(Received for publication, November 7, 1994)
From the
A 45-kDa polypeptide preferentially present in neuronal
membranes was previously identified as a subunit of a binding (or
receptor) protein for several phospholipase A variants with
neurotoxicity, including crotoxin, by chemical cross-linking
experiments (Yen, C.-H., and Tzeng, M.-C.(1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor
protein was completely suppressed by sufficient F22Y, a mutated bovine
pancreatic phospholipase A
generated by site-directed
mutagenesis of Phe
of the wild-type enzyme to Tyr. The
IC
of this inhibition was estimated to be 1
µM. In sharp contrast, the wild-type enzyme gave no effect
even at 50 µM. This mutation resulted in only minor and
localized structural perturbations with little effect on enzymatic
activity. Other phospholipase A
molecules capable of
competing with crotoxin for this binding invariably have Tyr at this
position. It was concluded that this Tyr residue is an important
determinant for the binding of a number of phospholipase A
variants to the 45-kDa receptor.
Proteins with phospholipase A (PLA
) (
)(EC 3.1.1.4) activity can be found in extracellular
secretion as well as inside the cells of many organisms. The
extracellular (secreted) PLA
variants exhibit a variety of
biological effects, including phospholipid metabolism, host defense,
signal transduction, neurotoxicity (presynaptic and/or postsynaptic),
myotoxicity, and alteration of coagulation, which may or may not be
related to hydrolysis of phospholipids. Despite large differences in
biological actions, the secreted PLA
chains from most
sources show high degrees of homology in the primary, secondary, and
possibly tertiary structures. A small number of these proteins,
including crotoxin from the South American rattlesnake Crotalus
durissus terrificus, act primarily at the presynaptic level to
cause synaptic blockade by inhibiting the release of neurotransmitters,
though most of them also produce postsynaptic toxicity and other
effects. To exert neurotoxicity, these PLA
neurotoxins
appear to bind to the synaptic membranes strongly, followed by
hydrolysis of the membrane phospholipids by the PLA
activity. Lack of such binding is apparently the reason why a
larger number of PLA
variants, such as pancreatic
PLA
, are not neurotoxic despite high degrees of enzymatic
activity. Strong binding to plasma membranes of other tissues may also
be essential for the actions of many other PLA
s on these
tissues (see (1, 2, 3, 4, 5, 6, 7, 8, 9) for recent
reviews).
By the use of photoaffinity labeling and chemical
cross-linking techniques, a few binding proteins have been identified
for some of these presynaptic
toxins(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
One subunit of the binding proteins for crotoxin and several other
neurotoxic PLAs is, as observed by us, a 45-kDa polypeptide
preferentially present in the neuronal membrane(13) . As one of
our approaches to understanding the structural basis for this binding,
we converted bovine pancreatic PLA
, which showed no
detectable binding to the synaptic membrane, into a mutant capable of
competing for the binding of crotoxin to this receptor by site-directed
mutagenesis. This report is the first, to our knowledge, to convert
pancreatic PLA
into a competitor of any neurotoxic
PLA
. This new way of using site-directed mutagenesis will
be useful for the study of other toxic proteins as well.
We labeled crotoxin with I without affecting
its neurotoxicity. After
I-crotoxin had been incubated
with the synaptic membrane fraction from the brain to reach maximal
binding, disuccinimidyl suberate or disuccinimidyl
dithiobis(propionate) was employed to cross-link the binding complexes.
Similar to results reported previously(13) , cross-linking with
the above cross-linkers resulted in a 60-kDa conjugate when analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography
(data not shown). Crotoxin is a complex of subunit A of 9,000 Da and
subunit B of 14,400 Da in noncovalent association. The subunit B is an
active PLA
(36, 37) .
