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Biochemical Society Transactions
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Received 17 December 1992
Recognition of complex carbohydrates by the macrophage mannose receptor
Maureen E. Taylor
Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX I 3QU,
U.K.
Introduction
T h e mannose receptor of macrophages and hepatic
endothelial cells mediates binding and internalization of glycoconjugates terminating in mannose,
fucose or N-acetylglucosamine [ 11. Recognition of
Abbreviations used: BSA, bovine serum albumin; CRD,
carbohydrate-recognition domain.
Volume 2 I
carbohydrates by the mannose receptor allows the
discrimination of self from non-self, since terminal
mannose and N-acetylglycosamine residues are
found rarely in mammalian cell-surface or serum
glycoproteins, but are a common component of the
cell surface of bacteria, fungi and parasites. T h u s
the mannose receptor can play a role in the innate
immune response against pathogenic micro-organ-
Carbohydrates, Shapes and Biological Recognition
isms by mediating opsonin-independent phagocytosis [2-41. Some endogenous glycoproteins
bearing high-mannose oligosaccharides, including
lysosomal enzymes [ 51 and tissue plasminogen activator [6], are also cleared by the mannose receptor.
These potentially harmful proteins are often
released from cells in response to pathological
events.
Structure of the mannose receptor
Mannose receptors have been isolated from macrophages [7, 81 and from placenta [9]. The receptor
from both sources is a glycoprotein consisting of a
single subunit with a molecular mass of about 175
m a . Mannose receptors from human placenta [ 101
and human monocyte-derived macrophages [ 111
have been cloned and sequenced. Clones from
these two sources have identical sequences, suggesting that the receptor isolated from placenta is
also macrophage-derived. The receptor is oriented
as a type I transmembrane protein (C-terminus
inside the cell) with a 41 amino-acid cytoplasmic
tail. The extracellular portion of the receptor consists of three types of cysteine-rich domains. The
N-terminal cysteine-rich domain shows no similarity to other known sequences. The second
domain resembles the type I1 repeats of fibronectin
[ 121. No specific function has been assigned to
the type I1 repeats in fibronectin or to related
domains found in other proteins, so it is not
possible to make predictions about the role of this
domain in the mannose receptor. The rest of the
extracellular part of the receptor consists of eight
segments showing sequence similarity to the Ca2+dependent carbohydrate-recognition domains (Ctype CRDs) of other animal lectins [ 131.
C-type CRDs are discussed in detail elsewhere in this colloquium. They bind various different saccharides in a calcium-dependent manner and
are characterized by 14 invariant residues and 18
highly conserved residues, spread over approximately 120 amino acids [ 141. They are associated
with a variety of effector domains in a large number
of proteins [15]. The mannose receptor is the only
protein known to have more than one CRD within a
single polypeptide.
Structure-function analysis of the
mannose receptor
Since the mannose receptor is known to bind
carbohydrates in a Caz+-dependent manner it is
reasonable to assume that the carbohydrate-binding
activity of the receptor resides within the CRD-like
segments. Each of the mannose receptor CRDs
contains most of the conserved residues necessary
for maintaining the CRD fold seen in the crystal
structure of the CRD of rat serum mannose-binding
protein [14]. However, it is not possible to predict
simply from examination of the sequences, which of
the CRDs have binding activity. Analysis of the
binding activity of portions of the receptor
expressed in vitro, in fibroblasts, in bacteria and in
insect cells was undertaken in order to answer the
following questions: (1) are the N-terminal cysteinerich domain and the fibronectin-type I1 repeat
necessary for binding and internalization of glycoproteins? (2) Which of the CRDs can bind to carbohydrates? (3) Does the multispecificity of the
receptor arise from each CRD having specificity for
different saccharides? (4) Are several active CRDs
necessary for high-affinity binding to oligosaccharides?
localization of the binding activity to
CRDS4-8
Initial experiments indicated that the N-terminal
cysteine-rich domain and the fibronectin type I1
repeat are not essential for binding and endocytosis
of glycoproteins. Fibroblasts expressing a truncated
receptor with an extracellular portion consisting of
CRDs 1-8 are able to bind and endocytose a neoglycoprotein ligand, mannose,,-bovine serum albumin (Man,,-BSA), as efficiently as fibroblasts
expressing the intact receptor [16]. The truncated receptor has the same affinity as the intact
receptor for three different mannose-terminated
ligands. These results indicate that the carbohydrate-binding activity of the receptor resides
within the 0 s . The function of the N-terminal
cysteine-rich domain and the fibronectin type I1
repeat remains unclear. It is possible that they bind
to a non-carbohydrate ligand.
