(Received for publication, June 16, 1995; and in revised form, August 31, 1995)
From the
The rabbit muscle (M)-type receptor for secretory phospholipases
A (sPLA
s) has a large extracellular domain of
1394 amino acids, composed of an N-terminal cysteine-rich domain, a
fibronectin-like type II domain, and eight carbohydrate recognition
domains (CRDs). It is thought to mediate some of the physiological
effects of mammalian sPLA
s, including vascular smooth
muscle contraction and cell proliferation, and is able to internalize
sPLA
s. Here, we show by site-directed mutagenesis that
OS
, a snake venom sPLA
, binds to the receptor
via its CRDs and that deletion of CRD 5 completely abolishes the
binding of sPLA
s. Moreover, a receptor lacking all CRDs but
CRD 5 was still able to bind OS
although with a lower
affinity. Deletion of CRDs 4 and 6, surrounding the CRD 5, slightly
reduced the affinity for OS
, thus suggesting that these
CRDs are also involved in the binding of OS
. The M-type
sPLA
receptor and the macrophage mannose receptor are
homologous and are predicted to share the same tertiary structure. p-Aminophenyl-
-D-mannopyranoside bovine serum
albumin, a known ligand of the macrophage mannose receptor, binds to
the M-type sPLA
receptor essentially via CRDs 3-6.
Secretory phospholipases A (sPLA
s) (
)are structurally homologous enzymes that have been
isolated from a large number of biological sources, including mammalian
tissues as well as insect and snake
venoms(1, 2, 3) . At least six different
sPLA
s have been found in mammalian tissues. The pancreatic
sPLA
and the inflammatory sPLA
are well
characterized enzymes, while the others have only been purified (4) or cloned (5, 6, 7) recently. The
pancreatic sPLA
has long been thought to act only as a
digestive enzyme(8) . However, the presence of this sPLA
in several non-digestive tissues has been
demonstrated(9, 10, 11) , and it is now
thought to play a role in airway and vascular smooth muscle contraction (12, 13) as well as in cell
proliferation(14) . The inflammatory sPLA
has been
purified and cloned from several sources (15, 16) and
is believed to play a central role in inflammatory processes (reviewed
in (17, 18, 19, 20) ). It is
secreted by a large number of cell types in which its expression is
strongly up-regulated by inflammatory cytokines. Its concentration in
various extracellular fluids is dramatically increased in several
inflammatory diseases. Moreover, this enzyme has potent proinflammatory
activities(21) .
sPLAs are also found in
abundance in snake and bee venoms. Besides their role in prey
digestion, the snake venom sPLA
s can have neurotoxic,
myotoxic, anticoagulant, and proinflammatory
effects(22, 23, 24, 25) . High
affinity receptors for these enzymes have been
characterized(26, 27, 28, 29) . They
are apparently involved in their biological effects. A first type of
sPLA
receptors called N-type sPLA
receptors
(for neuronal) has been initially identified in rat brain (26) and then in other tissues (28, 29) using
OS
, a novel neurotoxic sPLA
, purified from the
Taipan snake (Oxyuranus scutellatus scutellatus)
venom(26) . It recognizes several other neurotoxic
sPLA
s with high affinity while non-neurotoxic venom
sPLA
s display much lower affinities. A second type of
sPLA
receptor called M-type sPLA
receptor has
been initially characterized in rabbit skeletal muscle(27) ,
using OS
and OS
, another sPLA
purified from the Taipan snake venom(26) . Very
interestingly, this receptor binds the porcine pancreatic sPLA
as well as the human inflammatory sPLA
with high
affinities (30) , suggesting that these endogenous
sPLA
s might be its physiological ligands. M-type
sPLA
receptors were subsequently characterized in
fibroblasts and other tissues using the porcine pancreatic sPLA
as a ligand(14, 31) .
More recently, the
M-type sPLA receptor has been cloned in rabbit, bovine, and
human species(30, 32, 33, 34) . The
cloned receptors are homologous to the macrophage mannose receptor, a
protein involved in the endocytosis of glycoproteins and
microorganisms(35, 36) , as well as to DEC-205, a
protein recently cloned in dendritic cells and involved in the
presentation of antigens(37) . Interestingly, all of these
proteins, which may constitute a new family of receptors, are predicted
to share the same structural organization, i.e. a large
extracellular region composed of an N-terminal cysteine-rich domain, a
fibronectin-like type II domain, eight(30, 35) or ten (37) repeats of a carbohydrate recognition domain (CRD),
followed by a unique transmembrane domain and a short intracellular
C-terminal domain. This latter domain contains a consensus sequence for
the internalization of ligand-receptor complexes and is thought to
confer to these receptors their endocytic
properties(30, 31, 33, 37, 38) .
