W .E. Zimmer et al. Brain Research 881 2000 18 –27
21
was diluted directly into the intercellular solution. Data were included for analysis provided that the holding
current remained stable and while the number of observa- tions within each experiment varied, all groups contained a
minimum of four individual experiments.
2.6. Antibody preparation The synapsin I [20] and spectrin amino terminal [4,30]
antibodies have been previously described by our labora- tory. The synapsin binding domain antibody used in this
study was produced in rabbits by injection of the peptide NH2-AFNALIHKHRPDLID-COOH
which represents
amino acids 207 through 221 of the bSpIIS1 molecule. IgG fractions were purified from pre-immune or immun-
ized rabbits using a commercial Fab purification kit Pierce Chemical Co., Rockford, IL. Protein concentrations were
determined by absorbance at 280 nm and aliquots 500 mg were lyophilized and stored at 2808C until use. IgGs were
dissolved as stock solutions 2 mg ml in intracellular solution and then further diluted just prior to experimenta-
tion.
3. Results
3.1. bSpIIS1 synapsin binding domain is located near the actin binding domain of the molecule
We have previously elucidated the primary structure of mouse bSpIIS1. In these studies we noted a region of the
molecule adjacent to the actin binding domain that shared 87 identity with human and mouse bSpIS1 proteins [23].
This segment included amino acids 207 to 445 of the b-spectrins. Given the high degree of homology and the
location of this region proximal to the actin binding domain, we proposed that this segment may contain the
synapsin I binding site of the molecule. To test this hypothesis, we generated baculoviral expression vectors
Fig. 1. bSpIIS1 1–457 is expressed as a fusion protein in baculoviral-
containing the amino-terminal segment of bSpIIS1. Using
infected cells. The amino terminal 457 residues of the bSpIIS1 protein were expressed in a baculovirus system as described in Materials and
PCR we generated a cDNA fragment encompassing the
457
Methods. The spectrin peptide was expressed as a fusion protein
ATG methionine-initiation
codon through Val
of
containing a histidine tag-His — at the extreme amino terminus. Crude
6
bSpIIS1 and cloned this fragment into a baculovirus
extracts and fractions obtained by affinity chromatography were analyzed
shuttle plasmid, in frame with a His-tag [His ]. As shown
by SDS–PAGE Coomassie and Western analyses Western blot using
6
in Fig. 1, lysates from Sf21 insect cells infected with virus
an anti-bSpIIS1 peptide specific antibody, Ab 921. The arrows indicate the 58 KDa recombinant His-tagged bSpIIS1 1–457 protein.
derived from the spectrin shuttle plasmid exhibited marked expression of a |58 KDa peptide crude extract. This |58
KDa peptide remained bound to a nickel-agarose affinity binding to the |58 KDa peptide found in crude extracts of
column Ni-NTA resin, and was eluted from the column infected SF21 cells and the protein purified on nickel-
with buffer containing 100 mM imidazole 100 mM agarose columns Ni-NTA affinity chromatography. Thus,
Imidazole. To confirm that |58 KDa peptide was the these data establish that we have expressed and purified an
expected bSpIIS1 amino terminal domain, we performed amino terminal segment of the bSpIIS1 molecule consist-
Western blotting analyses utilizing a peptide specific ing of the actin binding domain [18] and the putative
antibody directed against amino acids 207 to 221 of the synapsin binding domain [23].
molecule. As shown by the Western blot in Fig. 1, this We next examined the interaction of synapsin I with our
spectrin antibody, termed Ab 921, demonstrated specific bSpIIS1 amino terminal peptide. For these experiments we
22 W
adapted a synapsin blotting assay originally described by Iga et al. [15]. In this assay, baculovirus-infected cells
expressing bSpIS1–457, in addition to purified fusion protein, and lysates prepared from erythroid and neural
tissues were fractionated by SDS–PAGE and transferred to nitrocellulose. The filters were incubated with synapsin I,
isolated from brain tissue, under conditions which allow the binding of synapsin to proteins on the filter paper.
Bound synapsin is then visualized using a synapsin-spe- cific antibody. Consistent with our earlier studies and those
reported by others [1,12,20,26,29,35], synapsin I exhibits a high degree of non-specific interactions by virtue of its
highly charged basic characteristic. At a concentration of 1.5 mg ml as used by Iga et al. [15] synapsin I exhibits
non-specific binding to proteins of RBC-membranes as well as standard molecular weight protein controls data
not shown. However under these conditions, we also detect binding to the bSpII 1–457, 58 KDa peptide. Thus,
we modified the assay to improve the specificity of synapsin I binding. We found that specific binding
occurred quickly, such that after |30–45 min non-specific interactions began to occur raising the background over
specific binding, and that non-specific interactions are effectively competed with the addition of other highly
charged, basic proteins like histones data not shown. In addition, we found that reducing the amount of synapsin I
in the incubation buffer by |100 fold significantly reduced background interactions thereby enhancing the specificity
of binding. To demonstrate the ability of the synapsin blot assay, we utilized it on membranes containing our ex-
pressed bSpII 1–457 protein and spectrins from red blood cell and brain tissue Fig. 2A. As shown by this assay,
synapsin I demonstrates specific binding with the beta subunit of brain spectrin and to our baculoviral expressed,
amino terminal segment of bSpIIS1. Therefore, the synap- sin I binding site on brain spectrin resides upon the
beta-subunit and is within the amino terminal |450 amino acids of this protein.
