From the Lipid Metabolism Unit and Nessel Gene Therapy Center,
Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
Macrophage scavenger receptors are trimeric
integral membrane proteins that bind a diverse array of negatively
charged ligands. They have been shown to play a role in the
pathogenesis of atherosclerosis and in host responses to microbial
infections. Earlier mutational studies demonstrated that the distal
segment of the collagen domain of the receptor was critically important
for high affinity ligand binding activity. In this study, mutations
spanning the entire collagen domain were generated and binding was
assayed in transfected cells, as well as in assays employing a
secreted, receptor fusion protein. Many of the distal, positively
charged C-terminal residues in the type II collagen domain of the
receptor, previously reported to be essential for binding at 37 °C,
were found not to be critical for binding at 4 °C. Conversely, more
proximally charged residues of the collagen receptor that have not been
previously mutated were shown to have substantial effects on binding
that were also temperature-dependent. These data suggest
that scavenger receptor ligand recognition depends on more
complex conformational interactions, involving charged residues
throughout the entire collagen domain, than was previously
recognized.
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INTRODUCTION |
Macrophage scavenger receptors exhibit a breadth of ligand binding
specificity that is unusual among cell surface endocytotic receptors.
This property may facilitate macrophage participation in a wide variety
of host defense and immunologic responses. Although most interest in
macrophage scavenger receptors has focused on their role in the uptake
and degradation of modified forms of low density lipoprotein
(LDL),1 (1, 2) an event
thought to be critical to the earliest stages of atherosclerotic plaque
formation, the discovery of scavenger receptor binding interactions
with lipopolysaccharide (3, 4), crocidolite asbestos (5), and
-amyloid fibrils (6, 7) suggests that their role in human diseases
and host immune responses may be a more expansive one. The discovery
that scavenger receptors may also participate in cell adhesion further
strengthens this hypothesis (8). An elucidation of the structural
features of scavenger receptors that confer their broad but yet
restricted binding specificity might, therefore, yield useful insights
into a variety of macrophage-associated host defense responses.
The cDNAs encoding two forms of the macrophage scavenger receptor,
now termed SR class A type I and type II receptors, were initially
isolated using bovine lung mRNA (9, 10). Subsequently, homologous
receptors of both types have been cloned from murine, human, and rabbit
tissues (4, 11-13). The amino acid sequence of the type I receptor
suggested that the protein could be divided into six domains (4, 9, 14)
(domain I, an N-terminal cytoplasmic tail (residues 1-50 in the rabbit
sequence); domain II, transmembrane domain-(51-76); domain III, a
spacer domain-(77-151); domain IV,
-helical coiled-coil
domain-(152-272); domain V, collagen domain-(273-344), and domain VI,
a cysteine-rich C-terminal region-(345-454) that is a highly conserved
domain found in a diverse group of proteins (11, 15, 16)). The type II
receptor differs from type I only in that it lacks the cysteine-rich C
terminus, instead possessing a 6-17-residue (varying with species)
C-terminal truncated tail. The type II receptor was shown to bind the
ligands traditionally used to define SR function (10) establishing that
the cysteine-rich domain could not be the major ligand binding region
of the scavenger receptor. As the known ligands for the SR are all
polyanionic, it was initially postulated that the SR collagen sequences
might serve as the receptor binding domain, because all 24 Gly-X-Y triplets of the bovine collagen region
would be predicted to be neutral, or positively charged, at physiologic
pH. Recent experimental work has provided support for that initial
hypothesis.
Acton et al. (17) reported that a scavenger receptor mutant
lacking the C-terminal 16 Gly-X-Y repeats of the
collagen domain of the bovine receptor was incapable of binding the
defining ligand for the scavenger receptor, acetylated LDL (AcLDL).
This mutation did not appear to disrupt other aspects of scavenger
receptor cell biology, such as receptor synthesis, trimerization,
post-translational modification, or cell surface stability. It was also
demonstrated to inhibit the activity of a co-transfected wild type
receptor. Similar findings were reported by Dejager et al.
(18) who also showed that collagen truncation mutants could inhibit the
activity of endogenous scavenger receptors, when cDNAs encoding the
truncation mutants were transfected into the mouse macrophage cell
line, P388D1. To identify more precisely the critical amino acids in the collagen domain, Doi et al. (19) generated a series of
deletion mutants as well as point mutations affecting the positively
charged residues found in the last six Gly-X-Y triplets of
the human collagen domain. These receptors were studied in degradation
and binding assays utilizing COS cells transfected with cDNAs
encoding the mutant forms of the receptors. Binding and degradation of
AcLDL was analyzed in these transfected cell lines. Convincing evidence was presented to support the contention that several positively charged
residues in the C terminus of the collagen domain were important for
the binding and degradation of modified lipoprotein ligands. The
authors (19) of this work concluded that Lys-337 is essential for
binding and that the four "lysine cluster in the most C-terminal
portion of the collagen is the ligand-binding domain of the scavenger
receptor."
In this paper, we report the results of binding studies, utilizing both
transfected cells and a direct receptor protein binding assay, to
perform a broader mutational analysis of the collagen domain of the
macrophage scavenger receptor. It is demonstrated that a scavenger
receptor/human IgG fusion protein can assemble into higher weight
oligomers, including the characteristic trimeric form of the native SR.
These fusion proteins, after purification by affinity column
chromatography, retain the ability to bind SR ligands with affinity
comparable to that of the native receptor expressed on the cell
surface. Whereas mutational studies of both the fusion proteins and
their transmembrane analogues confirm the importance of the C-terminal
collagen residues for ligand binding, they also indicate that other
residues outside this region are equally critical for receptor binding.
Importantly, the restoration of binding at 4 °C to several different
receptor proteins with mutations in charged residues, previously
thought to be critical to ligand interactions, provides strong evidence
that the prevailing paradigm for SR binding interactions is incomplete.
