(Received for publication, November 1, 1994; and in revised form, January 9, 1995)
From the
Specific membrane receptors for secretory phospholipases A (sPLA
s) have been initially identified with novel
snake venom sPLA
s called OS
and OS
.
One of these sPLA
receptors (muscle (M)-type, 180 kDa) has
a very high affinity for OS
and OS
and a high
affinity for pancreatic and inflammatory-type mammalian
sPLA
s, which might be the natural endogenous ligands of
PLA
receptors. Primary structures of OS
and
OS
were determined and compared with sequences of other
sPLA
s that bind less tightly or do not bind to the M-type
receptor. In addition, the binding properties of pancreatic sPLA
mutants to the M-type receptor have been analyzed. Residues
within or close to the Ca
-binding loop of pancreatic
sPLA
are crucially involved in the binding step, although
the presence of Ca
that is essential for the
enzymatic activity is not required for binding to the receptor. These
residues include Gly-30 and Asp-49, which are conserved in all
sPLA
s. Leu-31 is also essential for binding of pancreatic
sPLA
to its receptor. Many other mutations have been
considered. Those occurring in the N-terminal
helices and the
pancreatic loop do not change binding to the M-type receptor.
Conversion of pancreatic prophospholipase to phospholipase is essential
for the acquisition of binding properties to the M-type receptor.
Mammalian secretory PLAs (
)(sPLA
s) have been divided in two different
structural groups(1, 2) , including group I (the
pancreatic type) and group II (the inflammatory type) (reviewed in (3, 4, 5, 6) ). The pancreatic-type
sPLA
is particularly well characterized. Its
three-dimensional structure is known(7, 8) , and a
detailed characterization of its catalytic properties has been
described(3, 9) . This enzyme, which has long been
considered exclusively as a digestive enzyme(10) , has now been
localized in several tissues of non-digestive origin such as lung,
spleen, and plasma(11, 12, 13) . The
pancreatic-type sPLA
now appears to have a variety of other
cellular functions. It plays a role in cell proliferation(14) ,
and in smooth muscle contraction(15, 16) . The group
II sPLA
is present in abundance in synovial fluids and
plasma of patients with diverse inflammatory diseases and has been
proposed to play a key role in the pathogenesis of inflammatory
diseases (reviewed in (17, 18, 19, 20, 21, 22) ).
Its expression and secretion are induced by proinflammatory agents like
interleukin-1, interleukin-6, and tumor necrosis factor and are
inhibited by glucocorticoids (reviewed in (17, 18, 19, 20, 21, 22) ).
Other members of mammalian sPLA
s have been discovered very
recently(23, 24) .
sPLAs are also found
in abundance in snake and bee venoms (25, 26) . These
enzymes have conserved many important features with mammalian
sPLA
s, including a common catalytic mechanism, the same
calcium requirement, and very conserved primary and tertiary
structures(2, 6, 27, 28) . In
addition to their probable roles in the digestion of preys, snake venom
sPLA
s have evolved into extremely potent toxins displaying
neurotoxic, myotoxic, anticoagulant, and proinflammatory effects
(reviewed in (25) and (26) ). The diversity of the
pathophysiological effects of venom sPLA
s is probably
linked to the presence of specific high affinity receptors for these
enzymes(29, 30, 31, 32, 33) .
A first type of receptors initially identified in brain is called N
(for neuronal)-type PLA
receptors. It recognizes with high
affinity a large number of toxic sPLA
s including
OS
, a new highly neurotoxic sPLA
purified from
the Taipan snake venom, the bee venom sPLA
, and the
neurotoxic sPLA
CM-III from Naja mossambica
mossambica(29) . Nontoxic venom sPLA
s as well
as the porcine pancreatic sPLA
display very low affinities
for these receptors(29) . A second type of receptors initially
identified in rabbit skeletal muscle and thus referred to as M (for
muscle)-type PLA
receptors recognizes with very high
affinities OS
and OS
, a new non toxic
sPLA
also purified from the Taipan snake
venom(30) . This M-type PLA
receptor does not bind
the bee venom sPLA
or the CM-III sPLA
from N. mossambica mossambica(30) . This receptor binds the
porcine pancreatic group I sPLA
as well as the human
inflammatory group II sPLA
, both with fairly high
affinity(31) . It is now known that M-type as well as N-type
receptors have different subunit constitutions and are not exclusively
present in brain or muscle(32, 33) . It is believed
that these receptors are normal binding targets for endogenous
sPLA
s, which, by binding to them, could work as hormones or
growth factors. Molecular cloning of rabbit (31) and bovine (34) M-type PLA
receptors has recently been
achieved and has revealed that these receptors are structurally related
to the macrophage mannose receptor, a protein involved in the
endocytosis of mannose-bearing glycoproteins and
microorganisms(35, 36) .
