. In contrast, substitutions at nearby
residues, Gln
, Pro
, and Ile
did not affect the reactivity of any anti-
4 mAb representing
the known
4 epitopes. Homotypic cell aggregation triggered by
anti-
4 epitope A mAb was prevented in the transfectants expressing
mutated
4 Arg
-Asp
Asp residues,
while cell aggregation was fully achieved with either anti-
4
epitope B2 or anti-
1 mAb. Mutations at
4 residues
Gln
, Pro
, and Ile
did not
affect the homotypic cell aggregation of the transfectants expressing
these mutations. In addition, the adhesion of mutant
Arg
-Asp
4 transfectants to the
connecting segment-1-containing fibronectin-40 (FN-40) fragment of
fibronectin was diminished compared to wild type
4 transfectants,
as well as to other mutant
4 transfectants. This adhesion to FN-40
was restored when the activating anti-
1 TS2/16 mAb was present in
the adhesion assays. In contrast, adhesion to VCAM-1 was not affected
by mutations at Arg
-Asp
, nor at
Gln
, Pro
, and Ile
4
residues. Altogether, these results indicate that
4 residues
Arg
and Asp
are included in a region involved
in homotypic cell aggregation, as well as in adhesion to FN-40, but not
to VCAM-1.
The 4 integrins play an important role in leukocyte
extravasation during inflammation, lymphocyte traffic to lymphoid
organs during a normal immune response, and hematopoietic progenitor
cell adhesion to bone marrow stroma (reviewed in (1, 2, 3, 4) ). The
4 subunit
can associate with the
1 or the
7 chains conforming the
heterodimers
4
1 (VLA-4) and
4
7, respectively. VLA-4
interacts with the sequence EILDVPST within the alternatively spliced
connecting segment-1 (CS-1) (
)region of
fibronectin(5, 6, 7) , and to domains first
and fourth of vascular cell adhesion molecule-1
(VCAM-1)(8, 9, 10) . Interaction with other
sequences on fibronectin(11, 12, 13) , as
well as with thrombospondin(14) , requires VLA-4 to be
activated. VLA-4 also interacts with the bacterial coat protein invasin
in an activation-independent manner(15) .
4
7 known
ligands include the fibronectin CS-1 region, VCAM-1, and mucosal
addressin cell adhesion molecule
(MadCAM)(16, 17, 18, 19) .
In
addition to most types of leukocytes, VLA-4 is also expressed on
various nonhematopoietic tumor cells such as melanomas (20) and
during muscle differentiation at the stage of myotubes(21) .
The 4 subunit is expressed on nonlymphoid tissues in developing
mouse embryo(22, 23) , and it has been reported that
the absence of a functioning
4 gene results in defects in
placental and cardiac development, leading to embryonic
lethality(24) .
4
7 integrins are expressed on most
lymph node T and B cells(25) , on subsets of CD4
memory T cells(18) , and on lymphocytes present in
rheumatoid synovium(26) . The
4 subunit can be expressed
at the cell surface as an uncleaved 150-kDa form, or as proteolytically
cleaved fragments of 80 and 70 kDa, designated previously as
and
,
respectively(27) .
Most data accumulated so far on the in vivo role of the 4 integrins have come from studies
using anti-
4 mAb in animal models. Lung antigen
challenge(28, 29, 30) , experimental allergic
encephalomyelitis(31, 32) , ulcerative
colitis(33) , contact
hypersensitivity(34, 35) , and
diabetes(36, 37, 38) , are among the
processes where
4 integrins play a significant role. The
inhibitory effect of the anti-
4 mAb in these processes comes from
the blockade of VLA-4/ligand interactions, resulting in an inhibition
of leukocyte recruitment.
Functional epitope mapping of the 4
subunit with a wide panel of anti-
4 mAb revealed the existence of
three topographically distinct epitopes(39) . Epitope A
anti-
4 mAb were able to induce homotypic aggregation, blocked
partially adhesion to the CS-1-containing FN-40 fibronectin fragment
and did not inhibit adhesion to VCAM-1. Epitope B mAb were subdivided
into B1 and B2, both blocking adhesion to FN-40 and VCAM-1, with the
difference that B2 mAb also triggered homotypic cell aggregation, while
B1 mAb did not(39) . The only effect so far described for
epitope C mAb is the blocking of homotypic aggregation (39) .
