From the Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
Received for publication, January 17, 2000, and in revised form, February 8, 2001
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ABSTRACT |
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The macrophage mannose receptor mediates
phagocytosis of pathogenic microorganisms and endocytosis of
potentially harmful soluble glycoproteins by recognition of their
defining carbohydrate structures. The mannose receptor is the prototype
for a family of receptors each having an extracellular region
consisting of 8-10 domains related to C-type carbohydrate recognition
domains (CRDs), a fibronectin type II repeat and an N-terminal
cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments
performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the
receptor is a monomeric, elongated and asymmetric molecule. Proteolysis
experiments indicate the presence of close contacts between several
pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for
modeled receptor conformations are consistent with an extended
conformation with close contacts between three pairs of CRDs. The
N-terminal cysteine-rich domain and the fibronectin type II repeat
appear to increase the rigidity of the molecule. The rigid, extended
conformation of the receptor places domains with different functions at
distinct positions with respect to the membrane.
The mannose receptor of macrophages and liver endothelial cells is
the best characterized member of a family of multidomain cell-surface
receptors (1). Each member of the family shares the same overall domain
organization, with an extracellular region consisting of an N-terminal
cysteine-rich domain followed by a fibronectin type II repeat and 8 or
10 domains related in sequence to the C-type carbohydrate-recognition
domains (CRDs)1 of animal
lectins. The mannose receptor and one other member of the family,
endothelial receptor Endo-180, are true C-type lectins, binding
carbohydrates in a Ca2+-dependent manner (2,
3). In contrast, the C-type lectin-like domains of the M-type
phospholipase A2 receptor and the dendritic cell
receptor DEC-205 do not contain the conserved residues necessary for
Ca2+-dependent sugar binding, and these two
receptors do not bind carbohydrates (4-6). Each of the receptors
mediates endocytosis, but physiological ligands have only been
identified for the mannose receptor and the phospholipase
A2 receptor.
The mannose receptor acts as a molecular scavenger, binding and
internalizing pathogenic microorganisms and potentially harmful glycoproteins (7, 8). The multiple domains in the extracellular region
of the receptor allow recognition of a diverse range of glycoconjugate
ligands. Several of the eight C-type CRDs are involved in
Ca2+-dependent recognition of terminal mannose,
N-acetylglucosamine or fucose residues on the surfaces of
microorganisms or on the oligosaccharides of endogenous glycoproteins
(9, 10). The receptor plays a role in the immune response against
pathogens such as Mycobacterium tuberculosis and
Pneumocystis carinii. It also regulates levels of endogenous
proteins such as lysosomal enzymes and tissue plasminogen activator
released from cells in response to pathological events. In addition,
the mannose receptor binds terminal sulfated
N-acetylgalactosamine residues on oligosaccharides of the
pituitary hormones lutropin and thyrotropin and plays a role in
clearing these hormones from the circulation after they have acted on
their target cells (11, 12). The cysteine-rich domain of the mannose
receptor recognizes sulfated N-acetylgalactosamine by a
mechanism distinct from that of the C-type CRDs (13, 14).
Individual C-type CRDs of the mannose receptor show only weak affinity
for monosaccharides. High affinity binding to the receptor requires
multivalent interactions of oligosaccharides involving several CRDs (9,
10, 15). CRDs 4 and 5 form a protease-resistant ligand-binding core
sufficient to bind some ligands but CRDs 4-8 are required for high
affinity binding to natural ligands such as yeast mannan. It is likely
that the presence of multiple C-type CRDs within the single polypeptide
chain of the mannose receptor is important in determining specificity
as well as affinity for oligosaccharide ligands, because the spatial
arrangement of the CRDs must influence which ligands are able to
interact in a multivalent manner. The importance of the spatial
arrangement of C-type CRDs in defining ligand selectivity is
illustrated by serum mannose-binding protein, in which the orientation
of the three CRDs in each trimer is fixed so that mammalian
oligosaccharides cannot be recognized and inappropriate complement
fixation does not occur (8).
The mannose receptor is unusual when compared with other multidomain
cell surface receptors in that the domains that seem to be most
important for ligand binding, CRDs 4 and 5, are in the middle of the
polypeptide rather than at the end. The fifth of the eight C-type
lectin-like domains of the M-type phospholipase A2 receptor
is also most important for binding of secretory phospholipases (16).
One explanation for this phenomenon could be that the extracellular
regions of each of these receptors adopt a U-shaped conformation rather
than extending linearly from the cell surface (Fig. 1) (8). Such an
arrangement would put CRDs 4 and 5 furthest from the membrane.
This paper describes hydrodynamic analysis of the mannose receptor
combined with analysis of protease resistance. The results indicate
that the receptor is likely to adopt an extended conformation with
contacts between some domains.
