From the Center for Blood Research and Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115
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ABSTRACT |
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The M subunit of integrin Mac-1 contains
several distinct regions in its extracellular segment. The N-terminal
region has been predicted to fold into a
-propeller domain composed
of seven
-sheets each about 60 amino acid residues long, with the
I-domain inserted between
-sheets 2 and 3. The structure of the
C-terminal region is unknown. We have used monoclonal antibodies (mAbs)
as probes to study the dependence of the structure of different regions of the
M subunit on association with the
2 subunit in the
M/
2 heterodimer. All of the mAbs to the I-domain
immunoprecipitated the unassociated
M precursor and reacted with the
M subunit expressed alone on the surface of COS cells. By contrast,
four mAbs to the
-propeller domain did not react with the
unassociated
M precursor nor with the uncomplexed
M subunit
expressed on COS cell surface. The four mAbs were mapped to three
subregions in three different
-sheets, making it unlikely that each
recognized an interface between the
and
subunits. These results
suggest that folding of different
-propeller subregions is
coordinate and is dependent on association with the
2 subunit. The
segment C-terminal to the
-propeller domain, residues 599-1092, was
studied with nine mAbs. A subset of four mAbs that reacted with the
M/
2 complex but not with the unassociated
M subunit were
mapped to one subregion, residues 718-759, and five other mAbs that
recognized both the unassociated and the complexed
M subunit were
localized to three other subregions, residues 599-679, 820-882, and
943-1047. This suggests that much of the region C-terminal to the
-propeller domain folds independently of association with the
2
subunit. Our data provide new insights into how different domains in
the integrin
and
subunits may interact.
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INTRODUCTION |
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The integrin family of adhesion molecules participate in important
cell-cell and cell-extracellular matrix interactions in a diverse range
of biological processes (1). Integrins are noncovalently associated
/
heterodimers, with each subunit consisting of a large
extracellular domain (>100 kDa for
subunits and >75 kDa for
subunits), a single transmembrane region, and a short cytoplasmic tail
(50 amino acids or less, except for the
4 subunit) (1). The
adhesiveness of integrins is dynamically regulated in response to
cytoplasmic signals, termed "inside-out" signaling (2-4). The
leukocyte integrin subfamily consists of four members that share the
common
2 subunit (CD18) but have distinct
subunits,
L
(CD11a),
M (CD11b),
X (CD11c), and
d for LFA-1, Mac-1, p150, 95, and
d/
2, respectively (5-7). The leukocyte integrins mediate a range of adhesive interactions that are essential for normal immune
and inflammatory responses (5).
Although the overall structure of integrins is unknown, several
structurally distinct domains in the extracellular portions of both and
subunits have been predicted or identified. The N-terminal
region of the integrin
subunits contains seven repeats of about 60 amino acids each (8) and has recently been predicted to fold into a
-propeller domain that consists of seven
-sheets, with each
-sheet containing four anti-parallel
-strands (9). The leukocyte
integrin
subunits (10), the
1 (11) and
2 (12) subunits of the
1 subfamily, and the
E subunit (13) of the
7 subfamily contain
an inserted domain or I-domain of about 200 amino acids that is
predicted to be inserted between
-sheets 2 and 3 of the
-propeller domain (9). The three-dimensional structure of the
I-domain from the Mac-1, LFA-1, and
2
1 integrins has been solved
and shows that it adopts the dinucleotide-binding fold with a unique
divalent cation coordination site designated the metal
ion-dependent adhesion site (14-17). The integrin
subunits contain a conserved domain of about 250 amino acids in the
N-terminal portion. This domain has been predicted to have an
"I-domain-like" fold (14, 18, 19). Very little is known about the
structure of the C-terminal half of the extracellular portions of both
and
subunits. Electron microscopic images of integrins reveal that the N-terminal portions of the
and
subunits fold into a
globular head that is connected to the membrane by two rod-like segments about 16 nm long corresponding to the C-terminal portions of
the
and
extracellular domains (20-22). This would suggest that
the C-terminal portions of both subunits are quite extended.