The purpose of the present
paper is to identify the specific region in the structure of
sPLAs that is responsible for the interaction with M-type
PLA
receptors, (i) using the structures of OS
and OS
, the two venom sPLA
s that have
initially served to discover PLA
receptors (29, 30) and (ii) using a series of mutants of the
pancreatic sPLA
, one of the endogenous sPLA
s
that associates with M-type PLA
receptors.
Automated
Edman degradation of S-pyridylated sPLAs and of
purified peptides were performed with an Applied Biosystem sequencer
(model 477A) equipped with an on-line phenylthiohydantoin-derivative
analyzer (model 120A). C-terminal amino acid analysis was confirmed by
carboxypeptidase Y cleavage according to the manufacturer's
protocol (Boehringer Mannheim).
Molecular weights of OS and OS
were first determined by SDS-polyacrylamide
gel electrophoresis, indicating that OS
and OS
have apparent molecular weights of 14,900 and 15,500,
respectively, under reducing conditions. Molecular weights of OS
and OS
, as calculated from their sequences, are
14,108 and 13,331, respectively. Molecular weights of OS
and OS
, as determined by mass spectrometry analysis
using a laser desorption technique (Finnigan laser mat), are 14,088 and
13,325, respectively.
Figure 1:
Properties of sPLAs binding to
rabbit M-type PLA
receptors. Panel A, competition
experiments involving
I-OS
and unlabeled
sPLA
s for binding to rabbit skeletal muscle myotube
membranes. Membranes (2 µg of protein/ml) were incubated in the
presence of
I-OS
(25 pM) and various
concentrations of unlabeled sPLA
s. All results are
expressed as percentages of control made without unlabeled
sPLA
. 100% corresponds to a
I-OS
specific binding of 0.9 pM. 0% corresponds to
I-OS
binding measurement in the presence of
100 nM unlabeled OS
, which was below 15% of the
total binding. Panel B, Scatchard analysis of
I-OS
binding to rabbit skeletal muscle
myotube membranes in the absence or presence of various concentrations
of unlabeled porcine pancreatic sPLA
. Main panel,
binding data obtained at various concentrations of
I-OS
(5-350 pM) in the absence
(
) or the presence of 10 (
), 25 (
), 50 (
), and
100 (
) nM unlabeled porcine pancreatic sPLA
were plotted according to Scatchard. Inset, Cheng and
Prusoff plot of the
I-OS
K
values obtained from the above Scatchard analysis as a
function of porcine pancreatic sPLA
concentrations.
Scatchard plot analysis of saturation curves
performed with increasing concentrations of I-OS
in the presence of various concentrations of porcine pancreatic
sPLA
indicates that the porcine pancreatic sPLA
competitively inhibits the binding of
I-OS
(Fig. 1B) since the maximal binding capacity is
unchanged (B
= 1.56 ± 0.05 pmol/mg
of protein), while the K
values for
I-OS
vary from 22 pM (no addition of
porcine pancreatic sPLA
) to 260 pM (addition of
100 nM unlabeled porcine pancreatic sPLA
). An
apparent inhibitory constant (K
) of 8.7 nM for the pancreatic sPLA
was calculated from the inset of Fig. 1B.
Since Ca is an essential cofactor for the enzymatic activity of
sPLA
s(3, 7, 9) , it was of
interest to analyze its effect on sPLA
binding to M-type
PLA
receptors. Fig. 2A shows that the
binding of
I-OS
is unchanged in the absence
or in the presence of Ca
. These results contrast with
those previously obtained for N-type PLA
receptors for
which binding activity was found to be highly dependent on the presence
of Ca
(29) . The binding of the pancreatic
sPLA
to M-type PLA
receptors is also
essentially Ca
-independent (Fig. 2B)
since the K
values for the inhibition of
I-OS
binding are similar in the presence (2
mM CaCl
) or absence (2 mM EDTA) of free
Ca
.
Figure 2:
Role of Ca in sPLA
binding to M-type PLA
receptors. Panel A,
effects of free Ca
ions on
I-OS
binding to rabbit skeletal muscle myotube membranes. Free
Ca
concentrations were adjusted by buffering with 2
mM EDTA. Therefore, 0 Ca
-free concentration
corresponds to addition of 2 mM EDTA in the binding assay.
Total binding (
) and nonspecific binding (
) are shown.