Materials--
Sepharose 6B,
Expression and Purification of Mannose Receptor--
Production
of Chinese hamster ovary cell lines expressing the extracellular region
of the mannose receptor (MMR-S) and a fragment containing only the
eight CRDs (MMR1-8) has been described previously (18). Both cell
lines were maintained in Deglycosylation of Mannose Receptor Fragments--
MMR1-8 (0.2 mg) was treated with endoglycosidase H (20,000 units) in 1 ml of 50 mM sodium citrate, pH 5.5, or with peptide N-glycosidase F (10,000 units) in 1 ml of 50 mM
sodium phosphate, pH 7.5. Reactions were carried out at 37 °C
overnight. Enzymes were separated from MMR1-8 by anion-exchange fast
protein liquid chromatography on a Mono-Q column (Amersham Pharmacia
Biotech). Protein was loaded in 50 mM Tris-HCl, pH 8.0, and
eluted with a gradient of 0-0.5 M NaCl in the same buffer.
Matrix-assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry (MALDI-MS)--
Samples of MMR-S and MMR1-8 (100 pmol/µl) were analyzed on a FinneganMAT Lasermat mass spectrometer.
Sinapinic acid applied to the target at 7 mg/ml in 70% acetonitrile
containing 0.1% trifluoroacetic acid was used as the matrix. Ten
spectra were collected for each sample in positive ion mode, each
spectrum consisting of signals averaged from 10 to 30 consecutive laser
pulses. Dimeric bovine serum albumin ([M + H])+ = 132,860 Da) was used as a calibration standard.
Analytical Ultracentrifugation--
All experiments were carried
out at 20 °C in the An60Ti rotor of a Beckman Optima XL-A analytical
ultracentrifuge equipped with absorbance optics. Before analysis,
protein samples were dialyzed overnight against an appropriate buffer.
For equilibrium sedimentation experiments, five absorbance readings
were taken after samples were equilibrated for 24 h at 6,000 rpm
and a further five readings were made after 24 h equilibration at
7,000 rpm. Two further scans were taken after overspinning at 48,000 rpm to obtain background absorbance readings. Data from three different loading concentrations and the two different rotor speeds were analyzed
simultaneously using the non-linear curve-fitting program Nonlin.
Sedimentation velocity experiments were carried out at 30,000 rpm with
absorbance readings taken at 5-min intervals over 6 h. Data were
analyzed by the g(s*) method using Beckman
Instruments software. Sedimentation coefficients for MMR-S and MMR1-8
in the absence of Ca2+ were also calculated by the
second-moment method. Sedimentation coefficients were corrected for the
effects of buffer density and viscosity (19).
Chemical Cross-linking--
Mannose receptor fragments were
dialyzed into 0.1 M HEPES, pH 7.5, and 0.15 M
NaCl. In some experiments, 5 mM CaCl2 or 5 mM CaCl2 and 100 mM
Analytical Gel Filtration--
Analytical gel filtration
chromatography was performed on a 300 × 7.8-mm BioSep-S3000
column (Phenomenex). The column was eluted with 50 mM Tris
acetate, pH 7.5, at a flow rate of 0.5 ml/min. Thyroglobulin (669 kDa),
Analysis of Protease Resistance--
MMR1-8 and MMR-S were
subjected to limited trypsin digestion after adjustment of
Ca2+ concentration, following previously described
protocols (10). Briefly, MMR1-8 and MMR-S (0.25 mg/ml in 25 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 10 mM CaCl2) were incubated with increasing
amounts of trypsin for 30 min at 37 °C. Digestions were stopped by
addition of an equal volume of double strength sample buffer and
immediate heating to 100 °C for 5 min. Products were analyzed by
SDS-PAGE on 15% gels, stained with Coomassie Blue, and scanned using a Molecular Dynamics densitometer. Digestion products separated on 15%
mini-gels were subjected to N-terminal sequencing in a Beckman LF3000
protein sequencer following transfer to polyvinylidene difluoride
membranes (22).
Calculation of Size and Shape Parameters--
Partial specific
volumes for MMR-S and MMR1-8 were calculated from their amino acid
sequences and estimates of the extent of glycosylation (see below)
using the method of Cohn and Edsall (23) and published values of
partial specific volumes of amino acids and monosaccharides (19).
Diffusion coefficients (D) for MMR-S and MMR1-8 were
calculated from the Stokes radii determined by analytical gel
filtration. Stokes radii were also determined from molar frictional
coefficients calculated using sedimentation coefficient values and the
molecular masses determined by mass spectrometry (19). Stokes radii
obtained from sedimentation coefficient values and from analytical gel
filtration were used to calculate frictional ratios
(f/f0) (19). Axial ratios (a/b) were
calculated from frictional ratios for prolate ellipsoids of revolution
(24). Hydration of 0.28 g of H2O per g of protein was
assumed (19). Dimensions of MMR-S and MMR1-8 were calculated from
their axial ratios using Equations 14-19 of Tanford (25).
Hydrodynamic Modeling--
The program HYDRO was used to
calculate sedimentation and diffusion coefficients for models of
different receptor conformations (26). Models of MMR-S and MMR1-8 were
formulated as a series of contiguous spheres. Each domain was
represented by a sphere with a volume equal to that calculated from a
molecular surface generated with the program GRASP (27). Spheres of 30 Å diameter were used for the CRDs, based on the crystal structures of
the C-type CRD of rat mannose-binding protein A and mannose receptor CRD-4 (28, 29). The fibronectin-type II repeat was represented by a
sphere of 24 Å, based on the solution structure of the second of these
domains from fibronectin (30), while the cysteine-rich domain was
modeled as a sphere of 32 Å based on its crystal structure (14).