Temperature-dependent binding activity alterations,
demonstrated in both proximal and distal collagen subdomain mutants,
suggest that these mutations substantially alter the conformation of
the receptor, rather than simply effect a loss of ionic receptor-ligand
interactions. These data make it clear that the current model of SR
binding, which postulates an ionic interaction between ligand and
receptor, involving the distal segment of the collagen domain, cannot
account for the full complexity of the binding behavior of this
receptor.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Lipoproteins--
All cell culture incubations
were at 37 °C in 95% air and 5% CO2. COS M7 cells were
maintained in Dulbecco's modified Eagle's medium with 10% calf
serum. For cell degradation assays, native and mutant forms of pXRSR2
(13), a plasmid containing the rabbit type II scavenger receptor in
pCDNA-1 (Invitrogen, San Diego), were transfected into the COS
cells using the DEAE-dextran method described by Aruffo and Seed (20).
The ability of the transfected cells to degrade 125I-AcLDL
in the presence or absence of inhibitors was measured 72 h after
transfections. All degradations were conducted at 37 °C for 5 h
as described previously (13). Lipoproteins used in these assays as well
as for the ligand blots and binding assays were prepared by methods
previously detailed (21). Iodination of the lipoproteins was
accomplished by a modified iodine monochloride reaction (22).
Plasmid Constructs and Mutagenesis--
The overall structure of
the proteins used in this study are schematically depicted in Fig. 1.
By using the polymerase chain reaction, a nine amino acid epitope
sequence, TETSQVAPA, encoding a C-terminal epitope of the rhodopsin
receptor (23) was generated as an in-frame addition to the C terminus
of the rabbit type II SR using pRABSR2 (13) DNA as the template. The
polymerase chain reaction primers contained 5' BamHI and 3'
XbaI sites, and these sites were used to ligate the epitope
sequence into wild type pRABSR2 that had been digested with
BstYI and XbaI. The epitope sequence encoded a
translational stop codon immediately distal to the final alanine of the
epitope sequence. The epitope sequence was then transferred to the type
II SR COS cell expression plasmid, pXRSR2 (13). These two plasmids,
pXRSR2 and pXRSR2-epi, were then used to generate mutant receptor
cDNAs by the oligonucleotide site-directed mutagenesis method of
Kunkel (24).
Fusion proteins were generated by creating a BamHI site in
pXRSR2 immediately distal to the putative receptor transmembrane sequence via the polymerase chain reaction. This fragment was then
ligated in frame to the Fc portion of a human IgG cDNA
that contained a BamHI site immediately distal to the IgG
sequence. This construct was generated in a CDM-8 COS cell expression
derivative, pCD5spe
C71, kindly provided by Dr. Brian Seed
(Massachusetts General Hospital, Boston). A similar construct was made
using the epitope containing scavenger receptor sequences, whose
creation is described above.
To generate mutant forms of the fusion protein, mutations were first
generated in pXRSR2 or pXRSR2-epi by the Kunkel method and then
transferred to the human IgG-SR fusion construct by cleavage with
ApaLI, an enzyme with a restriction site in the coiled-coil region of the SR and a second site in the origin of replication sequence found in both pCD5spe
71 and pCDNA-1. All mutations were sequenced from the start of the collagen domain to its terminus using the dideoxy chain termination method and Sequenase (U. S. Biochemical Corp.).
Antibodies, Immunoblots, and Ligand Binding Studies--
The
rhodopsin epitope antibody, ID4 (23), was kindly provided by Frank
Kowalkowski (Massachusetts General Hospital, Boston). The monoclonal
anti-fusion protein antibody, ASAb-1, was generated in Balb/c mice by
injection of purified fusion protein. Splenic fusions and hybridoma
generation were performed according to the protocols of Harlow and Lane
(25).
Following separation by SDS-polyacrylamide gel electrophoresis,
proteins were transferred to nitrocellulose membranes in a Bio-Rad
Mini-Transblot apparatus, using 25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol as the transfer buffer.
Transfers were performed at 150 mA for 2 h. After transfer, the
membrane was blocked for 1 h in PBS, pH 7.4, 1% Tween, and 10%
dried milk (Buffer A), washed × 2 with Buffer A minus the milk
(Buffer B), and then incubated for 30 min with first antibody diluted
in Buffer A. For ID4 (protein A purified from mouse ascites fluid), a
dilution of 1:1000 was employed; the supernatant from hybridoma
7180-112 (ASAb-1) was used at a 1:5 dilution. The unbound first
antibody was removed with 4 washes of Buffer B. A 30-min incubation
with a 1:1000 dilution of horseradish peroxidase-labeled anti-mouse antibody (Sigma) in Buffer A was then performed. The membrane was then
washed 4 × with Buffer B and developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) according to the manufacturer's (Sigma) instructions.
Ligand blots were performed according to a modification of the
procedure of Daniel et al. (26). AcLDL was substituted for LDL, and the binding and wash buffers were substituted 50 mM NaCl for 90 mM NaCl. Rabbit anti-human LDL
antibody (Biomedical Technologies, Inc., Stoughton, MA) was used at a
1:5000 dilution in conjunction with a peroxidase-conjugated goat
anti-rabbit antibody (Sigma) diluted 1:1000. Development with DAB was
done as for the immunoblots. For quantitative ligand binding assays,
125I-AcLDL was used (1 × 106 cpm per
filter strip) to bind to nitrocellulose strips on which fusion proteins
had been transferred. Equal amount of protein, as measured by a
modified Bradford assay (Pierce), were loaded in each lane of the gel
prior to transfer. All binding assays were done in triplicate.
For saturation binding studies, receptor-coated plastic wells (Terasaki
plates) were employed. Plates were coated initially with 0.5 µg of
fusion protein, in PBS, pH 7.4, for a minimum of 4 h at 4 °C.
The wells were then blocked with 0.25% BSA, 0.05% Tween, and 0.02%
azide for 4 h. Wells were washed with PBS and then incubated with
125I-AcLDL for 4 h at 4 °C. Competition was
performed in the presence of unlabeled AcLDL (400 µg/ml) or poly(I)
(100 µg/ml). After washing the wells three times with PBS, the wells
were excised and counted in a gamma counter.