Binding conditions are as in Fig. 1. Panel B,
competition experiments with
I-OS
and the
porcine pancreatic sPLA
in the presence of 2 mM CaCl
or EDTA. Binding conditions are as in Fig. 1.
Figure 3:
Sequence alignment of OS,
OS
, porcine pancreatic sPLA
(49) and
CM-III sPLA
from N. mossambica
mossambica(50) . The common numbering system defined by
Renetseder et al. is used(51) . Amino acid residues
found in all active group I sPLA
s sequenced so far are
indicated in the consensus
sequence(2, 27) .
Sequences of OS and OS
are aligned with
those of the porcine pancreatic sPLA
and of the CM-III
sPLA
in Fig. 3. These four sPLA
s are all
group I sPLA
s but have very different binding properties
for M-type PLA
receptors (Fig. 1A). The
highest similarity between these four sPLA
s is in the
Ca
-binding loop (residues 25-35) and in the
second large
-helical segment (residues 40-57), which
contains 2 out of the 4 amino acids implicated in the catalytic
network(9, 28) . Another large region of homology is
found in a region between residues 91 and 111, which contains aspartic
acid 99, another perfectly conserved residue involved in the catalytic
network(9, 28) . In addition, all these four
sPLA
s have the amino acid residues (Fig. 3) found
conserved in all group I sPLA
s sequenced so
far(2, 27) . A detailed comparison of these four
sPLA
s failed to reveal obvious amino acid residues that
could be responsible for the binding to M-type PLA
receptors. The overall homology is almost the same between
OS
and the porcine pancreatic sPLA
(50%
identity), OS
(54% identity), or the CM-III sPLA
(51% identity). Therefore, the identification of amino acid
substitutions that determine the binding of sPLA
s to M-type
PLA
receptors requires different approaches.
Figure 4:
Stereoview of the -carbon backbone of
the porcine pancreatic sPLA
. Positions of the N-terminal (Nt) and C-terminal (Ct) residues are shown. All the
amino acid positions that have been analyzed are marked using the one-letter code. Position of mutations (W3F, S7R, M8L, M20L,
H17D, Y69F or -K, E71N, and
62-66) and of amino acid
substitutions occurring in iso-PLA
(alanine 12, histidine
17, methionine 20, and glutamic acid 71 changed to threonine, aspartic
acid, leucine, and asparagine, respectively) that are without effect on
binding activity are indicated by small balls. Conservative
mutations obtained by replacing all lysines for arginines which do not
affect binding activity are not indicated by small balls because it is quite possible that if these lysines were changed to
neutral or negatively charged residues, an effect on binding might
occur. The mutant
62-66 was obtained by the deletion of
residues 62-66 (residues shown in pink) and the
simultaneous introduction of mutations D59S, S60G, and
N67Y(44) . Position of mutations affecting the affinity of the
pancreatic enzyme (G30S, L31R, -S, -T or -W, and D49K) are indicated by largerballs. Localization of Ca
is
indicated by a purple ball.
The first large N-terminal -helix and the short
-helix
that follows in the structure are both located at the surface of the
sPLA
molecule (Fig. 4). This region of the
pancreatic sPLA
molecule contains residues involved in
interfacial binding, substrate specificity and formation of the
hydrophobic channel (28, 53) . Analysis of the binding
properties of four different mutants in this region of the enzyme and
of the iso-PLA
suggests that this region does not contain
structural determinants essential for the binding activity to M-type
PLA
receptors (Table 1). Indeed, mutation of
tryptophan 3 to phenylalanine (W3F; (38) ) does not strongly
modify the K
value as compared with the
wild-type recombinant sPLA
(Table 1). The presence of
different residues at position 3 in OS
, OS
, and
the CM-III sPLA
confirms that residues at this position
have probably no role in the binding step. Likewise, a mutation of
serine 7 to an arginine (S7R; (39) ) does not significantly
change the K
value as compared with the
wild-type recombinant enzyme (Table 1). This is consistent with
the observation that serine 7 is replaced by an asparagine in OS
and a phenylalanine in OS
, which bind well to M-type
PLA
receptors, but also by an asparagine in the CM-III
sPLA
, which does not bind to this receptor. Similarly, a
pancreatic mutant in which methionines 8 and 20 have been replaced by
leucines (M8L/M20L; (40) ) still displays an affinity (K
= 18 nM) similar to that of
the recombinant wild-type enzyme (K
= 15
nM), indicating that replacement of methionines by leucines at
these positions does not change the binding properties (Table 1).