Linker regions between the domains were represented as four spheres of
diameter 5 Å. These dimensions were based on the average length and
volume calculated for stretches of 10 amino acids taken from six
different regions of the rat mannose-binding protein A structure
(28).
Hydrodynamic and proteolysis experiments were carried out on two
purified soluble fragments of the mannose receptor in order to examine
the shape, oligomeric state and domain arrangement of the receptor. One
fragment (MMR-S) consists of the whole extracellular domain of the
receptor, while the other (MMR1-8) consists of just the eight C-type
CRDs without the cysteine-rich domain and the fibronectin type II
repeat (Fig. 1). MMR1-8 was analyzed in
order to determine possible effects of the two N-terminal domains on the conformation of the receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-methylmannopyranoside and molecular weight markers
for gel filtration were obtained from Sigma.
Bis(sulfosuccinimidyl)suberate was obtained from Pierce.
Endoglycosidase H and peptide N-glycosidase F were from New
England Biolabs. Methotrexate, dialyzed fetal calf serum, and all media
were obtained from Life Technologies. Mannose-Sepharose was prepared
using the divinyl sulfone method (17).
-minimal essential medium without
nucleosides or deoxyribonucleosides, supplemented with 10% dialyzed
fetal calf serum and 0.5 µM methotrexate. Following growth to confluence, cells expressing MMR-S were changed into serum-free medium (Chinese hamster ovary-S-SFM II) without nucleosides, supplemented with 50 mM HEPES, pH 7.55, 0.11 µM CaCl2, and 5 µM methotrexate. Cells expressing MMR1-8 were kept in serum-containing medium after growth to confluence. In each case, medium was changed and
collected every 3 days. MMR-S and MMR1-8 were isolated from the medium
by affinity chromatography on mannose-Sepharose as described previously
(18). MMR1-8 was separated from trace serum contaminants by
cation-exchange fast protein liquid chromatography on a Mono-S column
(Amersham Pharmacia Biotech). Protein was loaded in 20 mM
MOPS, pH 6.0, and eluted with a gradient of 0-0.5 M NaCl in the same buffer. MMR-S isolated from serum-free medium required no
further purification. Pure protein fractions were concentrated if
necessary using a Centricon-30 micro-concentrator (Millipore).
-methylmannoside were added. Aliquots were incubated at room
temperature for 1 h with different concentrations of
bis(sulfosuccinimidyl)suberate before analysis by SDS-gel
electrophoresis on 5% polyacrylamide gels.
-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), and carbonic anhydrase (29 kDa) were used as
standards, with RNase S peptide used as a marker of the included volume
of the column. Values for the Stokes radius (RS) of
protein standards were calculated from their known diffusion
coefficients (20). Values for the Stokes radii of MMR-S and
MMR1-8 were determined from their elution positions (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Domain organization of the mannose
receptor. Representation of the mannose receptor in two possible
conformations. Left, extended. Right, U-shaped.
The arrows indicate expressed extracellular portions of the
receptor, MMR-S and MMR1-8, analyzed in this study.
Hydrodynamic Analysis of the Mannose Receptor-- Comparison of the calculated peptide molecular masses for MMR-S and MMR1-8 with the molecular masses determined by MALDI-MS (Table I) allows estimation of the extent of glycosylation of these mannose receptor fragments. The mass difference is about 20,000 Da for both MMR-S and MMR1-8. There are eight potential sites for N-linked oligosaccharides on MMR-S and seven on MMR1-8. Four sites for O-linked oligosaccharides have also been identified based on protein sequencing data (2). The additional mass in each case was attributed to the presence of seven N-linked biantennary complex oligosaccharides and four O-linked disaccharides of GalNAc and GlcNAc. Partial specific volumes of 0.714 cm3/g for MMR-S and 0.717 cm3/g for MMR1-8 were calculated using these estimates of glycosylation (19, 23).
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Sedimentation velocity analysis of MMR-S and MMR1-8 by analytical
ultracentrifugation was used to determine sedimentation coefficients.
Data were analyzed by the g(s*) method in which the distribution of sedimentation coefficients is calculated. In the
absence of Ca2+, a narrow symmetrical peak is obtained for
each receptor fragment indicating the presence of a single species
(Fig. 2). The sedimentation coefficients
obtained (Table I) are lower than would be expected for globular
proteins of the same mass as the mannose receptor fragments, indicating
that the receptor is asymmetric. Equilibrium sedimentation experiments
show that the receptor fragments are monomeric in the absence of
Ca2+. The apparent molecular mass as a function of receptor
concentration remains constant across the sample cell (Fig.