Protein Purification--
cDNAs encoding fusion proteins
were transfected into COS cells as described above. 24 h after
transfection, the cells were extensively washed with PBS, and the COS
cell media were replaced with a serum-free substitute (Opti-MEM from
Life Technologies, Inc.). 48-72 h subsequent to this, the media were
collected; phenylmethylsulfonyl fluoride (0.5 mM final
concentration) was added to it, and the media were then chilled to
4 °C. After chilling, the media were adjusted to 100 mM
Tris before binding to 0.5 g of protein A (Sigma) that had been
equilibrated in the same buffer. Binding was performed on a shaking
platform overnight at 4 °C. The protein A was then packed into an
Amersham Pharmacia Biotech 10/10 column. By using the Amersham
Pharmacia Biotech FPLC system, the column was then washed with 10 ml of
100 mM Tris, pH 8.0, followed by 10 ml of 10 mM
Tris, pH 8.0. Protein was eluted from the column in 100 mM
glycine, pH 3.0, using on-line UV absorption monitoring at 280 nm. The
eluate was immediately buffered with one-tenth volume of 1.0 M Tris, pH 8.0, to which phenylmethylsulfonyl fluoride was
added (0.5 mM final concentration). The protein eluate was stored at
20 °C until used.
Ligand Binding Analysis--
Data from saturation binding
studies were analyzed using a modified version of the Ligand program
(RADLIG; G.A. McPherson, Biosoft, Ferguson, MO). Curve fitting was done
using both a one-site and a two-site binding model. The molecular
weight of apolipoprotein B (518,000 daltons) was used to estimate the
molarity of AcLDL protein.
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RESULTS |
Proteins Generated in the Study--
The SR fusion
proteins created for this study are depicted in Fig.
1. These proteins were encoded by rabbit
type II cDNAs from which the N-terminal cytoplasmic tail and
transmembrane domain sequences had been removed. They were replaced
with a signal sequence and Fc domain derived from human
IgG. Wild type scavenger receptor cDNAs (for use in cell
degradation studies) and fusion receptor cDNAs were also modified
so as to encode a rhodopsin epitope sequence at the C terminus of the
protein (Fig. 1). The epitope tag was originally designed to assay
receptor expression on the surface of transfected cells. Although the
epitope tag was readily detected under the denaturing conditions used
for the immunoblots, its detection on intact cells was unreliable,
perhaps indicating that access to this C-terminal tag is blocked when
the SR is folded into its native, trimeric structure. The demonstration
by Resnick et al. (27) that the extended C terminus of the
type I scavenger receptor appears to fold into a globular domain
tightly juxtaposed to the fibrous strands of the collagen domain
provides some support for this hypothesis. The nine amino acid epitope
sequence did serve two other purposes as follows: 1) to identify any
unintended frameshift mutation that could have occurred in the
mutagenesis process and that might have eluded detection by DNA
sequencing; 2) to permit detection of the epitope-containing proteins
by immunoblot. To ensure that the binding of ligands to the SR fusion
proteins was dependent only on the presence of the SR sequences within the fusion, a truncated protein (IgGS), containing only the human IgG
Fc fragment, was also generated. Mutant receptors
cDNAs, corresponding to the fusion proteins, but retaining their
cytoplasmic and transmembrane domains, were also generated for use in
cellular degradation experiments following transfection into COS cells.

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Fig. 1.
A, schematic representation of proteins
employed in this study. The wild type protein for the study is the type
II rabbit scavenger receptor encoded by the cDNA cloned by Bickel
and Freeman (13). The six domains of the receptor are not depicted to
scale. Proteins containing human IgG have had their scavenger receptor
cytoplasmic tail and transmembrane sequences replaced by an
Fc fragment from human immunoglobulin. The nine amino acid
epitope tag, represented by the single letter amino acid
code TETSQVAPA, derives from bacterial rhodopsin and is
recognized by the monoclonal antibody, ID4 (23). The inset
photo shows media collected from COS cells transfected with the
expression plasmid, pCDNA-1, containing the wild type fusion
cDNA (IgG-SR) or no insert (Mock). Cells were
labeled with 587 µCi of [35S]methionine per 10-cm
plate, washed in serum-free media, and then incubated in serum-free
media for 2 h. Media were bound to 0.5 g of protein A, washed
with 10 ml of 0.1 M Tris, pH 8.0, then washed with 10 ml of
0.010 M Tris, pH 8.0, and finally eluted with 0.1 M glycine, pH 3.0, in 0.5-ml aliquots. The aliquots were
adjusted to pH 8.0 with 1 M Tris, pH 8.0, containing the
protease inhibitor phenylmethylsulfonyl fluoride. The eluate was
electrophoresed under reducing conditions on an 8% SDS-polyacrylamide
gel overlaid with a 4% stacking gel, and autoradiography was
performed. B, collagen domain alignment of SR proteins. The
collagen domains of the bovine (bo), human (hu),
murine (ms), and rabbit (ra) are aligned. The
collagen triplets (Gly-X-Y) containing charged
residues are in bold. Four subdomains, containing positively
charged residues, were arbitrarily assigned the names CCM (collagen
charge mutation)-1 through CCM-4. The bottom half of the
figure lists each of the wild type and mutant proteins used in the
study and the mutations they contain. All of the charge mutants
converted a positively charged amino acid (Lys or Arg) to an uncharged
residue. Proteins that contain the rhodopsin epitope tag have an E
appended to their name.
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Protein Purification and Characterization--
After DEAE-dextran
transfection of COS cells with the receptor cDNAs, secreted
proteins were collected in a serum-free medium and then purified by
protein A column chromatography as described under "Experimental
Procedures." Fig. 1 shows an autoradiogram of the column eluate that
resulted from this procedure following [35S]methionine
labeling of COS cells transfected with the wild type fusion cDNA.
