(Received for publication, August 30, 1995)
From the
Synaptotagmins I and II are inositol high polyphosphate series
(inositol 1,3,4,5-tetrakisphosphate (IP), inositol
1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate)
binding proteins, which are thought to be essential for
Ca
-regulated exocytosis of neurosecretory vesicles.
In this study, we analyzed the inositol high polyphosphate series
binding site in the C2B domain by site-directed mutagenesis and
compared the IP
binding properties of the C2B domains of
multiple synaptotagmins (II-IV). The IP
binding
domain of synaptotagmin II is characterized by a cluster of highly
conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324).
Among these, three lysine residues, at positions 327, 328, and 332 in
the middle of the C2B domain, which is not conserved in the C2A domain,
were found to be essential for IP
binding in synaptotagmin
II. When these lysine residues were altered to glutamine, the IP
binding ability was completely abolished. The primary structures
of the IP
binding sites are highly conserved among
synaptotagmins I through IV. However, synaptotagmin III did not show
significant binding ability, which may be due to steric hindrance by
the C-terminal flanking region. These functional diversities of C2B
domains suggest that not all synaptotagmins function as inositol high
polyphosphate sensors at the synaptic vesicle.
Synaptotagmin is an integral membrane protein of secretory
vesicles, abundant in neural and some endocrine cells(1) . The
structure of synaptotagmin is characterized by two copies of highly
conserved repeats homologous to the C2 regulatory region of protein
kinase C in the cytoplasmic domain(2) . The importance of the
C2 domain of synaptotagmin in Ca-regulated exocytosis
is shown by several studies. A recombinant protein of the C2A domain
inhibited Ca
-regulated exocytosis of a large dense
core vesicle in PC12 cells(3) . Injection of basic peptides
from the central region of both C2 domains into the squid giant
presynaptic terminal also inhibited neurotransmitter
release(4) . The studies of mutants of Caenorhabditis
elegans(5) , Drosophila(6, 7) ,
and the knock-out mouse (8) also indicated that synaptotagmin
plays a key role in Ca
-regulated exocytosis.
In
our previous study, we characterized synaptotagmin II as an IP(
)or rather an inositol-high polyphosphate series
(IP
, inositol 1,3,4,5,6-pentakisphosphate, and inositol
1,2,3,4,5,6-hexakisphosphate, referred to as IHPS) binding
protein(9) . Deletion analysis of synaptotagmin II clearly
showed that about 30 amino acids of the central region of the C2B
domain were essential for IHPS binding(10) . This binding
domain includes a sequence corresponding to the squid Pep20 peptide,
which is essential for neurotransmitter release(4) , suggesting
that IHPS has some effect on neurotransmitter release. Our recent study
of the squid giant presynapse supported this suggestion. Microinjection
of the members of IHPS into the squid giant presynaptic terminal is
shown to block synaptic transmission without affecting the presynaptic
Ca
current(11) , whereas inositol
1,4,5-trisphosphate shows no effect, suggesting that IHPS is a potent
modulator of neurotransmitter release.
Recently, two novel isoforms designated synaptotagmins III and IV were isolated(12, 13) . Multiple isoforms of the proteins involving vesicular trafficking, such as the syntaxin family (14) and the synaptobrevin family(15, 16) , are not always functionally similar as indicated by in vitro protein interaction(17, 18) . Our interest is whether multiple synaptotagmin isoforms are functionally diversified or not.
In the present study, we first determined the essential
residues for IP binding in synaptotagmin II by
site-directed mutagenesis. Then, we tested IP
binding to
the C2B domains of synaptotagmins III and IV. Our data indicate that
synaptotagmin III cannot bind IP
despite the presence of a
putative IP
binding sequence. On the basis of these
results, we discuss the functional differences in C2 domains in
multiple synaptotagmin isoforms.
Mouse synaptotagmin IV cDNA (amino acids 1-425) was also obtained by reverse transcriptase-PCR from mouse cerebellum (sense 5`-ACATGGCTCCTATCACCACC-3` and antisense 5`-AGCTAACCATCACAGAGCAT-3` (13) ). Cycling conditions were the same as in the case of synaptotagmin III described above. The PCR product was subcloned into pT7 Blue T-vector and completely sequenced.