Utilizing the
phosphorothioate method(24) , we produced mutants of bovine
pancreatic PLA by site-directed mutagenesis of a chemically
synthesized gene for the wild-type protein(25) . When the
bovine pancreatic PLA
mutant F22Y, in which the Phe
of the wild type is replaced by Tyr(35) , was present
during the binding period, the subsequent formation of the 60-kDa
radioactive conjugate was suppressed with an IC
of 1
± 0.1 µM as estimated from the curve in Fig. 1and three other separate experiments. In sharp contrast,
the wild-type PLA
purified from the bovine pancreas gave no
effect even at a concentration as high as 50 µM (Fig. 1). The wild-type pancreatic enzyme produced by
cloning techniques and another mutant F22A, which has Ala at residue
22, were also without effect at the highest concentration used.
Figure 1:
Effects
of bovine pancreatic PLA and the F22Y mutant on the
cross-linking of
I-crotoxin to synaptic membranes from
the brain. Various amounts of the F22Y mutant (
) or the wild-type
bovine pancreatic PLA
(
) together with
I-crotoxin (7 ng) were incubated with the synaptic
membrane fraction (50 µg) from the guinea pig brain. After
cross-linking, the counts of radioactivity in the 60-kDa conjugate were
measured. The data are expressed as percentages of the counts in the
absence of unlabeled PLA
.
The
one- and two-dimensional NMR spectra of F22Y and the wild type are
almost identical except for the obvious changes arising from the new
phenolic OH group and a 0.19 ppm change in one of the three chemical
shifts of Phe, which is in close proximity to residue 22,
forming the second half of the Phe
-Phe
aromatic sandwich ( Fig. 2and Fig. 3and Table 1). Similarly, except right around the mutated residue, the
NMR spectra of F22A are perturbed only slightly(35) . The
enzymatic activity of the two mutants is also comparable with that of
the wild-type enzyme(35) . Among the many mutants we have
analyzed, F22Y is most similar to the wild type structurally and
kinetically. Hence it is unlikely that the inhibitory effect of the
F22Y mutant is due to the hydrolysis of membrane phospholipids. In
addition, we have chosen the assay condition that minimizes the
enzymatic activity by using a solution containing 10 mM Sr
, 0.5 mM EGTA, and no
Ca
, as it has been shown that Sr
is
antagonistic to Ca
(see (1, 2, 3, 4) for reviews), which is
required for the enzymatic activity of secreted PLA
.
Moreover, F22Y was equally effective in suppressing the formation of
the 60-kDa conjugate when the experiments were performed at 4 °C to
completely arrest the enzymatic activity. We thus conclude that the
F22Y mutant blocked the formation of the radioactive conjugate by
competing the binding of
I-crotoxin to the binding
protein.
Figure 2:
One-dimensional proton NMR spectra of
pancreatic PLA and its F22Y mutant in D
O.
Spectra of samples of 1.5 mM protein in 300 mM NaCl
and 50 mM CaCl
, pH 4.0, were recorded at 500 MHz
and 37 °C. Free induction decays from 200 scans were processed with
gaussian multiplications (line broadening, -5; gaussian
broadening, 0.1). WT, wild type.
Figure 3:
Phase-sensitive nuclear Overhauser effect
spectroscopy proton NMR spectra in DO at 500 MHz. Samples
are as described in the legend of Fig. 2. The mixing time was
200 ms. A 4096
512 matrix in the time domain was recorded and
zero-filled to a 4096
2048 matrix prior to multiplication by a
gaussian function (line broadening, -3; Gaussian broadening,
0.1). Chemical shifts for the indicated spin systems are given and
assigned in Table 1.
However, because the binding affinity of the F22Y mutant was not high relative to that of crotoxin (<10 nM), there must be other residues also involved in binding and thereby in neurotoxicity. Judging from the affinity of F22Y, we would not expect it to be neurotoxic. All mice injected with the F22Y mutant, either intraperitoneally or intracisternally, lived and behaved normally even at a dose of 18 mg of protein/kg of body weight. We have iodinated the F22Y mutant and attempted to demonstrate its binding to the synaptic membrane directly. Specific binding was not evident, apparently because the affinity is too low.