Previous studies using mAbs1
as probes have shown that the structure of specific domains in LFA-1
requires association of the L and
2 subunits. mAbs to the
2
subunit conserved domain do not react with the unassociated
2
subunit, whereas mAbs to the regions preceding and following this
domain do, indicating that the structure of the conserved domain is
dependent on association with the
L subunit (23). mAbs to the
I-domain react with the unassociated
L subunit (24). This finding
together with the fact that the I-domain can be expressed as an
isolated domain (14, 16, 25, 26) show that the I-domain assumes a
native structure independently of the
2 subunit. By contrast, two
mAbs (S6F1 and TS2/4) mapped to the N-terminal region of the
-propeller domain, and one mAb (G-25.2) that maps to a region of 212 amino acids with 159 amino acids located in the
-propeller domain
and the remainder in the C-terminal region, do not recognize the
L subunit in the absence of association with the
2 subunit (24). Another mAb (CBRLFA-1/1) that maps to a region overlapping the I-domain
and
-propeller domain reacts weakly with the uncomplexed
L
subunit. These results indicate that at least one region in the
-propeller domain is dependent on association with the
2 subunit
for mAb reactivity, and it has been suggested that the most likely
explanation is that folding of the
-propeller domain is not
completed until after association with the
subunit (24). Since mAbs
specific for the region of the
L subunit C-terminal to the
-propeller domain have not been described, it is not known whether
folding of this region is dependent on association with the
subunit.
In this study, we have used mAb probes to study the structure of the
Mac-1 subunit in the presence and absence of association with the
2 subunit. We have studied the
-propeller domain, the I-domain,
and the extensive region C-terminal to the
-propeller domain.
Compared with the previous studies on LFA-1, our studies on the
-propeller domain are more definitive, since mAb specificity is
defined to individual amino acid substitutions between mouse and human,
and mAb to epitopes that are widely separated in the predicted
-propeller structure all show a dependence on
subunit association for reactivity. Furthermore, we employ a panel of mAbs that
defines four different subregions within the C-terminal region of the
subunit. The results show that epitopes in three of these regions
have a native structure in the absence of
subunit association,
whereas a fourth epitope is dependent on association with the
subunit. Thus, much of the C-terminal region of the
M subunit
appears to assume a native fold independently of association with the
2 subunit.
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MATERIALS AND METHODS |
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Cell Lines-- U937, a human monoblast-like cell line, was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 µg/ml gentamicin, and 50 µM 2-mercaptoethanol (complete medium). COS cells (SV40-transformed monkey kidney fibroblasts) were maintained in RPMI 1640 supplemented with 10% FBS and 50 µg/ml gentamicin. Human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mM glutamine, and 50 µg/ml gentamicin.
mAbs--
The following murine mAbs against the M subunit of
human Mac-1 were previously described: OKM1, OKM9 (27), TGM-65 (28), CBRM1/1, CBRM1/2, CBRM1/29, CBRM1/20, CBRM1/32, CBRM1/10, CBRM1/16, CBRM1/17, CBRM1/18, CBRM1/23, CBRM1/25, CBRM1/26, and CBRM1/30 (29).
All these mAbs were used as ascites except for CBRM1/29 that was used
as concentrated hybridoma supernatant. CBRN1/6 and CBRN3/4 against the
M subunit of Mac-12 were
used as hybridoma supernatant. TS1/18 and CBRLFA-1/2 against human
leukocyte integrin
2 subunit were described previously (30, 31) and
used as purified IgG.