The purpose of this paper is to identify by site-directed
mutagenesis techniques the specific domain in the large extracellular
part of the rabbit M-type sPLA receptor (1394 residues)
that is responsible for the binding of sPLA
s.
Figure 1:
Schematic
representation of native and mutated rabbit M-type sPLA receptors. All the constructs have been obtained by deletion. The
deleted domains are indicated by lines. The names of the
mutants have been chosen to indicate which part of the protein has been
deleted.
X stands for a mutant in which the X domain has
been deleted;
NF lacks the N-terminal cysteine-rich and
the fibronectin-like type II domains (residues 34-211);
12, CRDs 1 and 2 (residues 219-502);
14, CRDs 1-4 (residues 219-796);
X-5, all CRDs but CRD 5 (residues 219-796 and
938-1375);
34, CRDs 3 and 4 (residues 510-796);
5, CRD 5 (residues 795-937);
56, CRDs
5 and 6 (residues 796-1096);
58, CRDs 5-8 (residues
796-1375);
6, CRD 6 (residues 797-1096); and
68, CRDs 6-8 (residues 938-1375) (details are
described under ``Experimental Procedures''). The native
receptor is referred to as WT.
Figure 2:
Immunoblot of native and mutated rabbit
M-type sPLA receptors. Protein samples were separated on a
7.5% acrylamide gel, transferred electrophoretically to Hybond-C Extra
membranes, and subjected to an immunolabeling using an anti-rabbit
M-type sPLA
receptor guinea pig antiserum. The lanes are named according to the mutant receptor that has been loaded,
and MOCK corresponds to membranes from mock-transfected COS
cells. Because mutants in which large regions have been deleted were
not labeled as well as the wild-type receptor, various amounts of
membranes (3-120 µg of proteins) have been loaded in the
different lanes.
Figure 3:
Indirect immunostaining of COS cells
expressing native and mutated M-type sPLA receptors. COS
cells were transfected with an expression plasmid containing the cDNA
coding for the native receptor (A) or mutated receptors
X-5 (B),
58 (C), and
68 (D).
The cells were stained with guinea pig anti-M-type sPLA
receptor antiserum and then with anti-guinea pig fluorescein
isothiocyanate-conjugated whole goat IgG as described under
``Experimental Procedures.''
Figure 4:
Equilibrium binding experiments of I-OS
to wild-type (WT) and M-type
sPLA
receptor mutants. A, saturation curves of
I-OS
to membranes of cells transfected with
the wild-type receptor (
, total binding;
, specific binding;
, nonspecific binding). B, Scatchard plot of the
specific binding shown in panel A. C and D,
Scatchard plots calculated from saturation curves of
NF and
X-5 mutants, respectively.
Deletion of
CRDs 1-4 (12,
34, and
14 mutants) only results in
2-6-fold reduced affinities for
I-OS
(Table 1). This suggests that the sequence comprising CRDs
1-4 is involved but is not essential for
I-OS
binding to the receptor.
Deletion of
CRDs 6-8 (6 and
68 mutants) also leads to lower
affinities of
I-OS
for the receptor (Table 1). These deletions decreased the affinity for
I-OS
by 10- and 12-fold, respectively. These
changes indicate that the CRDs 6-8 sequence is also not crucially
involved in
I-OS
binding, although CRD 6 is
likely to play a role in
I-OS
binding.
A
more dramatic effect was observed in different mutants lacking CRD 5
(58,
56, and
5, see Table 1), demonstrating that
this CRD is directly involved in
I-OS
binding. All these mutants have completely lost their ability to
bind
I-OS
. It was then interesting to test
whether CRD 5 expressed alone was able to bind
I-OS
. The
X-5 construct, in which all
CRDs but CRD 5 have been deleted, was still able to bind
I-OS
, although with a 40-fold reduced
affinity (Table 1).
Taken together, these data show that the
main domain of the extracellular region of the rabbit M-type
sPLA receptor involved in
I-OS
binding is CRD 5, while CRDs 3, 4, and 6 provide further
interactions to increase the affinity of
I-OS
.