Under reducing conditions, a single predominant band of approximate
molecular mass of 95 kDa was visualized (Figs. 1 and
2B). The expected molecular
weight of the fusion protein is the sum of the mass of the IgG
fragments (approximately 30 kDa) plus the mass of the SR's
extracellular domains. The extracellular domains of the type II SR
would be expected to have a mass of 57-64 kDa (the highly homologous
intact bovine type II receptor was previously shown to have a mass of
between 65 and 72 kDa (28) and the two SR domains removed in the fusion
construct have a calculated mass of 8.3 kDa). Thus, the 95-kDa fusion
protein migrated on an SDS-polyacrylamide gel in the expected size
range. The yield of fusion protein varied between 4 and 12 µg per
10-cm transfection plate, 72 h after transfection, with most
transfections typically yielding 6-8 µg per plate. COS cells
continued to secrete fusion proteins for at least 120 h from the
time of transfection, making it possible to obtain additional protein
with subsequent media collections. Minimal non-scavenger receptor
protein contamination of the protein A-purified preparations was
visible after [35S]methionine labeling (Fig. 1 and data
not shown). A protein of approximately 50 kDa (Fig. 2) was consistently
evident on Coomassie staining of electrophoresed proteins. This protein
had no ligand binding activity (Fig.
3A) and was subsequently
determined to be residual bovine IgG, deriving from the calf serum in
which the COS cells were originally cultured. More extensive washing and depletion of calf serum of IgG before use eliminated this contaminant (see Fig. 4).

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Fig. 2.
Oligomerization and glycosylation of fusion
proteins. A, wild type and mutant scavenger receptor fusion
proteins assemble into high molecular mass oligomers of approximately
400, 300, and 180 kDa. 5-10 µg of each affinity chromatography
purified fusion protein was electrophoresed in an 3-10%
SDS-polyacrylamide along with protein size markers (Sigma) of the
indicated molecular weight. Proteins were fixed in methanol/acetic acid
containing Coomassie Blue, destained in methanol/acetic acid without
the dye, and then photographed. B, monomeric scavenger
receptor fusion proteins migrate with an apparent size of 95 kDa, 30 kDa of which can be removed by deglycosylation. 5-10 µg of each
fusion protein was electrophoresed in an 3-10% SDS-polyacrylamide gel
in a loading buffer containing 5% -mercaptoethanol. In lanes
2-5, the fusion proteins were first deglycosylated, prior to
electrophoresis, using peptide-N-glycosidase F. Proteins
were fixed, stained, and photographed as described in Fig.
3A. The protein migrating between 45 and 66 kDa was not
visible on autoradiographs of media collected from
[35S]methionine-labeled cells, was recognized by
antibodies to bovine IgG, and was absent from cells incubated in serum
that had been depleted in bovine IgG by column chromatography (data not
shown). B-gal, -galactosidase; phos.b,
phosphorylase b; ovalb, ovalbumin.
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Fig. 3.
Ligand blots with fusion proteins.
A, scavenger receptor fusion proteins, containing wild type
collagen domains, when electrophoresed through SDS-polyacrylamide gels
and transferred to nitrocellulose, bind AcLDL. The gel depicted in Fig.
2A was transferred to nitrocellulose in 25 mM
Tris, pH 8.3, 192 mM glycine, 20% methanol as described
under "Experimental Procedures." 5 µg/ml of AcLDL was then
incubated with the filter in Buffer A (50 mM Tris, pH 8.0, 2 mM CaCl, 50 mM NaCl, 50 mg/ml bovine serum
albumin). After washing the filter with Buffer A (containing only 10 mg
of BSA mg/ml), the AcLDL bound to filter proteins was detected using a
rabbit anti-human LDL antibody (1:5000 dilution in Buffer A) followed
by binding with a goat anti-rabbit peroxidase-labeled second antibody
(1:1000 dilution in Buffer A). Color detection was performed using DAB
according to the manufacturer's (Sigma) instructions. B,
binding of AcLDL to scavenger receptor fusion proteins is competed by
poly(I). A Bio-Rad Mini Transblot apparatus was used to transfer wild
type and mutant fusion proteins using the methods described in Figs. 2
and 3. AcLDL binding and detection of bound ligand were also performed
as described in A. For the competition experiment, 400 µg/ml poly(I) was added to the binding solution containing
AcLDL.
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Fig. 4.
Immunoblot of SR fusion proteins.
A, detection of fusion proteins using an anti-IgG monoclonal
antibody. Gel electrophoresis and transfer were performed as described
in Figs. 2-3 and under "Experimental Procedures." A 1:5 dilution
of the supernatant taken from a hybridoma secreting the anti-human IgG
antibody, ASAb-1, was used to bind to the filter. After washing,
detection was accomplished using a peroxidase-labeled anti-mouse
antibody and DAB. B, detection of epitope-tagged fusion
proteins using an anti-rhodopsin antibody. The anti-rhodopsin epitope
monoclonal antibody, ID4(23), was used to detect proteins which had
been electrophoresed and transferred to nitrocellulose as described in
the previous figures. Detection of ID4 was accomplished with a
peroxidase-labeled anti-mouse antibody and DAB.
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SDS-polyacrylamide gels, run without (Fig. 2A) or with (Fig.