DNA Constructs and Mutagenesis--
The human wild-type M
subunit cDNA was subcloned in the expression vector pCDNA3.1+
(Invitrogen, Carlsbad, CA) as
described.3 For generating
human-mouse
M chimeras, a SacII site was created immediately after the stop codon (nucleotides 3532-3534). By
specifically primed reverse transcription of murine spleen mRNA
(CLONTECH, Palo Alto, CA) from approximately 50 nucleotides downstream of the stop codon, the first strand of the mouse
M cDNA (33) was generated with Moloney murine leukemia virus
reverse transcriptase (Stratagene, La Jolla, CA). By using this as a
template for PCR, a 2-kilobase pair mouse
M cDNA fragment
covering nucleotides from the SfiI site (nucleotide 1688) to
the stop codon and having a SacII site immediately after the
stop codon was made. This mouse
M SfiI-SacII
fragment was used to replace the corresponding human
M
SfiI-SacII fragment to generate the initial
chimeric
M cDNA encoding the N-terminal 529 residues of human
sequence and the remaining C-terminal sequence from mouse. Using this
initial chimeric construct as template, eight human-mouse
M chimeras
with a variable mouse C-terminal portion were generated by overlap
extension PCR (34, 35). Briefly, outer primers for overlap PCR were
just 5' to the SfiI site and 3' to the SacII
site, and the first set of reactions was carried out using the human
wild-type
M and the initial chimeric construct as templates. After
the overlap extension reaction, the chimeric products were digested
with SfiI and SacII, and the
SfiI-SacII fragments were swapped into the human
wild-type
M in vector pCDNA3.1+. Human to mouse individual amino
acid substitutions in the region from amino acids 718-759 of human
M were made by overlap extension PCR (34, 35). Briefly, the
overlapping primers contained the desired mutations, and the outer
primers were 5' to the SfiI site and 3' to the
NdeI site, respectively. The overlap extension PCR products
were digested with SfiI and NdeI and swapped into
human wild-type
M in expression vector pEFpuro (36).
Transient Transfection--
COS cells were transfected by the
DEAE-dextran method (36) with the M cDNA alone or were
co-transfected with the wild-type or chimeric
M and
2 cDNA.
The wild-type and chimeric
M cDNA were in plasmid pCDNA3.1+,
and the
2 cDNA was contained in plasmid pEF-BOS (36). Three days
after transfection, COS cells were detached with Hanks' balanced salt
solution supplemented with 5 mM EDTA for flow cytometric
analysis. 293 cells were transfected with the calcium phosphate method
(37, 38). Briefly, 7.5 µg of wild-type or mutant
M cDNA in
plasmid pEFpuro and 7.5 µg of
2 cDNA in plasmid pEF-BOS were
used to transfect one 6-cm plate of 70-80% confluent cells. Two days
after transfection, cells were detached with Hanks' balanced salt
solution, 5 mM EDTA for flow cytometric analysis.
Flow Cytometry-- COS cells and 293 cells were washed twice with L15 medium containing 2.5% FBS (L15/FBS) and resuspended to 1-2 × 106 cells/ml in the same medium. 50 µl of the cell suspension was incubated with an equal volume of the primary antibody (20 µg/ml purified mAb, 1:100 dilution of mAb ascites, or 1:2 dilution of hybridoma supernatant in PBS) on ice for 30 min. Cells were then washed three times with L15/FBS and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (heavy and light chain, Zymed Laboratories, San Francisco, CA) for 30 min on ice. For staining with mAb CBRM1/20 that requires Ca2+,3 the primary and secondary antibodies were diluted in PBS supplemented with 1 mM Ca2+. After washing, cells were resuspended in cold PBS and analyzed on a FACScan (Becton Dickinson, San Jose, CA).
Radiolabeling, Immunoprecipitation, and Gel Electrophoresis-- For metabolic labeling, U937 cells were plated in four 10-cm Petri dishes and induced with PMA for 3 days as described previously (39). Cells in each dish were washed twice with methionine-free RPMI 1640 medium and labeled with 0.625 mCi of [35S]methionine in 5 ml of methionine-free RPMI 1640 containing 15% dialyzed FBS. After incubation at 37 °C for 30 min, cells in two dishes were washed twice with cold PBS and lysed by addition of 3 ml of lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.24 TIU/ml aprotinin, and 10 µg/ml each of pepstatin A, antipain, and leupeptin) and incubation for 30 min at 4 °C with gentle agitation. For chase labeling, 5 ml of complete medium supplemented with 100 µg/ml unlabeled methionine was added to each of the remaining dishes, and incubation at 37 °C was continued for 16 h. The chase-labeled cells were lysed identically to pulse-labeled cells, and lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C.