An in vitro-binding assay was used to determine which of the CRDs are able to bind sugars
[ 161. Portions of DNA coding for one or more
CRDs were fused to codons specifying the preproinsulin signal sequence in an SP64 expression
vector and transcribed in vitro. The RNAs produced were translated in vitro in the presence of
microsomes. The insulin signal sequence directs
translation products into the lumen of the microsomes, where disulphide bonds which are necessary for the production of an active CRD, can form.
Translation products were solubilized with detergent and tested for their ability to bind mannose
immobilized on Sepharose. The results are summarized in Figure 1.
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Figure I
Carbohydrate-binding activity of CRDs in the macrophage mannose receptor
470
The structure of the CRD-containing portion of the receptor IS
summarized at the top with the activity of constructs containing subgroups of CRDs shown below. Activity was measured
by retention on mannose-Sepharose following in vitrotranscription and translation [ 161. Adapted from [ I71 with permission.
Binding of MMR domains to Man-Sepharose
following in vitro translation
BINDING
DOMAINS
1 2 3
4 5 6
4 5 6
4 5
4
5
5 6
5 6
6
7 8
7 8
7
7 8
+
+
+
+
+
+
-
A segment of the receptor consisting of CRDs
1-3 does not bind to mannose-Sepharose, whereas
a segment consisting of CN>s 4-8 has binding
activity. O f CKIIs 4-8, only CRD 4 can bind to
mannose-Sepharose in the absence of other CKI)s.
CKI) 5 alone does not show binding activity. However, a segment of the receptor consisting of CRDs
5-8 does show binding activity. Since the removal
of CKI) 5 results in loss of binding, it can be concluded that CRD 5 contributes to the binding
activity of the receptor. A similar argument can be
made for CRI) 7, since a fragment consisting of
CKI)s 5-7 binds to mannose-Sepharose, but CKDs
5-6 do not bind. CRDs 1-3 and CRDs 6-8 failed to
bind to fucose. N-acetylglucosamine and glucose, as
well as to mannose.
The results of the in vitro-translation studies
suggest that CRI)s 1-3 do not bind carbohydrates
and that the binding activity of the receptor resides
in CRI)s 4-8.IIowever, since this is not a quantitative assay, it was necessary to confirm the results by
further expression studies in fibroblasts [ 161. Cells
expressing a truncated receptor consisting of
domains 4-8 are able to endocytose "'1Man,,-BSA as efficiently as cells expressing the
Volume 2 I
intact receptor. This truncated receptor has the
same affinity as the intact receptor for three different ligands. Cells expressing a truncated receptor
consisting of CRLIs 5-8 can also endocytose "'IMan,,-HSA, but at a much slower rate than cells
expressing CIWs 4-8 or the intact receptor, highlighting the importance of CRI) 4.Cells expressing
CRDs 6-8 are not able to internalize "'IMan,,-HSA, indicating that CKI) S is also essential
for binding and endocytosis.
Saccharide-binding activity of CRD 4
Since CRD 4 was the only domain found to show
carbohydrate-binding activity in the absence of
other CRDs, this domain was produced in a bacterial expression system so that its binding properties could be characterized further [ 161. Correctly
folded CRI) 4 was purified from the bacteria by
affinity chromatography on mannose-Sepharose.
Purified CRI) 4 immobilized on a microtitre plate
binds 1251-Man,,-HSA in a saturable fashion. The
affinity of the purified domain for various monosaccharides and glycoproteins was determined,
based on their ability to inhibit binding to "'1Man,,-HSA. CKI) 4 binds to N-acetylglucosamine,
fucose and glucose as well as to mannose with dissociation constants in the millimolar range. The
intact receptor also has specificities for these monosaccharides with dissociation constants in the millimolar range. Thus a single CRD can mimic the
monosaccharide-binding properties of the whole
receptor. Multispecificity for mannose, fucose, Nacetylglucosamine and glucose is also shown by the
CRD of rat serum mannose-binding protein, which
has recently been crystallized in complex with an
oligosaccharide [ 181. It is likely that the interaction
between CRD 4 and monosaccharides will be very
similar to that seen for the mannose-binding protein, since the residues involved in ligating the sugar
are completely conserved [ 191. However, CRD 4
binds only poorly to glycoproteins, such as invertase and mannan, and thus cannot account for the
binding of the receptor to natural ligands. As indicated by the expression studies in fibroblasts and in
vitro. other CRDs in the 4-8 segment must contribute to the binding of oligosaccharides.
Binding to multivalent ligands
Hydrodynamic studies of the receptor show that it
is a monomer in solution and crosslinking studies
indicate that it is a monomer in the membrane [20].