Site-directed mutagenesis of GST-STII-C2B (mut3, mut4, mut5, mut8, and mut9) was carried out with a mutan-K kit (Takara Shuzo) as described previously(10) . All other mutant GST fusion proteins including deletion mutants (mut13) were produced by two-step PCR techniques as follows(19) . In the case of GST-STII-C2B-mut1(K322Q), for example, the right and left halves of the C2B domain were separately amplified with two pairs of oligonucleotides (primer A, 5`-CGGATCCGAGAAGGAAGAGCCAGAGAA-3` and mutagenic primer C, 5`-GCTTAAGTCTCTGACCGTTC-3` (right half); mutagenic primer D: 5`-GCTTAAGAAGAAGAAGACGACAGT-3` and primer B, 5`-CGAATTCATGTCGGACCAGTGCCGCA-3` (left half)). The two resulting PCR fragments were digested with AflII (underlined), ligated to each other, and reamplified with primers A and B. The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin II (K322Q) was subcloned into the BamHI-EcoRI site of pGEX-2T and verified by DNA sequencing.
Since we were also able to introduce the AflII site into the C2B domain of synaptotagmin III without amino acid substitutions (Fig. 5B, arrowhead), we could produce the chimera from synaptotagmins II and III by two-step PCR techniques. GST-STII/III-C2B-a contains amino acids 267-323 of mouse synaptotagmin II followed by amino acids 480-549 of mouse synaptotagmin III. GST-STIII/II-C2B contains amino acids 425-479 of mouse synaptotagmin III followed by amino acids 324-393 of mouse synaptotagmin II. GST-STII/III-C2B-b, containing amino acids 267-344 of mouse synaptotagmin II followed by amino acids 500-549 of mouse synaptotagmin III, was also produced by the same techniques.
Figure 5:
IP binding properties of
multiple synaptotagmins. A, schematic representation of
synaptotagmins II, III, and IV is shown at the top, where the
transmembrane region (TM) and the two C2 repeats are shown. Below, construct of the chimera and deletion mutants of the
GST fusion protein are shown. B, alignment of putative
IP
binding domains of the synaptotagmin family. Residues
that are identical in the three sequences are shaded. Residue numbers are given on both sides. Asterisks indicate the most essential residues for IP
binding to
synaptotagmin II as determined from Fig. 2. Arrowhead indicates chimeric point between synaptotagmin II and III. C, [
H]IP
binding to GST
fusion proteins from C2 domains of synaptotagmins III and IV. GST
fusion proteins (5 µg) were analyzed by
[
H]IP
binding assay as described
under ``Materials and Methods.'' The data are means ±
S.E. of three measurements, normalized to 100% for binding to
GST-STII-C2B.
Figure 2:
Mutational analysis of the IP binding domain. A, the amino acid sequence (top) is the putative IP
binding domain of
synaptotagmin II as determined previously(10) . Q indicates the substitution of Gln for Lys or Arg, and a dash indicates the deletion. Shaded boxes indicate the most
important residues for IP
binding. B, mutant
GST-STII-C2B (5 µg) were analyzed by
[
H]IP
binding assay as described
under ``Materials and Methods.'' The data are means ±
S.E. of three measurements, normalized to 100% for binding to
GST-STII-C2B.
Immunoblot analysis was carried out with these domain-specific antibodies as follows: proteins were transferred to a polyvinylidene difluoride membrane (Millipore), blocked with 2% skim milk, 0.05% Tween 20 in phosphate-buffered saline, incubated with anti-STII-C2A (or C2B) polyclonal antibody (2.3 µg/ml), and incubated with peroxidase-labeled goat IgG against rabbit IgG. Immunoreactive bands were visualized with 3,3`-diaminobenzidine.