For surface labeling, COS transfectants (2 × 106 cells) were washed three times with PBS and resuspended in 1 ml of PBS. The cells were surface-labeled with 1 mCi of Na125I using two IODO-BEADS (Pierce) following the manufacturer's instructions. The labeled cells were washed three times with PBS containing 10% FBS and once with PBS and lysed as described above. For immunoprecipitation, cell lysates were precleared by addition of 1/10 volume of recombinant protein G agarose (50% suspension in PBS) (Life Technologies, Inc.) and incubation at 4 °C for 2-3 h with agitation. The precleared lysates were split into 250-µl aliquots, and to each aliquot, 2.5 µl of mAb ascites or 10 µg of purified mAb or 250 µl of mAb supernatant was added, and the final volume was adjusted to 500 µl with lysis buffer. After incubation overnight at 4 °C, followed by centrifugation at 12,000 rpm for 10 min at 4 °C to remove protein aggregates, the antigen/antibody mixture was incubated with 50 µl of protein G-agarose beads for 1.5-2 h at 4 °C with agitation. Beads were washed three times with lysis buffer and once with lysis buffer without detergent. For immunoprecipitation with mAb CBRM1/20, lysis buffer and wash buffer were supplemented with 1 mM Ca2+. Bound proteins were eluted from beads with 50 µl of Laemmli sample buffer by heating for 5 min at 100 °C, and the immunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis (40). The gels were processed for fluorography for [35S]methionine-labeled proteins or autoradiography for 125I-labeled proteins.Secondary Structure Prediction--
The amino acid sequences
between the -propeller domain and the transmembrane segment of 36 integrin
subunits (9) were aligned with ClustalW, and then the
alignment was iteratively refined using default settings with PRRP and
the Gonnet amino acid substitution matrix, and an evolutionary tree was
prepared with PHYLP (41). The
M and
IIb subunits fall in
different branches of this tree, each of which is well populated. One
branch containing 11 subunits most closely related to human
M,
i.e. murine
M, human
D, and
X, murine and human
L, human and rat
1, and bovine, human, and mouse
2, were
realigned with one another using PRRP. They are 21-70%,
= 34% identical to human
M. Another branch
containing the 17 subunits most closely related to human
IIb,
i.e. hamster, human, and mouse
3, human and
Xenopus
5, chicken and human
6, mouse
7, chicken
and human
8, and chicken, human, mouse, and Pleurodes
V, and YMA1
of Caenorhabditis elegans, were realigned in a separate
group. They are 20-38%,
= 28% identical to
human
IIb. The alignments in MSF format, with gaps in human
M and
human
IIb removed to increase prediction accuracy, were separately
submitted for secondary structure prediction to PHD
(42).4 Smaller subgroups
containing a higher degree of relationship to
M (6
subunits,
with 27-70% identity to
M) or to
IIb (9
subunits, with
33-38% identity to
IIb) gave very similar predictions but with a
slightly lower correlation between the
M and
IIb predictions.