3, A and B)
indicating that MMR-S and MMR1-8 are monomeric at all protein
concentrations. Apparent molecular masses are 173,000 ± 7000 Da
for MMR-S and 165,000 ± 4000 Da for MMR1-8. These values
correspond closely to the values of 174,500 Da and 146,700 Da
determined for MMR-S and MMR1-8 by mass spectrometry. Thus, analytical
ultracentrifugation indicates that the mannose receptor is monomeric in
the absence of Ca2+ and sediments at a rate consistent with
an asymmetric, extended conformation.
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Because at least some of the C-type CRDs of the receptor bind Ca2+, sedimentation experiments were also carried out in the presence of 5 mM CaCl2. However, the results indicate that both MMR-S and MMR1-8 aggregate under these conditions. On sedimentation velocity analysis, a broad asymmetric peak is obtained for each receptor fragment, indicating the presence of multiple species (Fig. 2). Average sedimentation coefficient values of 10.02 ± 0.11 S and 9.01 ± 0.08 S were obtained from three independent sedimentation velocity experiments for MMR-S and MMR1-8, respectively. These average values are considerably higher than those obtained in the absence of Ca2+ (Table I), although overlap in the two curves indicates that a proportion of the protein in Ca2+ is monomeric (Fig. 2). Equilibrium sedimentation analysis confirms that aggregation of MMR-S and MMR1-8 occurs when Ca2+ is present. The apparent molecular mass of each receptor fragment increases as a function of concentration throughout the sample cell (Fig. 3, C and D). The fact that the curves calculated at different loading concentrations do not all overlap indicates that at least some oligomeric forms do not interconvert, consistent with aggregation. Cross-linking experiments confirm the ultracentrifugation data, with monomeric receptor fragments forming at least tetramers on addition of Ca2+ (data not shown). In the absence of Ca2+, cross-linking of monomers does not occur.
Previous studies on the intact mannose receptor have suggested that the
receptor is monomeric (10). Detergent-solubilized receptor sediments on
a sucrose gradient as a single band at a position corresponding to a
monomer. Furthermore, no evidence of oligomerization was obtained in
cross-linking studies in the presence of 10 mM
Ca2+ (10). Thus, aggregation seen in the presence of
Ca2+ is unlikely to be an indication of the natural state
of the receptor. Binding of oligosaccharides on one receptor molecule
by the CRDs of another molecule could cause Ca2+-induced
association. For example, high mannose oligosaccharides or hybrid
oligosaccharides with terminal mannose or
N-acetylglucosamine residues could act as ligands for CRDs 4 and 5. Analytical ultracentrifugation and cross-linking carried out on
receptor fragments treated with endoglycosidase H to remove high
mannose and hybrid oligosaccharides, or carried out in the presence of
-methylmannoside in an attempt to saturate ligand-binding sites,
resulted in a decrease in the amount of aggregation (data not shown).
Combination of endoglycosidase H treatment and working in
-methylmannoside decreased aggregation more than either strategy
used separately. These results indicate that Ca2+-induced
aggregation is largely due to the presence of terminal sugar residues
on one receptor molecule being bound by another receptor molecule,
although aggregation could not be completely abolished. Because
endoglycosidase H treatment leaves a single GlcNAc residue that could
still bind to the mannose receptor, removal of all N-linked
oligosaccharides by peptide N glycanase F treatment was attempted.
Unfortunately, this treatment resulted in aggregation of receptor
fragments under all buffer conditions (data not shown).
Because Ca2+-induced aggregation could not be completely eradicated, data obtained in the absence of Ca2+ were used for analysis of the conformation of the receptor. Under physiological conditions, Ca2+ would be bound by some of the CRDs of the mannose receptor. Studies with other C-type lectins indicate a local change in the position of the loops forming the sugar-binding site but no overall conformational change in the CRD in the absence of Ca2+ (31). While it is possible that Ca2+ could mediate CRD-CRD interactions within the mannose receptor, there is no evidence for such Ca2+-dependent interactions in this receptor or in other C-type lectins. The overall conformation of the receptor is therefore not expected to differ in the presence or absence of Ca2+. This conclusion is supported by the data obtained from sedimentation velocity experiments (Fig. 2) which show overlapping peaks in the g(s*) analysis, indicating that monomers sediment at similar rates in the presence and absence of Ca2+. Thus the only change occurring on moving from a Ca2+-free to a Ca2+-containing buffer is a change in the oligomeric state.
Analytical gel filtration was used to determine the Stokes radii and
diffusion coefficients for MMR-S and MMR1-8. Each receptor fragment
eluted as a sharp peak indicating the presence of a single species
(Fig. 4). Values for Stokes radii and
diffusion coefficients obtained from gel filtration for MMR-S and
MMR1-8 in the absence of Ca2+ are shown in Table I. Values
for Stokes radii were also calculated from the sedimentation
coefficients using the molecular masses determined by MALDI-MS. Values
obtained by the two different methods are in close agreement, and were
thus averaged for calculation of the frictional ratios
(f/f0) (Table I). The frictional
ratios obtained for MMR-S and MMR1-8 are considerably greater than
1.00, indicating that the shape of the receptor deviates substantially from that of a sphere. Axial ratios of 8.5:1 and 6.1:1 calculated for
MMR-S and MMR1-8 modeled as prolate ellipsoids of revolution indicate
that the receptor is elongated. Dimensions calculated from the axial
ratios are 306 Å × 36 Å for MMR-S and 232 Å × 38 Å for MMR1-8.