Figure 1:
Effect of specific antibodies against
the C2 domain on IP binding to synaptotagmin II. A, purified GST-STII-C2A (a, 0.5 µg/lane),
GST-STII-C2B (b, 0.5 µg/lane), and solubilized mouse
cerebellar membrane proteins (c, 5 µg/lane) were analyzed
by SDS-polyacrylamide gel electrophoresis. Proteins were visualized by
amido black (left), and immunoblotting was carried out with a
polyclonal antibody raised against each C2 domain to check its
specificity (middle and right). Note that
anti-STII-C2A slightly reacted with GST-STII-C2B, whereas anti-STII-C2B
specifically recognized GST-STII-C2B. Numbers on the left indicate positions of molecular weight markers. B,
[
H]IP
binding activity of
GST-STII-C2B (5 µg); C, purified synaptotagmin II (0.7
µg) in the presence of various concentrations of IgG: anti-STII-C2A (open circles), anti-STII-C2B (closed circles), and
normal rabbit IgG (triangles). The data are means ±
S.E. of three measurements.
By use of these two antibodies, we
confirmed that the C2B domain was the authentic IP binding
domain in synaptotagmin II purified from mice cerebella. In the
presence of the anti-STII-C2B antibody, the IP
binding
activity of both GST-STII-C2B and purified synaptotagmin II was
completely inhibited dose dependently (Fig. 1, B and C). In contrast to anti-STII-C2B, the effect of anti-STII-C2A
antibody was very weak. The reduction of IP
binding
activity by anti-STII-C2A antibody may be due to the cross-reactivity
with the C2B domain (Fig. 1, A and B).
Although anti-STII-C2A antibody has weak inhibitory effect on
IP
binding to synaptotagmin II, this antibody inhibits
Ca
/phospholipid binding to the C2A domain of
synaptotagmin II. (
)The normal rabbit IgG had almost no
effect.
Figure 3:
Comparison of putative IHPS binding
domains. Alignment of the central region of two C2 domains of mouse
synaptotagmin II according to (2) . The top is the
sequence of the C2A domain corresponding to the IHPS binding domain (middle(10) ). Shaded boxes indicate the most
important residues for IP binding as shown in Fig. 2. The bottom sequence is that of the mutant
GST-STII-C2B (K327Y, K328E, and K332H). Amino acid residue numbers are shown on both sides.
To evaluate the mutational effect on IP binding
activity, Scatchard analysis of IP
binding to mutant GST
fusion proteins was performed (Fig. 4A), and the
dissociation constants (K
) and B
are
shown in Fig. 4B. In the case of one mutation, the K
values of these mutants were also divided into
three groups: almost no change (mut1 and -2), twice as high (mut3, -4,
and -8), and three to four times as high as that of normal GST-STII-C2B
(mut5, -6, and -7), while B
remained unchanged
(mut5-9) or increased slightly (mut1-4). GST-mut9-11,
double mutations of important residues, showed still lower affinity for
IP
, below one-fifth of that of GST-STII-C2B, and the
B
value of GST-mut9 and -mut11 was two-thirds of that of
the GST-STII-C2B. All these data suggest that three Lys residues at
positions 327, 328, and 332 of synaptotagmin II are responsible for the
high affinity binding to IP
, but Lys at position 322 and
Arg at position 323 have no effect.
Figure 4:
IP binding properties of
mutant GST-STII-C2Bs. A, Scatchard analysis of IP
binding to mutant GST-STII-C2B: mut2 (closed circles),
mut3 (closed squares), mut6 (open circles), mut10 (open squares), mut11 (triangles). Dashed line indicates the normal GST-STII-C2B as previously
reported(10) . B, parameters of IP
binding
to mutant GST-STII-C2Bs. dash, not determined because of lack
of IP
binding activity.
The B values of
these mutants shown in Fig. 4B indicate
substoichiometric binding of IP
to GST fusion proteins.
This substoichiometric binding was also observed in the case of
purified cerebellar synaptotagmin II previously described(9) .
This may result from instability of an IP
sensitive form of
synaptotagmin II because purified synaptotagmin II easily loses its
IP
binding by repetitive freezing and thawing or short
storage at 4 °C.