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RESULTS |
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mAbs to the -Propeller Domain and a Subset of mAbs to the
C-terminal Region Do Not React with the Unassociated
M
Subunit--
To study whether folding of the
M subunit is dependent
on association with the
2 subunit, we examined the expression of mAb
epitopes on the unassociated
M subunit. Eighteen mAbs that have
previously been mapped to different regions in the
M subunit were
used (29)2 (Fig. 1). Previous
studies on leukocyte integrin biosynthesis have shown that the
and
subunit precursors are initially unassociated in the endoplasmic
reticulum and that transport to the Golgi apparatus and processing from
high mannose N-linked carbohydrates to complex carbohydrates
are dependent on the formation of
and
complex (39, 43, 44). We
therefore examined whether mAbs to the I-domain, to the
-propeller
domain, and to the C-terminal region immunoprecipitated the
unassociated
M precursor (
'M). All mAbs immunoprecipitated the
mature
M subunit with molecular size of about 170 kDa from the
lysate of cells pulse-labeled with [35S]methionine for 30 min and chased for 16 h (Fig. 2,
lower panel). The
M subunit was complexed with the
2
subunit as shown by co-immunoprecipitation of the
2 subunit with the
M subunit. However, mAbs differentially precipitated the
'M
precursor, which is slightly smaller than the mature
M subunit from
the pulse-labeled cells (Fig. 2, upper panel). There was
little or no
'M precursor associated with the
2 precursor (
'2)
in the pulse-labeled cells, since no detectable
'2 over background
was co-precipitated by mAbs to the
M subunit, but
'2 was
precipitated with mAb CBRLFA-1/2 to the
2 subunit (upper
panel, lane 18). All mAbs to the I-domain precipitated
'M
(upper panel, lanes 2-6). By contrast, three mAbs (CBRN1/6, CBRN3/4, and CBRM1/20) to the
-propeller domain did not precipitate
'M (upper panel, lanes 7, 8, and 21). mAb
CBRM1/32 to the
-propeller domain did not precipitate the
M/
2
complex or
'M from cell lysates (data not shown), suggesting that
its epitope is sensitive to detergent extraction. Five mAbs (OKM1,
CBRM1/10, CBRM1/23, CBRM1/25, and CBRM1/26) to the C-terminal region
precipitated
'M (upper panel, lanes 9 and 10 and 14-16), whereas four other mAbs (CBRM1/16,
CBRM1/17, CBRM1/18, and CBRM1/30) precipitated no to very little
'M
(upper panel, lanes 11-13 and 17). Thus, epitopes of mAbs to the I-domain are expressed on the unassociated
M
precursor, whereas epitopes of
-propeller domain mAbs and a subset
of mAbs to the C-terminal region are not.
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Epitope Mapping of mAbs to the C-terminal Region and to the
-Propeller Domain of the
M Subunit--
The finding that a
subset of mAbs to the C-terminal region does not react with the
unassociated
M subunit suggests that the structures of certain
subregion(s) in this C-terminal 493-amino acid segment may be dependent
on association with
2. To localize such subregion(s), as well as
subregion(s) that fold independently of
2 association, epitopes of
the nine mAbs to the C-terminal region were mapped using human-mouse
M chimeras. The chimeras were generated by progressively replacing
the human sequences from the C terminus with the corresponding
sequences from mouse
M (Fig. 5) and
were co-expressed with human
2 in COS cells. mAb reactivity with
chimeric
M/
2 was determined by immunofluorescent flow cytometry
(Table I). All chimeras were expressed on
the surface in association with the human
2 subunit, with levels of
cell-surface chimeric
M/
2 complex comparable with that of wild-type
M/
2 complex. In addition, all chimeric
M/
2
complexes were stained with mAbs to the
-propeller domain (Table I
and data not shown), showing structural integrity of the
-propeller domain despite the C-terminal region swapping. The results from epitope
mapping are summarized in Table I and Fig.
6. A 41-amino acid sequence (residues
718-759) was required for epitopes of the four mAbs (CBRM1/16,
CBRM1/17, CBRM1/18, and CBRM1/30) that did not react with the
unassociated
M subunit. The epitopes of five mAbs that reacted with
the unassociated
M subunit were mapped to three other subregions as
follows: OKM1 to a region immediately following the
-propeller
domain (residues 599-679); CBRM1/10, CBRM1/25, and CBRM1/26 to a
region from residues 820 to 882; and CBRM1/23 to a region from residues
943 to 1047. Thus, mAb epitopes that map to one subregion (residues
718-759) require association of
M with
2, whereas epitopes
localized in three other subregions (residues 599-679, 820-882, and
943-1047) are independent of the
2 subunit.