We were also aware that residue 22 of the
pancreatic PLA of the rat is Tyr. If the rat enzyme blocked
I-crotoxin from forming the radioactive conjugate, our
conclusion would be further substantiated. We therefore purified the
PLA
from the rat pancreas and then investigated its effect
on the conjugation. As expected, the rat pancreatic PLA
could completely inhibit the generation of the 60-kDa band,
although the potency (IC
= 10 µM) was
lower than that of the F22Y mutant of the bovine PLA
. This
may be due to the differences between the rat and the bovine enzymes at
other areas. As would be expected, we found that the rat PLA
was nontoxic when tested as described above.
Although further
work is needed to extend our findings for a generalization concerning
the binding of the neurotoxic PLAs, it would be useful to
put forward some suggestions for further testing. It has not been
possible for us to study the binding of all variants of
PLA
, but available data seem to indicate that the 45-kDa
polypeptide may be a common binding protein, or one of its subunits,
for most neurotoxic PLA
s, and strong binding to this
polypeptide appears to be linked to neurotoxicity (see (1) for
review; (11) and (13) ). Significantly, examination of
the aligned sequences of the PLA
chains from various
sources (38, 39) revealed that the active PLA
chain of each of the PLA
neurotoxins has Tyr at the
position corresponding to Phe
of the bovine enzyme in
their aligned sequences (it is position 21 for many PLA
chains, including subunit B of crotoxin), whereas those with Phe
are nontoxic or marginally toxic. Our present results would suggest
that Tyr
in these neurotoxic PLA
variants is
also an important determinant for such binding and that other as yet
undefined determinants are also involved. On such ground, one may
resolve the seeming conflict that, though a small number of
PLA
s with Tyr
exhibit low toxicity, most of
them are potent toxins. With Tyr
in its PLA
chain,
-bungarotoxin is to date the only neurotoxic
PLA
that does not bind to the 45-kDa polypeptide. This can
be explained by steric hindrance due to another polypeptide covalently
bonded to its PLA
chain, an interpretation supported by the
insusceptibility of this residue to chemical modification(40) .
As to the other determinants for binding, a definitive answer has not
been obtained for any of the PLA
variants, but some
information is available (see (41) for review). Based on
comparisons of the amino acid sequences of the PLA
variants
with presynaptic toxicity and those of the nontoxic ones, basic
residues around position 59, at position 69, around position 76, and at
position 93 (or 94) and the segments of residues 1-7,
68-85, and 80-110 (all numbered with respect to pancreatic
PLA
according to (42) ) have been proposed to be
involved in binding(43, 44) . From the variations in
amino acid sequences of the three ammodytoxins, Tyr
,
Arg
, and Lys
have been suggested to
participate in binding(45, 46) . Chemical modification
and other studies indicate that Trp
, Tyr
,
Tyr
and Tyr
of notexin, Tyr
of
the PLA
chain of
-bungarotoxin, and Asn
,
Met
, Lys
, and Lys
of Pa-11 are
probably needed for their neurotoxicity, but binding experiments have
not been performed except for some modified
Pa-11(40, 47, 48, 49, 50) .
Extension of the site-directed mutagenesis studies that we have
reported here will hopefully provide a clear-cut answer.
In summary,
we have identified Tyr as one of the important
determinants for the neurotoxicity of PLA
proteins. The
Phe
of bovine pancreatic PLA
is located at the
B-helix, away from the active site and exposed to solvent. It does not
play an important structural role because mutations at this position
cause only minor and localized structural perturbations. The F22Y is a
silent mutation catalytically, but it acquires the ability to compete
with crotoxin (a neurotoxic PLA
) for binding to its
receptor protein with an IC
value of 1 µM.
Although such a binding affinity is still too low for in vivo neurotoxicity (the affinity of crotoxin for binding to the same
receptor protein is <10 nM), the results have demonstrated
a dramatic effect that can possibly be induced by a point mutation and
have shed light on how the small PLA
protein (14 kDa for
pancreatic enzymes) could give rise to hundreds of natural variants
displaying a great variety of pharmacological actions.
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