A precise localization of the 4 residues involved in
VLA-4-mediated functions will contribute to the understanding of the
interactions between VLA-4 and its ligands and could help in the
designing of compounds aimed at blocking this interaction during
unwanted inflammatory processes. In the present work we have analyzed
the effect of amino acid substitutions at selected positions in the
amino-terminal end of the
4 integrin subunit, in the adhesion of
K562 cells expressing transfected mutant
4 to fibronectin and
VCAM-1, as well as in homotypic cell aggregation.
Figure 1:
A, surface probability profile of the
first amino-terminal 200 amino acids from the integrin 4 subunit.
The index of surface probability according to Emini (see Kyte and
Doolitle(52) ) for each
4 residue is plotted. The amino
acids subjected to site-directed mutagenesis are indicated. B,
schematic diagram of the amino-terminal end of
4 and site-directed
mutagenesis of the
4 cDNA. Shown are the double and single
mutations introduced on selected
4 residues and the designation of
the
4 transfectants. C denotes the cysteines included in
the first 200 amino-terminal amino acids.
Expression of 4
by the transfectants was analyzed by flow cytometry using anti-
4
mAb recognizing
4 epitopes A (HP1/1, HP1/3, HP1/7), B1 (HP2/1), B2
(HP2/4), and C (B-5G10)(39) . Wild type
4 transfectants,
here called 4M7, as well as QP(101-102)HL and I108M mutants,
expressed all
4 epitopes at comparable levels ( Fig. 2and Table 1). However, the RD(89-90)SA mutants consistently
showed a weaker staining with anti-
4 epitope A antibodies,
compared to the staining with antibodies recognizing epitopes B1, B2,
and C on the same cells ( Fig. 2and Table 1). On average,
the decrease in the mean fluorescence values obtained in the
RD(89-90)SA transfectants with the anti-
4 epitope A mAb
HP1/1 and HP1/7 were 52 and 45%, respectively, of those obtained with
the HP2/4 (epitope B2), and 48 and 38% compared with those obtained
with B-5G10 (epitope C). Flow cytometry analysis of G130R transfectants
showed only weak
4 expression detectable with the epitope C B-5G10
antibody, while no staining was detected with any anti-
4 antibody
in the DL(138-139)QV transfectants through all rounds of
transfections (Table 1).
Figure 2:
Flow cytometry analysis of K562 4
transfectants. 4M7 (wild type
4 transfectants), and the
4
mutant RD(89-90)SA, QP(101-102)HL, and I108M cells were
incubated at 4 °C for 40 min with saturating concentrations of
control P3X63 mAb, anti-
1 (TS2/16) mAb, or the
anti-
4 mAbs HP1/1, HP1/3, HP1/7 (Epitope A), HP2/1 (Epitope B1), HP2/4 (Epitope B2), and B-5G10 (Epitope C). After washing, cells were incubated at 4 °C
for 30 min with FITC-conjugated secondary antibody (Dako, Denmark) and
finally analyzed using a Coulter Epics XL flow
cytometer.
We next surface iodinated wild type
and mutant 4 transfectants, and after solubilization the cell
extracts were immunoprecipitated with anti-
4 antibodies
representing epitopes A, B1, and C, as well as with anti-
1
antibodies, followed by SDS-PAGE. The 4M7, QP(101-102)HL, and
I108M cells showed a characteristic pattern of
4 structural forms
in
4-K562 transfectants(27) . Thus, most of
4 is
expressed at the cell surface as a cleaved
form, with little expression of the uncleaved
form (Fig. 3). No differences in terms of
4 structure
and expression levels with the various anti-
4 antibodies were
found amongst these cells (Fig. 3). In contrast, the anti-
4
epitope A HP1/1 and HP1/7 immunoprecipitates from the RD(89-90)SA
transfectants showed a dramatic reduction in the amount of cell surface
4 subunit compared with that found in HP2/1 and B-5G10
immunoprecipitates, which were identical to their counterparts in the
other three transfectants (Fig. 3). Also, anti-
1
immunoprecipitates from RD(89-90)SA cells did not change with
respect to the anti-
1 immunoprecipitates from 4M7,
QP(101-102)HL, and I108M cells (Fig. 3). Together with the
flow cytometry analyses, these results indicate that
4 residues
Arg
-Asp
are contained within the region
corresponding to
4 epitope A.