Although a prolate ellipsoid is unlikely to be a close representation
of the shape of the receptor, these values give a first approximation
of its size.
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Identification of Protease-resistant CRD Clusters in the Mannose Receptor-- Proteolysis experiments were performed to investigate the extent of contact between neighboring CRDs. Examination of the amino acid sequence of the mannose receptor reveals linker regions of about 10-15 amino acids between CRDs (2). Each linker region contains at least one lysine or arginine residue and would be expected to be susceptible to trypsin digestion if the sites were accessible to the enzyme. Because individual C-type CRDs are resistant to proteolysis in the presence of Ca2+, protease digestion of the receptor would be most likely to occur at the linker regions (28). Extensive contacts between a pair of CRDs protect the linker from proteolysis, whereas linkers that adopt an extended conformation are susceptible to digestion.
The results of digesting MMR1-8 with trypsin in the presence of
Ca2+ are shown in Fig. 5.
Proteolysis yields distinct bands that can be identified based on
molecular weights and N-terminal sequences. Western blotting with
antibodies specific for CRD-4 and the cysteine-rich domain was also
used to confirm the identity of some
fragments.2 At low protease
concentrations, MMR1-8 is cleaved to yield two fragments consisting of
CRDs 1 to 6 and 7 to 8. Cleavage occurs at an arginine residue at the
start of the linker region between CRDs 6 and 7, releasing CRDs 7 to 8 with the linker region still attached. This result suggests that the
linker between CRDs 6 and 7 is extended or flexible. At higher trypsin
concentrations, the linker attached to CRD-7 is cleaved at a lysine
residue close to the start of CRD-7, leaving CRDs 7 and 8 connected. No
cleavage occurs between CRDs 7 and 8 even at high concentrations of
trypsin, indicating that these domains are in close contact. However,
some degradation of CRD 7 is observed, with cleavage of CRD 7 at an internal arginine residue, facilitating further degradation and release
of free CRD 8.
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Further digestion of the CRD 1 to 6 fragment occurs at higher concentrations of trypsin. Fragments consisting of CRDs 1 to 5, 1 to 3, and 4 to 6 appear transiently, but stable fragments consisting of CRDs 1 to 2 and 4 to 5 are the predominant products of digestion of CRDs 1 to 6. These results suggest that extensive contacts exist between CRDs 1 and 2 and confirm previous findings showing that CRDs 4 and 5 are in close contact (10). In contrast, the linkers between CRDs 2 and 3, between CRDs 3 and 4, and between CRDs 5 and 6 are exposed to the protease.
Digestion of MMR-S with trypsin in the presence of Ca2+ shows a very similar pattern to that obtained with MMR1-8 (Fig. 5). Delay is seen in the generation of the CRD 1 to 2 fragment, due to the cysteine-rich domain and the fibronectin type II repeat remaining attached to CRDs 1 and 2. This result suggests that the cysteine-rich domain and the fibronectin type II repeat, along with the fibronectin type II repeat and CRD 1 are in fairly close contact. However, the fragment consisting of CRDs 1 and 2 is seen at higher trypsin concentrations, and some intact cysteine-rich domain is detected, suggesting that proteolysis of the fibronectin type II repeat occurs.
The proteolysis studies show that there are three protease-resistant pairs of CRDs in the mannose receptor: CRDs 1 and 2, CRDs 4 and 5, and CRDs 7 and 8. Close contacts probably exist between the domains in each of these pairs. A conformation in which all the linker regions are extended, separating the domains like beads on a string can therefore be ruled out. However, the data suggest that CRDs 3 and 6 may not be in close contact with their neighboring domains. The linkers on either side of CRD-3 and of CRD-6 could be extended or flexible.
Modeling of Mannose Receptor Conformations--
Computer modeling
was used to identify conformations of the mannose receptor consistent
with the data described here. Using the program HYDRO, hydrodynamic
coefficients were predicted for various receptor conformations modeled
as arrays of contiguous spheres to represent the domains. Initially,
the program was used to predict hydrodynamic coefficients for a simple
U-shaped model with CRDs 4 and 5 at the apex and an extended model with
the domains arranged linearly (Fig. 6).
In each of these two models, neighboring domains are in contact with
each other. Comparison of the predicted hydrodynamic coefficients with
the values obtained experimentally for MMR-S and MMR-1-8 immediately
indicates that the U-shaped model can be ruled out. When modeled as a
U-shape, the receptor is quite compact, and this compact shape gives
predictions for sedimentation and diffusion coefficients that are much
higher than the values obtained by experiment. The experimental data fit much more closely to the extended model.