The K values
obtained for the inhibition of
I-OS
binding
by mannose-BSA to
NF and
12 mutants are very close to K
values obtained with the native receptor (Table 2). This demonstrates that the N-terminal cysteine-rich
domain, the fibronectin-like type II domain, and CRDs 1 and 2 are not
involved in the binding of mannose-BSA to the M-type sPLA
receptor. Conversely, mannose-BSA was not able to inhibit
I-OS
binding on the
14,
34, and
X-5 mutant forms of the M-type receptor. These results are in
accordance with those establishing the importance of CRD 4 for the
binding of mannose-BSA to the macrophage mannose receptor (42) . Since
I-OS
had no measurable
affinity on mutated receptors lacking CRD 5, it was not possible to
determine the importance of this latter CRD in the binding of
mannose-BSA to the M-type sPLA
receptor.
The affinity of
mannose-BSA for the 6 and
68 mutants of the M-type sPLA
receptor was decreased 13- and 19-fold, respectively, as compared
to its affinity toward the wild-type receptor (Table 2). This
suggests that CRDs 6-8 are not crucially involved in the binding
of mannose-BSA to the M-type sPLA
receptor but that these
latter CRDs contribute to the binding of mannose-BSA. The involvement
of these CRDs in the binding of a glycoprotein such as invertase and in
the binding of mannan has already been described in the case of the
macrophage mannose receptor(43, 44) . The CRD 4
structure of the macrophage mannose receptor is sufficient by itself to
bind invertase and mannan, but it does it with a weak affinity and
needs the presence of the CRDs 5-8 to bind with an affinity
identical to that of the native receptor (43, 44) .
Thus, as for the macrophage mannose receptor, the CRD 4 structure of
the M-type sPLA receptor is involved in the binding of
glycosylated ligands, but the CRD 5-8 structure is required to
confer a full affinity. Although the same CRDs appear to be involved in
the binding of mannose-BSA, several lines of evidence suggest that the
amino acid residues within these CRDs, which are implicated in the
binding process, are different in the two receptors. First, these two
receptors, although predicted to share a similar overall structural
organization, only display a low identity (28%) at the amino acid
level(30) . Second, the binding of mannose-BSA to the mannose
receptor is strictly Ca
dependent(42, 45) , while the binding of
sPLA
s to the M-type receptor is Ca
independent(46) . We also observed that mannose-BSA is
able to inhibit sPLA
binding in the absence of free
calcium. (
)In fact, residues predicted to be involved in
calcium and sugar binding in the CRDs of the mannose receptor are not
conserved in CRDs 4 and 5 of the rabbit M-type sPLA
receptor(30) . Third, although both receptors bind
mannose-BSA, they display different specificities for several other
glycoconjugates. For example, galactose-BSA, which is not a good ligand
for the mannose receptor(42) , binds to the M-type sPLA
receptor (30) while invertase and mannan, which avidly
bind to the mannose receptor(42, 43, 44) , do
not compete with
I-OS
binding to the M-type
receptor(30) . Taken together, these observations make it
unlikely that both types of receptors bind mannose-BSA through similar
residues within their respective CRDs 4-8.
The high conservation of
CRD 4 between different animal species (Table 3) has led to
suggest that this CRD may have a central role in sPLA binding(34) . However, the data shown here indicate that
CRD 5 rather than CRD 4 is most directly implicated in sPLA
binding.
Sequence comparisons show that among the CRDs of
sPLA receptors, CRD 5 is in fact the least conserved
between species (Table 3). The identification of CRD 5 as the
most important CRD for sPLA
binding might explain the
differences observed in the binding properties of different
sPLA
s to M-type receptors from different species (30, 33, 34) .
Recently, various
sPLA inhibitors have been discovered that are able to bind
sPLA
s(48, 49) with binding properties
(affinities and Ca
dependence) similar to those
observed for M-type sPLA
receptors(46) . They have
been purified from the blood plasma of different
snakes(48, 49) . Moreover, the pulmonary surfactant
protein A has been shown to recognize the sPLA
purified
from the Trimeresurus flavoviridis venom and to inhibit its
activity(50) . Very interestingly, the primary structure of all
of these proteins has sequence homology with several CRDs of the C-type
lectin family and hence with CRDs of M-type sPLA
receptors.
The different CRDs 5 of M-type sPLA receptors in
different species have a homology of at least 69% (Table 3),
while the similarity between these CRDs 5 and CRDs of sPLA
inhibitors is less than 20%. Sequence comparison between these
sPLA
inhibitors and CRDs of the M-type sPLA
receptors together with site-directed mutagenesis will probably
help to better define residues that, within CRD 5, are crucial for
sPLA
binding.