Figure 3:
Immunoprecipitation of wild type and
mutated forms of the 4 subunit. 4M7, RD(89-90)SA,
QP(101-102)HL, and I108M
4 transfectants were labeled with
I and solubilized, and the cell extracts were
immunoprecipitated with saturating concentrations of the anti-
4
mAbs HP1/1 or HP1/7 (epitope A), HP2/1 (epitope B1), B-5G10 (epitope
C), anti-
1 mAb TS2/16, or control mAb P3. Immunoprecipitates were
analyzed under nonreducing conditions by SDS-PAGE and autoradiography.
The migration of the different structural forms of the
4 subunit
and
1 subunit is indicated. The migration of the
5 integrin
subunit which is expressed by parental K562 is indicated, as obtained
by immunoprecipitation with the anti-
5 P1D6 mAb (not
shown).
Figure 4:
Loss
of homotypic cell aggregation in the RD(89-90)SA transfectants
induced by the anti-4 epitope A mAb HP1/7. 4M7, RD(89-90)SA,
and QP(101-102)HL
4 transfectants (10
cells/well) were incubated at 37 °C with the anti-
4
epitope A mAb HP1/7 or with the the anti-
4 epitope B2 mAb HP2/4.
Photographs were taken 3 h after the addition of
antibodies.
Figure 5:
Adhesion of 4 transfectants to FN-40.
4M7, RD(89-90)SA, QP(101-102)HL, and I108M cells were
labeled with BCECF-AM, incubated in the absence or in the presence of
the anti-
1 mAb TS2/16 (1/6 final dilution from culture
supernatant) for 20 min at 37 °C, and added to 96-well microtiter
plates coated with increasing concentrations of FN-40. After 20 min of
incubation at 37 °C, plates were washed, and adhesion was
quantified using a fluorescence analyzer. Each point represent the mean
of triplicate determinations, with a standard deviation of <10%.
K562 cells showed no adhesion at any concentration of FN-40 tested (not
shown).
When we analyzed cell adhesion to sVCAM-1, no significant differences among the 4M7, RD(89-90)SA, QP(101-102)HL, and I108M transfectants were observed, both in the absence and in the presence of TS2/16 (Fig. 6). In addition, the increment of cell adhesion to sVCAM-1 in the presence of TS2/16 was lower than that observed in the case of FN-40, and was observed mainly at lower sVCAM-1 concentrations (Fig. 6).
Figure 6:
Adhesion of 4 transfectants to
sVCAM-1. 4M7, RD(89-90)SA, QP(101-102)HL, and I108M cells
were labeled with BCECF-AM and processed for adhesion to increasing
concentrations of sVCAM-1 as in Fig. 5. Adhesion was carried out
at 37 °C for 20 min and quantified in a fluorescence analyzer. Each
point represent the mean of triplicate determinations, with a standard
deviation of <10%. K562 cells showed no adhesion at any
concentration of sVCAM-1 tested (not
shown).
In the present study we show that residues Arg and Asp
form part of the integrin
4 subunit
epitope A. Substitutions at these residues resulted in a diminished
capability of three anti-
4 epitope A mAb (HP1/1, HP1/3, and HP1/7)
to recognize the
4 subunit expressed by RD(89-90)SA
transfectants, which correlated with a decrease in cell adhesion to the
FN-40 fragment of fibronectin and lack of homotypic cell aggregation of
these transfectants. Anti-
4 mAb specific to epitopes B1, B2, and C
recognized the
4 molecule in the RD(89-90)SA transfectants
in a similar fashion to wild type
4 transfectants, and complete
homotypic cell aggregation in the RD(89-90)SA transfectants was
obtained using either anti-
4 epitope B2 or anti-
1 mAb,
indicating that the mutations did not affect the overall conformation
of
4. These results indicate that the
4 residues Arg
and Asp
are involved in the interaction of VLA-4
with FN-40, as well as in homotypic cell aggregation triggered by
epitope A anti-
4 mAb. It is unlikely that substitutions at
Arg
and Asp
modified nearby cysteine
disulfide bonds, since these residues do not appear to be in the middle
of such a bond, according to the
4 disulfide bond pairing
disposition shown earlier (54) , based in a comparison with the
IIb disulfide bond pairing previously assigned(55) .
Substitutions at residues Arg
and Asp
did not
affect the adhesion of RD(89-90)SA transfectants to VCAM-1,
indicating that these residues do not appear to be essential in the
interaction between VLA-4 and VCAM-1.