2B) reducing agent, in which wild type and mutant SR fusion
proteins have been separated by electrophoresis are depicted in Fig. 2. Protein was detected by Coomasie staining (A and
B). In the unreduced state, there appear to be four major
regions of protein staining of the wild type fusion protein. The lack
of discrete bands is due to variable glycosylation of the scavenger
receptor portion of the fusion proteins. These four regions have
approximate molecular masses, in descending order, of 400, 300, 180, and 100-130 kDa. The three higher weight proteins are multiples of the
95-kDa monomeric mass of the SR fusion protein and are presumed to be
tetramers, trimers, and dimers. The band migrating between 100 and 130 kDa may represent dimers of poorly glycosylated fusion proteins or proteolytic fragments of the higher molecular weight oligomers. Neither
contaminating bovine immunoglobulin nor the control human IgGS protein
binds AcLDL, as seen by the lack of lipoprotein staining on ligand
blots (Fig. 3A, lanes 4 and 7, respectively). As
previously shown by Penman et al. (28), the native SR
normally runs as a trimer and dimer under non-reducing conditions, and
the trimeric form is composed of a non-covalently linked monomer in
association with a disulfide-linked dimer. The putative tetrameric
protein present in Fig. 3A could represent the addition of a
monomer to the trimer, via a second disulfide linkage between the
cysteine residues present in the hinge region of the Fc
portion of the fusion protein, or a dimer of dimers. As Penman et
al. (28) showed, preparations of purified native SR proteins that
do not protect against oxidation frequently result in disulfide
linkages between all three chains of the trimer.
Under reducing conditions (Fig. 2B), the native fusion
protein migrates with the expected mass of approximately 95 kDa
(lane 8). After de-glycosylation, this protein has an
apparent mass of 60-65 kDa, indicating that glycosylation contributes
approximately 30 kDa to the mass of the fusion protein (lane
4). The epitope-containing fusion proteins (RT2E) migrate somewhat
more slowly in both the unreduced and reduced conditions, reflecting
the presence of their C-terminal nine amino acid epitope tag. The
migration of the human Fc fragment alone (IgGS) and an
epitope-containing mutant form of the fusion protein (CCM-2FE) are also
shown on these gels. The CCM-2FE migrates in a pattern identical to
RT2E. These data indicate that the SR fusion protein assembles into
higher molecular weight oligomeric structures, including those
typically seen with the native SR protein, and that charge mutations in
the collagen domain do not prevent the oligomerization process. These
data, in conjunction with the binding data presented below, establish that the native cytoplasmic tail and transmembrane domains of the SR
are not essential for the formation of a functional, oligomeric, rabbit
scavenger receptor, a finding confirming similar observations made by
Resnick et al. (5) in work with bovine and human SRs.
Fusion Protein Function--
The function of the SR fusion
proteins was tested using several different ligand binding assays.
These included ligand blots, in which the proteins were first
electrophoresed through polyacrylamide and then transferred to a solid
filter support (nitrocellulose). Ligand binding to the proteins could
then be measured either using a radiolabeled ligand or using a
non-labeled ligand whose binding could be detected immunologically.
Fig. 3, A and B, shows ligand blots in which the
demonstration of ligand binding was performed using the antibody
detection method. When ligand binding was conducted in the presence or
absence of the SR binding competitor, polyinosinic acid (poly(I)) (Fig.
3B), the specificity of ligand binding to the wild type SR
fusion protein, with and without the epitope sequence, was
demonstrated. No anti-AcLDL antibody staining is detected to the IgGS
mutant, either in the presence or absence of poly(I), indicating that
the human IgG fragment is not responsible for any of the binding of the
AcLDL to the SR fusion proteins. Similarly, there is little or no
binding evident with the CCM-2FE mutant. Several other mutants appear
to have diminished binding (e.g. CCM-3FE, CCM-1-(335,341))
when compared with that seen with the wild type proteins. These results
qualitatively demonstrate that charged residues throughout the entire
collagen domain of the SR fusion protein are critical for receptor
binding activity.
To confirm that the proteins that did not bind ligand were effectively
transferred to the nitrocellulose, without undergoing degradation,
immunoblots were performed using two antibodies (Fig. 4). ID4, a
monoclonal antibody to the rhodopsin epitope, and ASAb-1, a monoclonal
antibody raised to the wild type SR fusion protein, were used. Only the
epitope containing proteins (RT2E, CCM-2FE, CCM-3FE, and CCM-4FE) were
recognized by ID4, whereas all proteins were recognized by ASAb-1,
including the IgGS, indicating that its epitope recognition site
resides within the human IgG portion of the fusion protein. The
immunoblots demonstrate that the failure to detect AcLDL binding to
several of the mutant fusion proteins was neither due to a failure of
those proteins to be transferred to the nitrocellulose membrane nor to
protein degradation of the mutant receptors.
Whereas the ligand blotting methods permitted a qualitative assessment
of SR fusion protein binding activity, they did not permit any
quantitative assessment of function to be made. Two other binding
assays were employed in an effort to establish more quantitative
estimates of fusion protein function. In the first assay, equal amounts
of receptor proteins were electrophoresed and then transferred to
nitrocellulose filters. Radiolabeled AcLDL was then bound to the
filters at varying temperatures. Equally sized filter strips
representing each lane of the gel were then excised, and the bound
125I was subsequently counted. A second assay was used in
which the SR fusion proteins were bound to plastic wells (Terasaki
plates), with binding of 125I-AcLDL, and then measured in
the presence or absence of competing, unlabeled AcLDL. Saturation
binding studies were conducted to determine the affinity of
radiolabeled AcLDL for the purified proteins.
Filter binding assays were conducted at three different temperatures
(4, 22, and 37 °C) and are shown in Fig.
5A-C. At the lowest temperature, only the IgG control and CCM-2FE proteins had
binding activity less than 10% of the wild type proteins. All of the
CCM-1 proteins, as well as CCM-4FE and CCM-2-(317,325) retained greater
than 60-70% of wild type binding activity. The CCM-3FE protein,
however, did have significantly less binding activity than the RT2E
protein, indicating that mutations in this more proximal region of the
SR collagen domain could dramatically affect receptor binding. Somewhat
surprisingly, the effect of many of the mutations proved to be
temperature-dependent. As the binding reactions were
carried out at progressively higher temperatures, more of the mutants
lost their activity. At 22 °C, all of the CCM-1 mutations fell to
less than 50% wild type binding, and at 37 °C only CCM-2-(317,325)
retained more than one-quarter of the wild type activity. These data
indicate that mutations in charged residues throughout the collagen
domain can dramatically affect ligand binding but that the effects of
most of those mutations are temperature-dependent. Only the
triple charge mutant, CCM-2FE, was incapable of binding ligand at any
of the three temperatures, establishing the critical importance of
residues 317, 325, and 328 in receptor function. The
temperature-dependent effects of the other mutations
suggest that the receptor conformation in the collagen domain is a
critical feature of ligand binding and that the substitution of
non-charged residues for positively charged residues in this domain can
disrupt that conformation.