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Values for sedimentation and diffusion coefficient predicted for the simple extended model are still higher than the values derived experimentally. Addition of linker regions between the domains extends the model further and lowers the predicted sedimentation and diffusion coefficients. For MMR-S, inclusion of linkers on either side of CRDs 3 and 6 gives predicted values of sedimentation and diffusion coefficients that are almost identical to those obtained by experiment (Fig. 6). The very close agreement between the predicted hydrodynamic parameters and those derived from the experiment, combined with the fact that the proteolysis experiments suggest that only four linkers are likely to be extended, provides good evidence that this extended model approximates the actual conformation of the mannose receptor. Adding together the diameters of the spheres in this model gives an estimate of 380 Å for the length of the receptor. Inclusion of four linker regions in the U-shaped model still gives predicted values of sedimentation and diffusion coefficients that are substantially higher than the values derived from the experiments (Fig. 6). Other U-shaped models, in which the molecule is bent at different places, also yield predicted hydrodynamic coefficients that are much higher than the experimental values (data not shown).
For MMR1-8, inclusion of the four linkers in the extended model gives predicted sedimentation and diffusion coefficients that are slightly lower than those determined by experiment (Fig. 6). This result suggests that in the absence of the cysteine-rich domain and the fibronectin type II repeat, the eight CRD fragment may be somewhat flexible, so that it sediments faster than predicted for a rigid molecule. However, predicted coefficients for MMR1-8 in the U-shaped model with four linkers are still inconsistent with the experimental values, indicating that the portion of the polypeptide containing the eight CRDs must be extended.
As well as indicating that the receptor is extended with contacts
between some domains, the modeling studies also show that the receptor
must be fairly rigid. Flexibility allowing some bending in the receptor
would lead to a more compact molecule that would sediment and diffuse
faster than a rigid linear molecule. If the receptor were flexible, it
would have to be much longer for diffusion and sedimentation
coefficients to be as low as those determined. Such an increase in
length would require all the domains to be separated without contacts
between them, and the proteolysis studies indicate that this is not the case.
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DISCUSSION |
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A combination of hydrodynamic analysis and proteolysis experiments provides good evidence that the mannose receptor adopts a rigid, extended conformation with close contacts between some pairs of neighboring domains. A U-shaped conformation can be ruled out, as can a conformation in which the domains are spread out like beads on a string. The approximate dimensions calculated from the modeling studies suggest that the mannose receptor may project about 380 Å from the cell surface. These findings have a number of implications for the function of members of the mannose receptor family in endocytosis and cell adhesion.
With the mannose receptor in an extended conformation, the two C-type CRDs most important for binding glycoproteins, CRDs 4 and 5, are not outermost from the membrane. However, when the receptor is localized in fluid-filled clathrin-coated pits, it will be surrounded by ligand, allowing binding to CRDs 4 and 5 from the side of the molecule. An extended conformation of the receptor results in the cysteine-rich domain being projected furthest away from the membrane. The cysteine-rich domain would not need to be in this exposed position to allow binding of glycoprotein hormones in coated pits, but such an arrangement would be important if binding of cell- surface sulfated ligands by the cysteine-rich domain plays a role in directing macrophages to germinal centers during the immune response (32, 33). For comparison, CD8, E-selectin, and sialoadhesin, three proteins involved in cell-cell interactions, are predicted to extend about 200, 270, and 500 Å from the cell surface (34-36). Projection of the cysteine-rich domain 380 Å from the membrane would be sufficient to allow trans interactions between the mannose receptor and sulfated ligands on other cells.
The finding that six CRDs form pairs with close contacts between the two CRDs in each pair, while CRDs 3 and 6 appear to be separated from their neighboring domains by extended linker regions, is likely to be important in explaining how the mannose receptor can interact with both exogenous and endogenous oligosaccharides. While CRDs 4 and 5 could be close enough together to recognize mannose residues on a mammalian high mannose oligosaccharide, the extended linkers on either side of CRD 3 and CRD 6 would provide wider spacing between CRDs 4 and 5 and the accessory domains. Such an arrangement might be suitable for matching the more widely spaced arrays of sugars on the surfaces of pathogens. In serum mannose-binding protein, the sugar-binding sites in adjacent CRDs are 53 Å apart (37). The linker regions in the mannose receptor could enable a comparable separation of some of its CRDs. A large amount of flexibility between the CRDs would be energetically unfavorable for binding to oligosaccharides. However, having a limited amount of flexibility in the linker regions between some domains, while the position of other CRDs are fixed relative to one another by close contact, might be important in allowing the mannose receptor to recognize a diverse range of ligands.
Carbohydrate-mediated aggregation of soluble receptor fragments observed in the presence of Ca2+ probably results from interactions that would be less likely to take place with the receptor in the membrane. The soluble extracellular domain of the receptor adopts a rigid extended conformation, but without the constraints imposed by a membrane anchor, a high mannose oligosaccharide on any part of one receptor molecule is free to interact with CRDs 4 or 5 on another. However, under some circumstances, a high mannose or hybrid oligosaccharide might occur on some receptor molecules at the correct distance from the membrane to allow limited oligomerization to occur. In this way, post-translational modifications could alter the ligand-binding properties of the receptor. Cross-binding of receptor molecules is consistent with the proposal that some cell types might express some dimeric receptor able to bind glycoprotein hormones, but unable to bind mannose-terminated glycoproteins (12, 38).