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DISCUSSION |
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By using mAbs as probes, we have examined the structure of
different regions in the Mac-1 M subunit during biosynthesis and
M/
2 heterodimer assembly and after expression on the cell
surface. All five different mAbs to the I-domain reacted with the
unassociated
M subunit, confirming that the folding of the I-domain
does not require the
2 subunit. By contrast, four mAbs (CBRN1/6,
CBRN3/4, CBRM1/20, and CBRM1/32) that map to three different subregions in the
-propeller domain did not react with the unassociated
M
subunit (Fig. 6). CBRN1/6 and CBRN3/4 mapped to one or more of three
residues in the 3-4 loop of W4 (residues 421-425) (Fig. 8). CBRM1/20
is specific for three amino acid residues in the 1-2 loop of W5 and
two residues in the 3-4 loop of W63 (Fig. 8). The epitope
for CBRM1/20 includes two residues, Asn453 and
Asp457, that are predicted to coordinate with
Ca2+ in the 1-2 loop of W5, and binding of this mAb
requires Ca2+ with an EC50 of 0.2 mM.3 These mAbs did not immunoprecipitate the
unassociated
M precursor or react with the
M subunit expressed
alone on the surface of COS cells. mAb CBRM1/32 reacted with the
M/
2 complex expressed on the cell surface but did not react with
the
M subunit expressed alone on the cell surface. The epitope of
CBRM1/32 requires residue Arg-534 in the 2-3 loop of W6 (Fig. 8). One
possible interpretation of our results is that all three epitopes in
the
-propeller domain require the presence of the
subunit
because the
and
subunits associate with one another in each of
these regions, and each antibody binding site includes contacts with
both the
subunit and
subunit. If so, the contacts with the
subunit do not include any antigenic residues, because all mAb reacted
equally well whether the human or murine
subunit was associated
with human
M. Furthermore, we tested the chicken
2 subunit,
because 35% of the residues in the human and chicken
2 subunits
differ, as opposed to only 18% between the human and the mouse (45).
Amino acid differences between species are preferentially found on the
surface of proteins rather than buried. Although a substantial portion
of surface residues are expected to differ on the chicken and human
2 subunits, whether the chicken or human
2 subunit was present
did not affect mAb reactivity. The epitopes that were localized include
some that are quite distant. The Arg-534 residue recognized by CBRM1/32 mAb is on the upper surface of the
-propeller, whereas residues recognized by the CBRM1/20 and the CBRN1/6 mAb are on the lower surface
and point in opposite directions from one another. The C-
carbon of
the Arg-534 residue is predicted to be 30 ± 3 Å and 41 ± 3 Å distant from residues recognized by the CBRM1/20 and the CBRN1/6
mAb, respectively, and the C-
carbons of residues recognized by the
CBRM1/20 and the CBRN1/6 mAb are 23 ± 7 Å distant from one
another. The probability that three out of three different epitopes
would include surfaces from both the
and
subunits, even though
some epitopes are quite distant from one another, would appear to be
low. Because of this, and the lack of effect of the species origin of
the
subunit on mAb reactivity, we favor the interpretation that
association between the
subunit and
subunit is required for the
-propeller domain to assume its final three-dimensional structure,
i.e. to assume the correct fold. Our data are consistent
with the idea that there is an interface between the
subunit
-propeller domain and the
subunit, although we believe that the
interface is not necessarily associated with any of the epitopes we
have mapped. Conversely, a number of mAb to different epitopes in the
conserved domain of the integrin
subunit are not reactive in the
absence of the
subunit (23). Thus, the conserved domain of the
subunit is a candidate for association with the putative
-propeller
domain of the
subunit. Analogously, the G-protein
subunit
-propeller domain is not properly folded in the absence of
association with the G-protein
subunit (46).
A previous study on the LFA-1 -propeller domain used two mAbs (S6F1
and TS2/4) that map to the
L subunit N-terminal 57 amino acids,
i.e. to part of
-sheets W7 and W1, and one mAb (G-25.2) that maps to a 212-amino acid region spanning W5-7 of the
-propeller domain and part of the C-terminal region. These mAbs did
not react with the unassociated
L subunit (24) (Fig. 6). Another mAb (CBRLFA-1/1) that overlaps the I-domain and W3 of the
-propeller domain showed weak reactivity in the absence of the
2 subunit. It is
not known whether this mAb recognizes a boundary region between the I
and
-propeller domains. Taken together, the findings on LFA-1 and
Mac-1 demonstrate that multiple mAbs to different regions in the
-propeller domain do not react with the
subunit in the absence
of the
subunit and suggest that the
-propeller domain folds as a
unit and that this folding depends on association with the
subunit.