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Fig. 5.
Binding of 125I-AcLDL at varying
temperatures to fusion proteins immobilized on nitrocellulose
membranes. A, 4 °C binding. Equal amounts of fusion
proteins were electrophoresed through SDS-polyacrylamide gels and
transferred to nitrocellulose membranes as described in Figs. 2-3 and
under "Experimental Procedures." Individual lanes were excised and
separated and then bound to 125I-AcLDL (1 × 106 cpm) in Buffer A (50 mM Tris, pH 8.0, 2 mM CaCl, 50 mM NaCl, 50 mg/ml BSA) for 4 h
at 4 °C. Strips were washed four times in Buffer A containing only
10 mg/ml BSA and then counted in a gamma counter. The difference
between the binding to identically sized filter strips containing
fusion proteins and control strips, cut from the same filter but in
lanes lacking any protein, was determined, and this value is depicted
as specific binding in the graphs. Values represent the mean ± S.D. B, 22 °C binding. Binding studies carried out as
described in A were performed at 22 °C. C,
37 °C binding. Binding studies carried out as described in
A were performed at 37 °C. D, loss of binding
to a mutant protein at 37 °C is not due to degradation or
irreversible denaturation. The mutant CCM-1-(335,341), which showed a
marked temperature-dependent loss in ligand binding, was
used to assess the reversibility of this loss. Wild type fusion
proteins, which showed no loss of binding at increased temperatures,
were included for comparison. Fusion proteins transferred to filters as
described in the previous figures were incubated for 4 h at either
4 or 37 °C. Following this preincubation period, a 4-h binding with
125I-AcLDL (1 × 106 cpm), at either 4 or
37 °C, was then performed. Binding was assayed as described in
A.
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An alternative explanation for the temperature-dependent
loss in protein binding seen with many of the mutant receptor proteins was that these proteins were more readily susceptible to protein degradation at the higher temperatures. To test that possibility, a
temperature shift binding experiment was conducted. In this study, one
of the mutants (CCM-1-(335,341)) with the most dramatic temperature-dependent losses in binding was employed.
Binding was preceded by a 4-h incubation at either 37 or 4 °C,
followed by binding at either 37 or 4 °C. As seen in Fig.
5D, the mutant CCM-1-(335,341) had less than 20% of its
binding activity retained when both incubations were carried out at
37 °C, consistent with the data shown in Fig. 5C. When
the preincubation at 37 °C was followed by binding at 4 °C,
however, the protein had a similar binding activity to that seen when
both the preincubation and the binding were performed at the cooler
temperature. Temperature shifts had little or no effect on the two
wild-type proteins. This experiment demonstrated that the results of
the 4-h binding experiments conducted at 37 °C (Fig. 5C)
were not due to irreversible denaturation or degradation of the mutant
receptor protein.
Saturation binding curves for all of the mutants that retained
significant binding activity at 4 °C were performed (data not shown). The ligand binding dissociation constants for these purified proteins, presented in Table I, are in
close agreement to those established for the wild type receptor protein
expressed on Chinese hamster ovary cells (4). Modeling of the number of
receptor binding sites for AcLDL did not show a statistically
significant better fit of the data for a two-site model
versus a one-site model. There was also a very high error
rate in the estimate of the Kd of a potential second
binding site. In addition, the ligand used for these studies, iodinated
AcLDL, has not been demonstrated to represent a single molecular
species, making lower affinity interactions with the receptor difficult
to interpret. Thus, based on these studies, we cannot establish
unequivocally the number of receptor ligand binding sites, although
there does appear to be only one high affinity binding region. These
data indicate that the purified proteins, with nanomolar binding
affinities virtually identical to those found for receptors expressed
in vivo, are a useful surrogate for receptor studies
in vivo.
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Table I
Binding affinities of scavenger receptor wild type and mutant fusion
proteins
Listed are the apparent dissociation constants (Kd)
and maximal binding sites (Bmax) as estimated from
saturation binding studies. The values for the non-mutant receptors are
given for one-site and two-site models of receptor ligand binding
interactions at 4 °C and for a one-site model at 22 °C. The
mutant receptor values utilize only the one-site model at 4 °C. The
error in calculating the second site Kd and
Bmax values exceeded 1000%, making them unreliable.
Comparison of the data for one- and two-site models did not show a
statistically significant better fit for one model. All values are
expressed as nmol/liter concentration (1 µg/ml = 1.93 nM).
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As the fusion proteins were also capable of assembling into oligomers
not typically formed by the wild type scavenger receptor, it was
important to confirm that the results obtained with the purified
proteins were not due to any anomalies of the fusion protein structure.
Cell binding and degradation studies were, therefore, conducted using
transmembrane scavenger receptor mutants identical (in their
extracellular domains) to the SR portions of the fusion proteins
studied in vitro (Fig. 6). The
binding and degradation of AcLDL at 37 °C in COS cells transfected
with these mutant proteins provide corroborative evidence for the
importance of temperature on receptor activity. The fusion proteins
that bound poorly at 37 °C also produced transmembrane proteins with markedly reduced AcLDL degradation or binding at the same temperature. When cellular binding activity was measured at 4 °C, the binding was
reconstituted in those cells expressing proteins whose fusion analogue
also reconstituted binding at the lower temperature. The reconstitution
of binding at 4 °C indicates that intracellular mishandling or
degradation of the mutant proteins cannot account for the reduced
cellular binding and degradation demonstrated at 37 °C. Thus, the
cellular experiments provide an important corroboration of the validity
of the results obtained with the purified fusion proteins.