It is likely that other members of the mannose receptor family will also adopt an extended conformation, which might be important for roles of these receptors in cell adhesion. Like the mannose receptor, the phospholipase A2 receptor binds its ligands, phospholipases A2, predominantly through domains in the middle of the polypeptide (16). As with the mannose receptor, sequestering of the phospholipase A2 receptor into clathrin-coated pits would allow phospholipases A2 to bind from the side. A role in cell adhesion has also been proposed for the phospholipase A2 receptor as it has been shown to mediate cell spreading on collagens type I and IV through collagen binding by the fibronectin type II repeat (39). Examination of the sequence of Endo-180, the other member of the family that binds carbohydrate ligands in a Ca2+-dependent manner, suggests that CRDs 1 and 2 are responsible for this activity (3). Once again, although Endo-180 is an endocytic receptor, it binds strongly to collagen V, suggesting that it might also have a role in cell adhesion and making an extended conformation more important (40). It is interesting that the presence of the N-terminal cysteine-rich domain and the fibronectin type II repeat seem to confer extra rigidity on the mannose receptor, since these are the domains predicted to be involved in cell adhesion functions.
Conservation of the linker regions between domains is seen in the members of the mannose receptor family (3-6). Therefore it is likely that the same pattern of close contacts between some neighboring domains with extended linkers on either side of CRDs 3 and 6 will be found in each protein. As in the mannose receptor, such spacing of the domains might be important for allowing DEC-205 to interact with a diverse range of ligands. Although no ligands have yet been identified for DEC-205, there is a considerable amount of evidence to suggest that it is involved in enhancing antigen processing and presentation (6, 41). In such a role, it is probable that DEC-205 would need to interact with multiple different ligands, rather than being specific for a single ligand.
In all of the receptors in the mannose receptor family, a rigid,
extended conformation positions different domains at distinct distances
from the membrane. It is likely that the constraints imposed by this
conformation influences which ligands each receptor can bind.
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ACKNOWLEDGEMENTS |
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We thank Russell Wallis for help with mass spectrometry, analytical ultracentrifugation, and comments on the manuscript, Samuel Bouyain for help with using GRASP, and Kurt Drickamer for helpful discussions and comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Mitzutani Foundation for Glycoscience, Wellcome Trust Grant 041845, and the Glycobiology Institute endowment.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.
Supported by a Nuffield Foundation Undergraduate Vacation Bursary
(AT/100/98/0028).
§ To whom correspondence should be addressed: Glycobiology Institute, Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: 44-1865-275747; Fax: 44-1865-275339; E-mail: mt@glycob.ox.ac.uk.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M100425200
2 C. E. Napper and M. E. Taylor unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CRD, carbohydrate recognition domain; MMR, macrophage mannose receptor; MALDI-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; MOPS, 2-(N-morpholino)ethanesulfonic acid; RS, Stokes radius.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Taylor, M. E. (1997) Glycobiology 7, v-viii[Medline] [Order article via Infotrieve] |
2. |
Taylor, M. E.,
Conary, J. T.,
Lennarz, M. R.,
Stahl, P. D.,
and Drickamer, K.
(1990)
J. Biol. Chem.
265,
12156-12162 |
3. |
Sheikh, H.,
Yarwood, H.,
Ashworth, A.,
and Isacke, C. M.
(2000)
J. Cell Sci.
113,
1021-1032 |
4. |
Lambeau, G.,
Ancian, P.,
Barhanin, J.,
and Lazdunski, M.
(1994)
J. Biol. Chem.
269,
1575-1578 |
5. |
Ishizaki, J.,
Hanasaki, K.,
Higashino, K.,
Kishino, J.,
Kikuchi, N.,
Ohara, O.,
and Arita, H.
(1994)
J. Biol. Chem.
269,
5897-5904 |
6. | Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, M., and Nussenzweig, M. C. (1995) Nature 375, 151-155[CrossRef][Medline] [Order article via Infotrieve] |
7. | Drickamer, K., and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9, 237-264[CrossRef] |
8. | Weis, W. I., Taylor, M. E., and Drickamer, K. (1998) Immunol. Rev. 163, 19-34[Medline] [Order article via Infotrieve] |
9. |
Taylor, M. E.,
Bezouska, K.,
and Drickamer, K.
(1992)
J. Biol. Chem.
267,
1719-1726 |
10. |
Taylor, M. E.,
and Drickamer, K.
(1993)
J. Biol. Chem.
268,
399-404 |
11. |
Fiete, D.,
and Baenziger, J. U.
(1997)
J. Biol. Chem.
272,
14629-14637 |
12. |
Fiete, D.,
Beranek, M. C.,
and Baenziger, J. U.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11256-11261 |
13. |
Fiete, D.,
Beranek, M. C.,
and Baenziger, J. U.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2089-2093 |
14. |
Liu, Y.,
Chirino, A. J.,
Leteux, C.,
Feizi, T.,
Misulovin, Z.,
Nussenzweig, M. C.,
and Bjorkman, P. J.