mAbs to the C-terminal region of the M extracellular domain
differentially reacted with the unassociated
M subunit. Five mAbs
(OKM1, CBRM1/10, CBRM1/25, CBRM1/26, and CBRM1/23) reacted with both
the unassociated and the complexed
M subunit and were mapped to
three subregions. OKM1 mapped to a subregion immediately following the
-propeller domain, residues 599-679. CBRM1/10, CBRM1/25, and
CBRM1/26 mapped to amino acids 820-882, and CBRM1/23 mapped to
residues 943-1047. Within each of these subregions, there are multiple
differences between the mouse and human sequences (Fig. 7). Whether the
multiple mAbs that react with residues 820-882 recognize one or more
epitopes within this subregion is not known. Minimally, these data show
that three epitopes in three different subregions of the C-terminal
segment are independent of the
2 subunit. By contrast, four other
mAbs to the C-terminal region (CBRM1/16, CBRM1/17, CBRM1/18, and
CBRM1/30) only reacted with the
M/
2 complex. These mAbs did not
react with the unassociated
M precursor or with the uncomplexed
M
subunit expressed on the COS cell surface. All four mAbs were mapped to
residues Thr725 and, additionally, Ser728
and/or Ala729. Although CBRM1/16, CBRM1/17, CBRM1/18, and
CBRM1/30 did not react with the unassociated
M subunit, they reacted
with the human
M/mouse
2 and human
M/chicken
2 complexes as
well as with the human
M/human
2 complex (data not shown). Thus,
association with the
2 subunit may be required for this region to
assume its final structure. Although we believe that the interpretation that the
and
subunits both contribute to the antibody-binding site is less likely, either interpretation shows an important interaction with the
subunit for the region of residues 725-729. Overall, the results show that three out of four epitopes in the C-terminal region of Mac-1
M subunit are intact in the absence of
association with the
2 subunit. If these results are representative of the C-terminal region as a whole, our data would suggest that much
of this region folds independently of the
2 subunit. This is in
marked contrast to the
-propeller domain.
To place our results on the C-terminal region within a structural
framework, we predicted its secondary structure using the PHD program
(42) (Fig. 7). By using a phylogenetic tree based on an iteratively
refined alignment (41) of 36 subunit C-terminal region sequences,
two subfamilies were identified. These subfamilies were large and
contained members that were 1) sufficiently similar to one another to
allow accurate alignment and to not be too divergent in tertiary
structure, and 2) were sufficiently different from one another to
contain a large amount of sequence information, and hence optimize
prediction accuracy (42). An alignment of 11 subunits was used to
predict the secondary structure of human
M, and an alignment of 17 other
subunits was used to predict the structure of human
IIb
(Fig. 7). Since no sequences were shared between the two alignments,
and between the two groups there is only 16-21% sequence identity,
the predictions for
M and
IIb are largely independent of one
another.
In the C-terminal segment, a total of 30-34 -strands were
predicted. Of these, 22 were independently predicted in both
M and
IIb. Only 5
-helices were predicted, and in each case these were
predicted in only one of the two
subunits. Thus, the C-terminal region is predicted to form domains of the all
class. In this respect, it is similar to the
-propeller domain (9) and different from the I-domain which is of the
/
class (14, 16).
The disulfide bond topology of IIb has been chemically determined
(47, 48). The conservation of cysteines suggest that 5 of 6 disulfide
bonds are conserved in human
M, whereas one differs (Fig. 7). The
first disulfide in this region,
IIb C473-C484, is confirmed by the
sequence alignment of 36 integrin
subunits, since these two
cysteines are selectively absent in the chicken
6 subunit, and the
cysteines and the loop in between them are absent in
2 subunits and
E subunits. The cysteines corresponding to the last disulfide bond
in
IIb, Cys885-Cys890, are missing from
L
subunits. Otherwise, there is only one predicted difference between
disulfide bonds in
IIb and the leukocyte integrin
subunits. The
cysteine corresponding to
IIb Cys484 is missing in all
leukocyte integrin
subunits, and all leukocyte integrin
subunits contain a cysteine with no equivalent residue in
IIb,
i.e. Cys706 in
M. We predict that the
cysteines at
IIb position 473, although aligned by sequence, are
non-equivalent, i.e. that the cysteine in
M is involved
in a different disulfide bond, to Cys706 (dashed
line in Fig. 7).