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Fig. 6.
AcLDL binding and degradation to COS cells
transfected with wild type and mutant SRs. A,
125I-AcLDL degradation by COS cells expressing wild type
and mutant proteins. 2 µg of each cDNA encoding scavenger
receptors that retained their transmembrane and cytoplasmic tail
domains, but containing the mutant collagen domains used in the fusion
proteins, were transfected into COS using DEAE-dextran, as detailed by
Aruffo and Seed (20). 72 h after transfection, 5 µg of
protein/ml of 125I-AcLDL was then bound and degraded in
6-well dishes as described previously (21, 33). Nonspecific degradation
was assessed by performing the degradation in the presence of
competing, unlabeled AcLDL (400 µg/ml). Degradation results are
expressed as nanograms of AcLDL protein, degraded over 5 h of
incubation time, per mg of cellular protein. B, binding of
125I-AcLDL to transfected COS cells at 4 and 37 °C.
250,000 COS cells per well were transfected in 6-cm tissue culture
dishes as described in A. 48 h after transfection, the
medium was replaced with Dulbecco's modified Eagle's medium
containing 5% human lipoprotein-deficient serum containing 10 µg/ml
125I-AcLDL. Binding was conducted at the indicated
temperature for 4 h in the presence or absence of 200 µg/ml
AcLDL. An inhibitor of endocytosis was not used in these experiments,
thus permitting some ligand to be internalized during the course of
binding at the higher temperature. Media were then removed, and the
cells were washed 5 × with 50 mM Tris, 0.15 M NaCl, 2 mg/ml BSA, pH 7.4. A sixth wash with the same
buffer minus the BSA was then performed. Cells were then lysed in 1.5 ml of 0.1 N NaOH and the cell-associated
125I-AcLDL counted. Values represent the mean ± S.D.
of specific binding (counts bound in absence of unlabeled AcLDL minus
counts bound in presence of unlabeled AcLDL) from quadruplicate
transfections of the cells.
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DISCUSSION |
In these studies, we have generated mutants spanning the entire
collagen domain of the type II macrophage scavenger receptor. These
mutants were expressed as integral membrane proteins on the surface of
transfected COS cells, as well as purified, secreted fusion proteins.
The removal of the cytoplasmic tail and transmembrane domains of the SR
fusion proteins did not prevent the assembly of the receptor into
functional oligomers that retain wild type binding affinity. AcLDL
bound to the fusion proteins, containing wild type extracellular
domains of the scavenger receptor protein, when the receptor proteins
were either transferred to nitrocellulose membranes or used to coat
plastic wells. Saturation binding studies and Scatchard analysis
suggested that the receptor fusion proteins possessed a single high
affinity binding site with an apparent Kd of
approximately 1-4 nM (1.0 µg/ml = 1.93 nM), consistent with the estimates of SR binding affinities
suggested by previous studies conducted in transfected cell lines (4).
This high affinity binding is destroyed in a single mutant receptor
protein that simultaneously replaces the three basic residues at
positions 317, 325, and 328 of the C terminus of the collagen domain
with three non-charged amino acids. Table
II shows a comparison of the binding
activities of all the SR charge mutants reported to date (the data from
this paper combined with that reported by Doi et al. (19)).
Mutations in the subdomains of the collagen region that we termed CCM-3
and CCM-4, which had not been previously altered in SR binding studies,
also resulted in a significant decline in ligand binding at 37 °C
that was at least as profound as that found with the distal collagen
mutants. Several mutations in the collagen domain demonstrated a clear
dependence on temperature for their effect. This progressive loss in
binding, seen as temperatures were raised from 4 to 37 °C, occurred
without irreversible dissociation of the oligomeric structure of the
receptor, as binding could be restored by lowering the temperature
again to 4 °C. The temperature-dependent loss in binding
did suggest, however, that important conformational shifts in the
protein occurred at the higher temperatures.
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Table II
Comparison of binding of proximal and distal collagen mutants at 37 and
4 °C
Values in the table represent the percentage of binding compared to the
appropriate wild type control. As the bovine SR (used in Doi et
al. 19) study) has one less amino acid than the rabbit SR,
proximal to the collagen domain, the numbers used in this table have
been converted to the rabbit numbering system, e.g. bovine
Lys-337 is homologous to Lys-338 in the rabbit sequence. The data from
Doi et al. (19) derives from the graphic data presented in
their paper.
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The data presented in this paper call into question the prevailing
model of SR binding. Doi et al. (19) concluded that their mutational analysis demonstrated that Lys-337 (equivalent to Lys-338 in
the rabbit sequence) was essential for AcLDL binding and that "a
lysine cluster in the C-terminal collagen-like domain is the ligand-binding domain of the scavenger receptor." The authors of this
work constructed a computer graphic model that suggested that the
receptor might form a three-coil, positively charged groove responsible
for the interaction with polyanionic ligands. They suggested that
residues 327, 334, 337, and 340 (comparable to 328, 335, 338, and 341 in this study) were the critical ligand binding amino acids and that
they formed a region of positive charges that bound to the negatively
charged moieties on SR ligands. This conclusion, now widely accepted,
has led to several subsequent studies by different investigative groups
in which peptide models of SR binding have been constructed using only
the three or four terminal lysine cluster residues to generate
synthetic receptor analogues (29-32). Although these peptides can bind
to SR ligands, the affinities of the binding interactions appear to be
at least 10-fold lower than for the native receptor or for the fusion
protein reported in this work (32). Earlier work by Acton et
al. (17), using C1q collagen sequences that are unrelated to the
SR collagen domain, also showed that these collagenous molecules
retained considerable capacity for binding SR ligands. Thus, the
demonstration of lower affinity binding of SR ligands to synthetic
collagen peptides is not sufficient to establish that any one region
constitutes the authentic binding locus of the receptor. Finally,
established SR ligands, such as polyguanylic acid, have failed to bind
to peptide models based on the lysine cluster model (30), a finding that does not support some of the concepts presented in the initial description of the positively charged groove hypothesis (19).