To compare the putative IP binding domains of synaptotagmins II and III (Fig. 5B), two Lys residues were replaced by Arg in
synaptotagmin III (amino acid positions 477 and 482). Especially, Arg
at position 482 is a corresponding residue which is important for
IP
binding ability in synaptotagmin II (Fig. 2). To
investigate the effect of this substitution, two mutant GST fusion
proteins were produced (GST-STIII-C2B (R482K) and GST-STII-C2B
(K327R)). The R482K mutation could not rescue the IP
binding activity of GST-STIII-C2B, and the K327R mutation had no
effect on IP
binding of GST-STII-C2B (Fig. 5C). To further determine which region causes the
difference in IP
binding activity between synaptotagmins II
and III, we constructed three chimera proteins from synaptotagmins II
and III, named GST-STII/III-C2B-a, -b, and -STIII/II-C2B (Fig. 5A). GST-STIII/II-C2B showed strong IP
binding activity (65% of that of GST-STII-C2B), whereas the
others showed very weak activity (below 15% of that of GST-STII-C2B),
indicating that the loss of IP
binding activity was mainly
caused by one-third of the C-terminal of the C2B domain of
synaptotagmin III (Fig. 5C). This was confirmed by the
facts that GST-STIII-C2B(
C), in which one-third of the C-terminal
of the C2B domain of synaptotagmin III was deleted, showed significant
IP
binding activity (30% of that of GST-STII-C2B, Fig. 5C). These findings suggest that the IP
binding property varies in multiple synaptotagmins, although all
of them basically possess the IP
binding domain.
The present experiments using specific antibodies against the
C2A and C2B domain demonstrate that the C2B domain of synaptotagmin II
is the IP binding domain. Success in producing these
specific antibodies provided us important information: the C2A and C2B
domains have different structures and functions. This was supported by
three recent studies: 1) Ca
-dependent phospholipid
binding of the C2A domain and Ca
-independent
phospholipid binding of the C2B domain(10, 21) ; 2)
inositol polyphosphate binding to the C2B domain(10) ; and 3)
clathrin-assembly protein AP-2 binding to the C2B
domain(22, 23) .
Mutational analysis of the
IP binding domain of GST-STII-C2B clearly indicated that
the numbers and positions of the positively charged amino acids,
especially Lys at positions 327, 328, and 332, were responsible for
high affinity IP
binding. These three Lys residues were
conserved in the C2B but not in the C2A domain. Based on the recently
reported three-dimensional structure of the C2A domain, these Lys
residues are located in the
4 strand of the C2 key(28) .
However, more detailed structural analysis of the C2B domain will be
required to state the IP
bound form of the C2B domain.
The amino acid sequences of the central region of the C2B domain are
highly conserved among different species (4, 5, 24, 25) as well as isoforms (Fig. 5B). Nevertheless, synaptotagmin III cannot bind
IP. Chimeric and mutational analysis suggested that the
putative IP
binding domain of synaptotagmin III (amino acid
positions 470-501) has the potential to bind IP
, but
one-third of the C-terminal of the C2B domain prevented IP
from binding to this synaptotagmin. The meaning of this
functional difference in IP
binding to the synaptotagmin
family is not clear. However, it is possible that inositol
polyphosphate cannot modulate synaptotagmin III-protein interaction,
such as C2B-AP2 binding, which may be involved in synaptic vesicle
endocytosis(22, 23) .
On the basis of the available
data, we propose that multiple isoforms of synaptotagmins do not always
have the same roles in synaptic function. First of all, synaptotagmins
I and II are thought to be functionally equivalent because of their
high degree of sequence similarities (above 87% identity in the C2
domain), the conserved neurexin binding sequence, which was not in
synaptotagmins III and IV (22, 26) , and complementary
distribution(22, 27) . Second, synaptotagmin III is
distinguished from other isoforms (I, II, and IV) in respect of the
lack of inositol polyphosphate binding to the C2B domain and the unique
structural feature, namely, the insertion of about 150 amino acids
between the transmembrane and the C2 domains (Fig. 5A and (12) ). Third, the C2A domain of synaptotagmin IV
lacked Ca-dependent phospholipid binding properties
when liposomes consisting of phosphatidylserine and phosphatidylcholine
(1:2.5, w/w) were used(22) . Furthermore, the protein
interaction of synaptotagmin IV may be different from that of
synaptotagmins I and II based on the recent observation that a novel
protein that specifically interacts with synaptotagmin IV but not with
synaptotagmins I and II was isolated by a yeast two-hybrid system (29) .
In summary, we first identified the inositol polyphosphate binding site by mutational analysis of the C2B domain of synaptotagmin II and found that the C2B domains of multiple synaptotagmins are functionally different with respect to IHPS binding. Our findings suggest that the multiple synaptotagmins may have different roles in presynaptic function.