OS
and OS
have a leucine or a methionine
respectively at these positions; as expected, both bind similarly to
M-type PLA
receptors, but this residue is not essential
since the CM-III sPLA
also has a methionine in position 8
but does not bind to the receptor. The last mutant that provides some
information on the role of this part of the pancreatic sPLA
structure in binding activity corresponds to a partial conversion
of the pancreatic sPLA
major isoform to the iso-PLA
minor isoform by substitution of histidine 17 to an aspartic acid
(together with a substitution of glutamic acid 71 to an asparagine).
These mutations fail to produce drastic effects on the binding
properties (Table 1). Finally, the native iso-PLA
,
which is different from the major isoform pancreatic sPLA
at four positions(37) , shows a K
value of 22 nM very similar to that of wild-type porcine
pancreatic sPLA
(Table 1). Prophospholipase
(pro-PLA
), the enzymatically inactive porcine pancreatic
zymogen, has no measurable affinity for M-type PLA
receptors (Table 1).
The effect of various
mutations at position 31 has also been analyzed (42) . Leucine
31 in the pancreatic sPLA is located in the
Ca
-binding loop (Fig. 4) but is not involved
in the coordination of Ca
. Three-dimensional
structures of several sPLA
s have shown that residues at
position 31 are located at the surface of the enzyme, at the entrance
of the hydrophobic channel and of the active
site(7, 51, 54, 55, 56, 57) .
Leucine 31 of the pancreatic sPLA
also forms a part of the
interfacial binding surface and is then involved in the binding of
sPLA
to aggregated phospholipids(28, 53) .
Site-directed mutagenesis experiments on porcine pancreatic sPLA
have shown that replacements of leucine 31 by a tryptophan, an
arginine, an alanine, a threonine, a serine, or a glycine modified the
affinity of the enzyme for monomeric and aggregated phospholipids as
well as catalytic activity toward these substrates(42) .
Replacements of leucine 31 in the porcine pancreatic sPLA
by a serine (L31S) or a threonine (L31T) resulted in a 100-fold
decrease of the K
value relative to the
wild-type enzyme. Replacement by an arginine (L31R) resulted in a total
loss of binding activity (Table 1). Interestingly, the CM-III
sPLA
has an arginine in position 31 (Fig. 3) and
does not bind to M-type PLA
receptors (Fig. 1A). Conversely, a mutation to a tryptophan
(L31W) did not result in a decrease of binding activity (Table 1).
Lysine
residues have been implicated in the interfacial recognition site of
the enzyme, in the process of interfacial activation, and in the
``penetrating power'' of sPLAs into the lipid
bilayer (see (48) ). A porcine pancreatic mutant was
constructed in which all 9 lysines were replaced by arginines (All
K-R). This mutant displayed 68% residual activity on micellar
zwitterionic substrates, indicating that lysines are not essential for
catalytic activity(48) . This mutant of the pancreatic
sPLA
has nearly the same binding properties to M-type
PLA
receptors as the wild type pancreatic sPLA
(Table 1). Sequence comparison at positions corresponding
to lysines in the porcine pancreatic enzyme with other
sPLA
s shown in Fig. 3also indicates that these
lysines are not essential elements for the interaction with M-type
PLA
receptors.
This paper presents sequences of the two sPLAs
isolated from the venom of Oxyranus scutellatus scutellatus and called OS
and OS
, which have initially
served to demonstrate the existence of high affinity receptors (in the
0.01-0.1 nM range) for
sPLA
s(29, 30, 31, 32, 33) .
Comparison of these sequences with the pancreatic sPLA
, one
of the probable endogenous ligand of the M-type PLA
receptor, which binds with an affinity in the 1-10 nM range (14, 31, 34) and with the CM-III
sPLA
, which does not recognize the M-type PLA
receptor(30) , failed to provide indications on residues
involved in receptor recognition. Therefore, since pancreatic
sPLA
binds competitively with the high affinity ligand
OS
to the M-type receptor, since this enzyme as previously
indicated is the probable endogenous ligand and since its
three-dimensional structure is particularly well known, it appeared
that the best way to analyze the structure-function relationships of
the interaction of sPLA
s with their receptors was to use
pancreatic sPLA
mutants.
Possible candidates for an
interaction with the receptor were N-terminal helices, which are
located at the surface of the sPLA molecule. Mutations at
different positions (W3F, S7R, M8L, M20L, and H17D) in this region of
the pancreatic sPLA
failed to produce marked changes of the
affinity of the pancreatic sPLA
to M-type PLA
receptors.