The loss in receptor binding seen in our CCM-3 and CCM-4 mutants
establishes that residues as proximal as position 284, and no more
distal than 308, are potentially as important to SR binding as those in
the more C-terminal lysine cluster. Furthermore, the restoration of
binding to several mutants at 4 °C, in both the proximal and distal
collagen domain, is very strong evidence that the loss in ligand
binding seen at the higher temperatures was not due only to disruption
of ionic interactions between the receptor and ligand, but rather that
multiple, charged residue interactions within the receptor are required
for its binding conformation at higher temperatures. The similarity of
the apparent binding dissociation constants (Kd) for
the mutant receptors and the wild type receptor at 4 °C (Table I)
further suggests that the mutant receptors that can reassemble at the
lower temperature are capable of presenting a binding domain that does
not differ very substantially from that of the wild type protein. The
lower Bmax for the mutant receptors suggests
that not all of the mutant molecules can successfully re-establish a
binding conformation when the temperature is reduced. Interestingly,
the study by Doi et al. (19) also contains two mutants, both
involving the amino acid they propose is critical to binding (Lys-337),
that substantially reconstitute binding activity at a lower temperature
(Table II). This observation led them to conclude that binding studies
to the SR need to be conducted at 37 °C. Whereas we agree that the relevant binding temperature for understanding SR function in vivo is 37 °C, the binding studies at 4 °C are essential to
any understanding of the structural determinants of receptor function. The slight increase in binding affinity we noted in wild type receptor
interaction at higher temperatures (Table I) does provide some support
for the concept by Doi et al. (19) that the collagen strands
become more closely compacted at increasing temperature and that this
could account for improved binding.
It should be noted that for the two mutants that were common to our
work and that of Doi et al. (19), the binding data are not
entirely concordant. Binding to the single 335 mutant at 4 °C and to
the double (335,338) mutant at 37 °C gave similar results, but the
335 mutant binding at 37 °C and the double mutant activity at
4 °C yielded discordant results. Whereas there is no definitive explanation for these discrepancies, it is possible that the
differences in amino acid substituted (Doi et al. (19)
replaced 335 and 338 charged residues with alanines) could account for
the difference. The failure of the alanine substitutions to show
consistently better retention of binding activity in the two mutant
receptors makes this explanation, perhaps, somewhat less compelling.
Alternatively, a defect in endocytosis might explain the discrepant
values at 37 °C, as our cellular binding studies were not performed
in the presence of an endocytosis inhibitor. Thus, the cellular binding values we report at 37 °C also include the uptake of some ligand that has been internalized. If any of the collagen mutants lowered the
rate of endocytosis, this would result in an apparent lower binding
compared with wild type receptor. As our cell binding experiments
recapitulated the binding measured in the purified protein assays, and
as a wide range of mutants, studied at both 37 and 22 °C, share the
temperature-dependent binding behavior, we think the data
are unlikely to differ because of a difference in endocytosis. An
endocytosis defect would also not account for the difference in binding
measured at 4 °C with the double mutants. Thus, there is no entirely
satisfactory explanation for the differences in the binding behavior of
the two mutants reported in these two studies.
Currently available data make it challenging to propose a precise model
for SR ligand binding. It is clear that the collagen domain is required
for binding and that positively charged residues in both the proximal
and distal regions of that domain are necessary to maintain binding at
physiologic temperature. Inactivating charge mutants in both regions
can be silenced by reducing the temperature of binding, a finding that
is most consistent with the hypothesis that the mutated residues are
critical for stabilizing receptor conformation at higher temperatures.
The simplest explanation for this result is that the trimeric collagen
strands must adopt a precise conformation to achieve ligand binding and
that changes in charge, in either the proximal or distal collagen
domain, disrupt that conformation. In lower energy states, the
necessary binding conformation can be maintained without all of the
interactions that are mediated by the charged residues, but at
physiologic temperature, these interactions are essential to hold the
receptor binding domain together. It is possible that SR ligands
interact directly with many of the charged residues, spanning the
collagen domain, or only a few, clustered in one region. The effects of mutations in one region may serve merely to disrupt the conformation of
contact residues at a distant site. An alternative explanation for the
data presented in this study is that the temperature shifts employed
induced an alteration in the ligand conformation, either in conjunction
with or instead of alterations in the receptor conformation. Whereas
this remains a formal possibility, the diversity of receptor mutants
that restore activity, their widespread spacing across the entire
collagen domain, and the similarity of the affinity constants for
binding to mutant and wild type receptors at the lower temperatures all
suggest that this explanation is unlikely.
Although the relevant conformational interactions necessary to
establish SR ligand recognition could all reside within the collagen
domain, recent data by Resnick et al. (27) suggest the
intriguing possibility that high affinity SR binding might require the
cooperation of two SR domains. In an electron microscopy study, they
presented evidence indicating that the SR adopts a jackknife
configuration, with the collagen domain bent back in close apposition
to the
-helical coiled-coil domain (27). Our data, and the earlier
work cited above, are also consistent with the hypothesis that this
jackknife configuration is essential for the highest affinity binding.
Deletion of the collagen domain, or mutations in its charged residues,
could disrupt this two domain interaction, resulting in an unfolding or
dismantling of the jackknife and a consequent loss in binding activity.
This hypothesis is directly testable. As the ability of scavenger
receptors to bind a multitude of ligands, some of which are thought to
be critical to the pathogenesis of human diseases, is the defining
trait of this family of proteins, further studies of its distinct
mechanism of ligand recognition are warranted in order to clarify this
remarkable property.
We thank R. P. Aftring, N. Freedman, H. Kronenberg, M. Krieger, A. Rich, B. Seed, and E. Harlow for the
many helpful suggestions they offered. We are also grateful to Dr. Anne
Sarbinowski for technical assistance in raising the monoclonal
antibody, ASAb-1, used in this study.