Thus multiple carbohydrates in a single receptor
polypeptide must co-operate to achieve high-affinity
binding of complex ligands. In order to determine
Carbohydrates, Shapes and Biological Recognition
how the CRDs cluster to achieve this high-affinity
binding, internal fragments of the receptor were
produced using the baculovirus expression system
[ZO]. Fragments of the receptor consisting of CRDs
4-5,4-6 and 4-7 were purified from the medium of
insect cells by affinity chromatography on mannose-Sepharose and were used in proteolysis
studies and in assays to determine the affinities for
various ligands [201.
The solid-phase-binding assay described for
CRD 4 was used to determine the affinities of the
larger fragments of the receptor for Man,,-BSA and
for two natural glycoproteins, invertase and
mannan, which bear high-mannose oligosaccharides. The results obtained for the internal fragments
of the receptor and those obtained for CRD 4 alone
and for CRDs 4-8 expressed in fibroblasts are
compared in Figure 2, which shows a plot of affinities for the three ligands as a function of the number
of CRDs present. The largest increase in affinity is
seen when CRD 5 is combined with CRD 4, with
the increase being particularly marked for the two
natural glycoproteins. CRDs 4 and 5 together are
sufficient to match the affinity of the intact receptor
for Man,,-BSA and invertase, suggesting that these
two domains form a ligand-binding core essential
for high-affinity binding of multivalent ligands.
However, the affinity for mannan increases as more
CRDs are added. The full affinity of the intact
receptor for mannan is only reached when CRDs
4-8 are present, indicating that accessory domains
6, 7 and 8 are necessary for binding to some
ligands.
Proteolysis experiments support the idea that
CRDs 4 and 5 form a ligand-binding core, since
these two CRDs cannot be separated by treatment
with subtilisin [20]. Resistance to proteolysis in the
presence of Ca2+ is a common feature of C-type
CRDs [Zl]. However, the fact that proteolysis of a
fragment consisting of CRDs 4-5 does not result in
the release of individual CRDs suggests that these
two domains form extensive contacts and are not
simply linked by a flexible tether. Such an arrangement may fix the orientation of the binding sites
within these two CRDs.
Different modes of clustering of Ctype CRDs
.
MANNAN
The experiments described above indicate that the
mannose receptor achieves high-affinity binding to
oligosaccharides by clustering of several active
CRDs in a single polypeptide. Clustering of CRDs
to allow high-affinity binding to oligosaccharides is
a common feature of many C-type lectins. These
other lectins have a single CRD in each polypeptide
and thus must form oligomers to achieve clustering
of CRDs. The chicken hepatic lectin forms trimers
by association of identical polypeptides in the membrane [21, 221. The soluble mannose-binding
proteins also forni trimers. In this case identical
polypeptides each with a single CRD are held
together by the association of collagen-like domains
[23]. In the rat asialoglycoprotein receptor, two different polypeptides each with a single CRD form
hetero-oligomers in the membrane "241. Different
subunits of the asialoglycoprotein receptor bind to
different terminal galactose residues on a glycopeptide, indicating that clustering of CRDs may
determine specificity as well as affinity [25]. Analysis of the crystal structure of the mannose-binding
protein CRD bound to an oligosaccharide has
revealed why clustering of CRDs is necessary to
achieve high-affinity binding [ 181. The contact
between the sugar and the CRD in the crystal is
very limited, with each CRD binding to a single
sugar residue by ligation of two hydroxyl residues.
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Biochemical Society Transactions
472
Thus the interaction between a single CRD and a
sugar is weak and several CRDs are needed to bind
tightly to oligosaccharide ligands.
It is probable that the different forms of
clustering of CRDs seen in C-type lectins allows the
selection of different types of ligand. The CRDs of
each lectin must be arranged to match the geometric
configurations of their particular oligosaccharide
ligands. The arrangement of CRDs in the asialoglycoprotein receptor (Figure 3 ) may provide optimal binding to the clusters of terminal galactose
residues that are found in the desialated complex
oligosaccharide chains of serum glycoproteins
cleared by this receptor. In contrast the linear
arrangement of CRDs in the mannose receptor
(Figure 3 ) may be more suitable for high-affinity
binding of repeated polymers such as yeast
mannan. The asialoglycoprotein receptor binds
endogenous ligands with a limited set of structures,
while the mannose receptor must recognize a
diversity of foreign ligands. In the mannose receptor
the presence of multiple CRDs in addition to the
CRD 4-5 ligand-binding core may provide the
flexibility needed to interact with this diversity of
ligands.