Folds of the all- class as a general rule contain anti-parallel
-sheets (49). The vast majority but not all of the predicted
-strands in the
IIb and
M C-terminal regions are markedly
amphipathic with alternating hydrophobic and hydrophilic residues. We
therefore predict that the C-terminal region folds into 2-layer,
anti-parallel
-sheet structures, i.e.
-sandwich or
-barrel domains of which the Ig fold is one of many representatives.
The total length of the C-terminal region of about 500 residues, the
number of predicted
-strands, and the overall number and location of
disulfide bonds are appropriate for approximately four to six
-sandwich domains.
In IIb, a main chymotryptic cleavage site is located around
Asn570 (47, 48). Cleavage of cell-surface
IIb
3
releases a ligand binding complex containing an N-terminal fragment of
II
of 55 kDa ending at approximately Asn570, and an
85-kDa N-terminal fragment of
3 (50). This suggests that the region
around Asn570 is well exposed and may represent a domain
boundary region. It is interesting that four mAbs dependent on
subunit association map to essentially the same site in
M (Fig. 7).
The region preceding the chymotryptic cleavage site and following the
-propeller domain in
IIb contains one long range disulfide bond
(Cys490-Cys545), and six predicted
-strands.
In
M, the corresponding region contains two predicted long range
disulfide bonds (Cys639-Cys696,
Cys623-Cys706), and seven predicted
-strands. Based on these features, we predict that this region of
about 120 amino acids following the
-propeller domain, residues 599 to about 718 for
M, and 450 to about 570 for
IIb, folds into a
structurally independent domain. Consistent with this prediction, this
region in
M appears to fold independently of association with the
subunit, as shown with the OKM1 mAb. This contrasts with the
flanking N-terminal
-propeller domain and the flanking C-terminal
region from residues 725 to 729, to which mAbs CBRM1/16, CBRM1/17,
CBRM1/18, and CBRM1/30 map.
Our results together with other recent studies provide new insight into
how different domains in the integrin and
subunits may
associate. The I-domain is predicted to be connected to the upper
surface of the
-propeller domain (9). The
subunit
-propeller
domain and the
subunit conserved domain may associate, since both
are dependent on
and
subunit association for folding (23, 24)
(this study). The predicted
-sandwich/
-barrel domain that follows
the
-propeller domain and contains the OKM1 epitope, residues
599-718, is connected to the C terminus of strand 3 of W7 of the
predicted
-propeller domain and hence to the bottom of the
-propeller domain. The following subregion of the
M subunit, from
residues 725 to 729, may directly associate with the
2 subunit, or
its structure may be indirectly dependent on associations elsewhere with the
subunit. Other subregions in the C-terminal portions of
and
subunits might also participate in
and
subunit association as proposed for the
IIb
3 integrin (32,
51), while retaining similar conformations in the unassociated and complexed forms.
In summary, the results from this study suggest that proper folding of
the -propeller domain of the integrin
M subunit requires association with the
2 subunit, whereas the I-domain folds
independently of the
2 subunit. Much of the region C-terminal to the
-propeller domain folds prior to
subunit association. Our
results further advance the understanding of integrin structure and
provide information that will be useful in guiding studies leading to
the characterization of integrin three-dimensional structure.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA31799, a fellowship from The Cancer Research Institute (to C. L.), and a fellowship from The Danish Natural Science Research Council (to C. O.).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: The Center for Blood
Research and Harvard Medical School, Dept. of Pathology, 200 Longwood
Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3232; E-mail:
springer{at}sprsgi.med.harvard.edu.
1 The abbreviations used are: mAb, monoclonal antibody; FBS, fetal bovine serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; hu, human; mo, mouse.
2 S. Q. Na and T. A. Springer, unpublished data.
3 C. Oxvig and T. A. Springer (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4870-4875.
4 Available on-line at the following address: http://www.embl-heidelberg.de./predict protein/.
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