(2000)
J. Exp. Med.
191,
1105-1115 |
15. |
Mullin, N. P.,
Hitchen, P. G.,
and Taylor, M. E.
(1997)
J. Biol. Chem.
272,
5668-5681 |
16. |
Nicolas, J.-P.,
Lambeau, G.,
and Lazdunski, M.
(1995)
J. Biol. Chem.
270,
28869-28873 |
17. | Fornstedt, N., and Porath, J. (1975) FEBS Lett. 57, 187-191[CrossRef][Medline] [Order article via Infotrieve] |
18. | Simpson, D. Z., Hitchen, P. G., Elmhirst, E. L., and Taylor, M. E. (1999) Biochem. J. 343, 403-411[CrossRef][Medline] [Order article via Infotrieve] |
19. | Laue, T. M., Bhairavi, D. S., Ridgeway, T. M., and Pelletier, S. L. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E. , Rowe, A. J. , and Horton, J. C., eds) , pp. 90-125, Royal Society of Chemistry, Cambridge, UK |
20. | Sober, H. A. (1970) Handbook of Biochemistry: Selected Data for Molecular Biology , The Chemical Rubber Co., Cleveland, OH |
21. | Laurent, T. C., and Killander, J. (1964) J. Chromatogr. 14, 317-330[CrossRef] |
22. |
Matsudaira, P.
(1987)
J. Biol. Chem.
262,
10035-10038 |
23. | Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids and Peptides as Ions and Dipolar Ions , pp. 370-381, Reinhold, New York |
24. | Harding, S. E., and Cölfen, H. (1995) Anal. Biochem. 228, 131-142[CrossRef][Medline] [Order article via Infotrieve] |
25. | Tanford, C. (1961) Physical Chemistry of Macromolecules , John Wiley, New York |
26. | Garcia de la Torre, J., Navarro, S., and Carrasco, B. (1997) HYDRO User's Manual, Version 5 , Universidad de Murcia, Murcia, Spain |
27. | Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve] |
28. | Weis, W. I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W. A. (1991) Science 254, 1608-1615[Medline] [Order article via Infotrieve] |
29. |
Feinberg, H.,
Park-Snyder, S.,
Kolatkar, A. R.,
Heise, C. T.,
Taylor, M. E.,
and Weis, W. I.
(2000)
J. Biol. Chem.
275,
21539-21548 |
30. | Pickford, A. R., Potts, J. R., Bright, J. R., Phan, I., and Campbell, I. D. (1997) Curr. Biol. 5, 359-370 |
31. | Ng, K. K.-S., Park-Snyder, S., and Weis, W. I. (1998) Biochemistry 37, 17965-17976[CrossRef][Medline] [Order article via Infotrieve] |
32. | Martinez-Pomares, L., Kosco-Vilbois, M., Darley, E., Tree, P., Herren, S., Bonnefoy, J.-Y., and Gordon, S. (1996) J. Exp. Med. 184, 1927-1937[Abstract] |
33. |
Leteux, C.,
Chai, W.,
Loveless, R. W.,
Yuen, C.-T.,
Uhlin-Hansen, L.,
Combarnous, Y.,
Jankovic, M.,
Maric, S. C.,
Misulovin, Z.,
Nussenzweig, M. C.,
and Feizi, T.
(2000)
J. Exp. Med.
191,
1117-1126 |
34. |
Hensley, P.,
McDevitt, P. J.,
Brooks, I.,
Trill, J. J.,
Field, J. A.,
McNulty, D. E.,
Connor, J. R.,
Griswold, D. E.,
Kumar, N. V.,
Kopple, K. D.,
Carr, S. A.,
Dalton, B. J.,
and Johanson, K.
(1994)
J. Biol. Chem.
269,
23949-23958 |
35. |
Boursier, J. P.,
Alcover, A.,
Herve, F.,
Laisney, I.,
and Acuto, O.
(1993)
J. Biol. Chem.
268,
2013-2020 |
36. | Crocker, P. R., Kelm, S., Hartnell, A., Freeman, S., Nath, D., Vinson, M., and Mucklow, S. (1996) Biochem. Soc. Trans. 24, 150-156[Medline] [Order article via Infotrieve] |
37. | Weis, W. I., and Drickamer, K. (1994) Structure 2, 1227-1240[Medline] [Order article via Infotrieve] |
38. |
Roseman, D. S.,
and Baenziger, J. U.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9949-9954 |
39. | Ancien, P., Lambeau, G., and Lazdunski, M. (1995) Biochemistry 44, 13146-13151 |
40. |
Behrendt, N.,
Jensen, O. N.,
Engelholm, L. H.,
Mortz, E.,
Mann, M.,
and Dane, K.
(2000)
J. Biol. Chem.
275,
1993-2002 |
41. |
Mahnke, K.,
Guo, M.,
Lee, S.,
Sepulveda, H.,
Swain, S. L.,
Nussenzweig, M.,
and Steinman, R. M.
(2000)
J. Cell Biol.
151,
673-684 |