Conclusions
Structure-function analysis of the mannose receptor has revealed that the carbohydrate-binding
Figure 3
Different modes of clustering of C-type CRDs seen in
the macrophage mannose receptor and the asialoglycoprotein receptor
The macrophage mannose receptor (left) contains eight CRDs
in a single polypeptide. The two CRDs that form the ligandbinding core are shaded. The N-terminal cysteine-rich domain
is represented by the vertical box and the fibronectin type II
repeat by the horizontal box. In the asialoglycoprotein receptor
(right) three polypeptides, each containing a single CRD, associate in the membrane to form a trimer.
MACROPHAGE MANNOSE
RECEPT0 R
8
ASIALOGLYCOPROTEIN
RECEPTOR
activity of the receptor is located in CRDs 4-8.
CRDs 4-5 form a protease-resistant ligand-binding
core sufficient to bind some ligands with high
affinity, but accessory CRDs 6-8 are also required
for high-affinity binding of other ligands. A consequence of the organization of the receptor is that
both the valency and the geometry of glycoconjugates are important determinants of the binding
affinity. Further structural work is required to determine how each of the CRDs interacts with sugars
and how the CRDs are arranged spatially to match
the geometric configurations of particular oligosaccharide ligands.
I thank Kurt Drickamer and Raymond Dwek for comments on the manuscript. The work described here was
supported by Grant GM42628 from the National Institutes of Health. The Glycobiology Institute is supported
by Monsanto.
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Carbohydrates, Shapes and Biological Recognition
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The role of mannose-binding protein in host defence
John A. Summerfield
Department of Medicine, St Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine,
London W2 INY, U.K.
Mannose-binding protein
Human serum contains mannose-binding protein
(MBP), a calcium-dependent C-type lectin, secreted
by hepatocytes, which binds glycoproteins terminating in mannose or N-acetylglucosamine. MRP
occurs in serum as a mixture of oligomers of 9-18
identical polypeptide chains of 32 kDa [I-51. On
binding to a mannose-rich surface, MRP activates
complement through the classical pathway [6].
MRP binds C l r and Cls to form C1 esterase, which
cleaves C 2 and C4, which in turn form a complex
on the mannose-rich surface to form C 3 convertase.
C 3 convertase cleaves C 3 to form C3b which then
binds to the surface and opsonizes the ligand.
Recently MBP has been shown to be identical with
Ra-reactive factor [7]. Ra-reactive factor is a complement-activating bactericidal protein which binds
to Ra-chemotype strains of bacteria and yeasts.
MRP is encoded by four exons each of which
code for different functional domains of the molecule [8,9]. Exon 1 encodes the signal sequence of a
secreted protein, a cysteine-rich domain and seven
copies of the motif Gly-Xaa-Yaa, which is typical of
a collagen domain. The junction between exon 1
and exon 2 encodes the sequence Gly-Gln-Gly.
Exon 2 encodes a further 12 Gly-Xaa-Yaa collagen
repeats. Exon 3 encodes a short ‘neck‘ domain and
exon 4,the largest exon, encodes the carbohydratebinding domain.
The structure of the high-molecular-weight
oligomers of MBP that are found in serum can be
inferred from the sequence of the MBP gene.
Trimers of MBP polypeptides associate by forming
Abbreviation used: MHI’, mannose-binding protein.
a triple helix between their collagen domains. The
interruption of the collagen motif by the sequence
Gly-Gln-Gly between exons 1 and 2, by analogy
with Clq, is probably the site where the triple-helical chains of MBP appear to bend on electronmicroscopy [ 101. The triple helices are stabilized by
disulphide bridges between the cysteine-rich
domains. Three to six of the trimers assemble by
disulphide bridges into oligomers. This gives the
final MBP oligomer the appearance of a bunch of
flowers, where the flower heads are the carbohydrate-binding domains and the stalks are the
collagen domains.
MBP is an acute phase protein [8, 113. The 5’
flanking sequence of the MBP gene contains a heatshock-promoter sequence and two glucocorticoidresponsive promoter sequences typical of acute
phase protein genes.
Immunodeficiency caused by defective
opsonization
Infants of 6-18 mth with this immunodeficiency
syndrome suffer repeated bacterial and fungal infections [ 12, 131. The immunodeficiency is present in
about 25% of children with frequent unexplained
infections. Children with the defect suffer about
twice as many severe infections as matched control
children [ 141. The infections may be severe and five
deaths have been reported. Usually relatives of the
immunodeficient infants suffered repeated infections while infants. There is a high frequency of
atopy in the children and their families [14]. This
immunodeficiency is common with an estimated
frequency, judged by tests of opsonic function, of
5-7% [13, 15, 161.
The sites of infection are varied; otitis media,
chronic diarrhoea and meningitis are the most
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