Interestingly, the Arg
4 residue posses the highest surface index probability of
the
proteolytic N-terminal fragment. The
4
proteolytic cleavage site located between residues
Lys
-Arg
and Ser
(27) , also shows a very high surface index probability,
probably reflecting a surface exposure to proteases. This suggests that
the Arg
and Asp
residues indeed may be on the
surface of the
4 subunit, which could account for their functional
relevance.
The diminished adhesion of RD(89-90)SA cells to
FN-40 in the absence of TS2/16 was in average 40% with respect to wild
type 4 transfectants. This result is in accordance with a previous
report showing that adhesion to FN-40 could be partially inhibited (up
to 60%) by anti-
4 epitope A mAb(39) . The same studies
indicated that
4 epitopes B1 and B2 were involved in the
interaction of VLA-4 with FN-40, which could be completely blocked by a
panel of anti-
4 mAb. Using
4 murine/human chimeric constructs
expressed in mammalian cells, it has recently been shown that epitope A
mAb mapped to the most
4 N-terminal 100 amino acids in one
case(56) , whereas another report found that residues
1-38 contained the epitope A(57) . Moreover, they
reported that B1 and B2 epitopes mapped to
4 residues
152-203(56) , while the other obtained different sites
for the B1 (
4 195-268) and B2 (
4 108-182)
epitopes(57) . In spite of these differences which could be
solved by performing point mutations, it is clear that a region
C-terminal to residue 108 contains the main site of
4 involved in
the interaction with the CS-1 domain of fibronectin, and that, as
demonstrated in this report, additional binding sites are found in a
region which includes residues Arg
and Asp
.
Our data confirm that these residues do not form part of the B1 and B2
epitopes. The possibility of the presence of several sites in
4
capable to interact with fibronectin could result in a potential
strengthening of the VLA-4-mediated adhesion. It is conceivable that
4 residues Arg
and Asp
might form part
of an
4 domain interacting with another site contained inside the
FN-40 fragment of fibronectin, such as the H1 site (58) , and
that perhaps these interactions might be differentially regulated.
The finding that 4 residues Arg
and Asp
are included in the region promoting homotypic cell aggregation
agrees with the
4 epitope mapping studies mentioned
earlier(56) . Homotypic cell interaction mediated by VLA-4
might play a relevant role in processes such as extracellular matrix
invasion by melanoma cells during metastasis, where homotypic
interactions between these cells results in a reduction of
invasion(20) . In this regard, it has been reported that the
4 integrin chain can directly interact with
4
1 and
4
7(59) , suggesting that cells could use the
4
integrins to interact in a homotypic manner. The results from the
present work do not distinguish between a role of Arg
and
Asp
4 residues in the initial steps leading to cell
aggregation, or as part of the
4 molecule directly interacting
with other
4 integrins or with unknown ligands.
Residues
Gln and Pro
are also included in an
4
region with high surface probability index. However, substitutions at
these residues did not affect binding of any anti-
4 mAb to the
4 subunit expressed by the QP(101-102)HL transfectants, nor
altered the adhesive functions of these cells. Transfectants expressing
the
4 subunit with mutations at residue Gly
showed
only minor reactivity with anti-
4 epitope C mAb, while no stable
transfectants were obtained with mutations at residues Asp
and Leu
. Both Asp
and Gly
residues are highly conserved among integrin
subunits,
which could suggest that alteration at this region of the
4
subunit might result in an improper folding of the molecule and/or an
inability to associate with the
1 subunit.
Previous studies
have shown that mutations at the 4 putative divalent cation sites
(included in the region between residues 281 and 414) resulted in a
reduction of VLA-4 interaction with both CS-1/fibronectin and
VCAM-1(60) . Our results show that a region included within the
first N-terminal 100 amino acids of the integrin
4 subunit
participates in certain VLA-4-mediated cell adhesion functions, namely
cell aggregation and binding to fibronectin. The
4 residues
involved in the interaction of VLA-4 with VCAM-1, as well as those
residues forming part of the epitope B1 and B2 involved in adhesion to
CS-1, remain to be identified. The results from the present study
provide an insight into the integrin
4 regions involved in the
interaction of VLA-4 with its ligands. This information could be useful
in future designing of products aimed at interrupting these
interactions in several inflammatory and autoimmune pathologies.