Another interesting property of pancreatic
sPLA is the existence of a pancreatic loop at residues
62-66, which is found in some other sPLA
s such as
OS
. Deletion of this loop does not significantly alter (a
decrease by a factor of
3) the affinity of the pancreatic
sPLA
to M-type PLA
receptors. The idea that
this loop is not essential for binding activity is supported by the
fact that OS
, which lacks this loop, binds to M-type
PLA
receptors with an affinity much better than that of the
pancreatic sPLA
( Fig. 1and Table 1).
Lysine residues are of course exposed at the surface of the
pancreatic sPLA(48) . Their replacement by
arginines, i.e. other positively charged residues, does not
significantly alter the affinity (a decrease by a factor of
2),
indicating that these amino acid side chains are not crucially involved
in the interaction. However, it cannot be eliminated that other types
of mutations not conserving the charges on the side chains could have
had a more drastic effect. Nevertheless, sequence comparisons shown in Fig. 3indicate that none of the lysines of the pancreatic
sPLA
but lysine 121 is conserved in the structures of
OS
and OS
, which both bind extremely well to
M-type PLA
receptors. It might be the change of lysine 121
plays some role (which would remain partially conserved after a
mutation to an arginine) in the interaction with the receptor.
The
-wing (residues 74-85, Fig. 3) of the pancreatic
sPLA
is probably not involved in the interaction with the
receptor since this part of the sPLA
differs considerably
in the three-dimensional structures of group I (7, 8, 54, 57) and group II
sPLA
s (51, 56) and since both groups of
sPLA
s bind to M-type PLA
receptors including
the human group II sPLA
(31) .
The most evident
domain of pancreatic sPLA involved in the binding with the
M-type receptor is the Ca
-binding loop. This part of
the enzyme, as its name indicates, is involved in Ca
binding, which is essential for catalytic activity (28, 53) . It contains glycine 30 and aspartic acid
49, which are present in all active sPLA
s sequenced so far,
including the evolutionary distant bee venom
sPLA
(2, 27, 55) . Mutations of
glycine 30 or of aspartic acid 49 drastically decreased binding
activity by factors of
8 (G30S) to
100 (D49K) (Table 1). Clearly, residues 30 and 49 are essential for binding
activity to M-type PLA
receptors, but their presence is not
sufficient to confer binding since other sPLA
s such as the
CM-III sPLA
and the bee venom sPLA
, which do
not bind to the M-type PLA
receptors ( Fig. 1and (30) ), also have these residues in their sequence ( Fig. 3and (55) ). Very drastic effects on binding
activity have also been observed by mutation of leucine 31 to residues
such as serine, threonine, or arginine (a decrease of the affinity by
more than 100 times). Conversely, mutation of leucine 31 to a
tryptophan did not result into a dramatic decrease of the affinity but
rather into a moderate increase of the affinity (Table 1),
suggesting that the presence of aliphatic or aromatic residues at this
position 31 can also confer high affinity to M-type PLA
receptors. Several different residues are found in position 31 in
other sPLA
s sequences(2, 27) . This
position is occupied by a lysine in OS
and OS
.
It seems that while glycine 30 and aspartic acid 49 are essential for
binding, the identity of the side chain in position 31 will finally
determine whether this binding is possible or not. The CM-III
sPLA
has an arginine in position 31; it does not bind to
the M-type PLA
receptors, consistent with the observation
that the pancreatic sPLA
mutant with an arginine in this
position is inactive in binding (Table 1).
Since the
Ca-binding loop is obviously involved in the
sPLA
-M-type receptor interaction, it was of course
important to determine whether Ca
is important for
the association. Clearly, results presented in this paper (Fig. 2) show that Ca
is not important for the
interaction. The receptor association of the ligand seems to be even
better in the absence of Ca
, suggesting that the
preferred form of sPLA
for binding is the
Ca
-free form (Fig. 2). Conversely, the other
type of PLA
receptors, i.e. the N-type receptor,
requires Ca
for sPLA
binding(29) .
One of the questions that immediately
comes to mind is to know whether sPLA catalytic activity is
required for binding. At first sight, the two activities seem to be
related (Table 1) since mutations that cause the largest
decreases in catalytic activity also have a large impact in terms of
binding. This is because the Ca
-binding loop is
necessary for both functions. However, catalytic activity is clearly
not required, since it is absent without Ca
while
binding activity is preserved and even increased (Fig. 2).
The pancreatic sPLA originates from its prophospholipase
precursor. It is particularly interesting to see that while the
pancreatic sPLA
as well as its variant iso-PLA
(with changes in positions 12, 17, 20, and 71) binds fairly
avidly to M-type PLA
receptors, the prophospholipase does
not. Clearly the precursor needs to be converted to acquire a
conformation (58, 59, 60) that confers both
catalytic activity and binding activity to M-type PLA
receptors.