3.2. Synapsin binds bSpIIS1 to a region encompassing amino acid residues
211 –235
Fig. 2. Specificity of the synapsin I blotting analysis. A qualitative analysis of synapsin I binding was developed using a modification of the
The demonstration of synapsin I binding to the beta
blotting technique described by Iga et al. [15]. Protein to be examined
subunit of brain spectrin near the actin binding domain is
were separated by SDS–PAGE and transferred to nitrocellulose mem-
consistent with our earlier studies utilizing by low angle
branes. The membranes were incubated in buffer containing synapsin I
rotary shadowing electron microscopy [20]. Since, synap-
after which the blots were rinsed and the bound synapsin fixed to the protein on the membrane with 4 paraformaldehyde. The synapsin I was
sin I is the major phosphoprotein on the membrane of
then localized on the blot through Western blotting using a rabbit
synaptic vesicles [7,12,28], its interaction with the beta
125
synapsin I specific antibody and I-Protein A. Panel A shows a
subunit of brain spectrin, specifically bSpIIS1, may play a
Coomassie stained gel of molecular weight markers standards, the |58
key role in the release of neurotransmitters. To more
KDa fusion protein bSpII 1–457, red blood cell ghosts RBC and
precisely localize the bSpIIS1 synapsin I binding site, we
purified brain spectrin Brain Spectrin. Proteins from a parallel gel were transferred to nitrocellulose membranes and incubated in buffer con-
expressed the bSpIIS1 1–457 peptide and COOH-terminal
taining 0.015 mg ml synapsin I after which the bound synapsin I was
truncations of the peptide as GST-fusion proteins in
localized by Western blotting and autoradiography as detailed in Materials
bacteria. Using PCR we cloned the |1.3 Kb bSpIIS1
and Methods Panel B. The arrows point to the positions of proteins that
1–457 coding sequence in frame with a glutathione-S-
specifically bind synapsin I, the bSpIIS1 1–457 fusion protein and the
transferase GST sequence and isolated the expressed
beta-subunit of brain spectrin.
W .E. Zimmer et al. Brain Research 881 2000 18 –27
23
proteins by thio-agarose affinity chromatography. Recent proteins to bind brain synapsin I in blotting assays. GST-
experiments from our laboratory [30] demonstrated that spectrin fusion proteins containing the original 457 amino
peptide specific antibodies directed to the C-terminal acids specifically bound synapsin I, whereas no binding
segment of the putative synapsin binding site amino acids was observed using GST alone, further demonstrating that
417–428 disrupted synaptic transmission. Therefore we synapsin I binds the amino terminal domain of bSpIIS1.
formed multiple bSpIIS1 peptides that are COOH-trunca- Moreover, these data suggest that eukaryotic post-transla-
tions starting from the initial synapsin binding V 457 tional modifications of bSpIIS1 are not critical for synap-
peptide. Fig. 3A summarizes our strategy which truncates sin I binding. In addition, examination of synapsin blots
peptides by |25 amino acids through the potential synap- such as the one illustrated in Fig. 3B showed that all but
sin I binding domain. Each of the bacterially expressed the shortest of the bacterially derived proteins bound
bSpIIS1 fragments were successfully expressed and synapsin I compare Western and synapsin blotting of
purified data not shown. Moreover, Western analyses of bSpII-A210 with adjacent lanes. These qualitative data
the expressed proteins using an antibody generated to the suggest that the exact synapsin binding region of the
amino terminus of bSpIIS1, within the actin-binding bSpIIS1 molecule is within a small domain of the mole-
domain amino acid residues 8–24, detects bacterially cule between amino acids L211 and Q 235.
derived spectrin peptides of the correct molecular weight Fig. 3B. The |30 KDa difference in apparent molecular
3.3. Synaptic vesicle binding capability of bSpIIS1 is weight of the bacterially synthesized bSpIIS1 fragment
conferred by amino acids A 210 through Q235
containing amino acids 1–457 and that of the original bSpII 1–457 baculoviral recombinant protein Figs. 1 and
Blotting experiments Fig. 3 suggest that synapsin I 2 accounts for the addition of GST to the bacterial
binding is mediated primarily through a |25 amino acid proteins.
segment of the bSpIIS1 protein bounded by L211 and We next examined the ability of the GST-spectrin fusion
Q235. As synapsin I has been implicated to play a major
Fig. 3. Localization of the synapsin I binding site on bSpIIS1. The localization of the synapsin I binding site was accomplished using bacterially synthesized truncations of the bSpII 1–457 amino terminus and the synapsin I blotting technique. Panel A diagrams the 10 bSpII amino terminal peptides
expressed in bacteria. Each successive bacterial peptide was truncated by |25 amino acids so that with the 10 fusion proteins we could examine binding throughout the entire putative synapsin I binding domain. Each coding sequence was developed by PCR and cloned into a bacterial vector such that they
would be in frame with a GST coding sequence. Panel B shows that proteins recognized by an antibody directed to an amino terminal segment of bSpIIS1 amino acid residues 8–24 also demonstrated synapsin I binding except for the bSpII A210 protein. Proteins recognized by the antibody Ab 43, [30] are
shown with the brackets while the protein which specifically bind synapsin I are denoted by the asterisks. As shown in the top panel, the bSpII A210 fusion protein was recognized by the bSpIIS1 antibody but was found not to bind synapsin I lower panel.
24 W
Table 1
role in linking synaptic vesicles to brain spectrin [28,29],
Binding affinity and maximal binding capacity of synaptic vesicle —
we sought to quantitatively examine the binding of our
spectrin fusion protein interactions
bacterially expressed GST-spectrin fusion proteins with
V457 Q235
A210
isolated synaptic vesicles. We focused upon the two fusion
a
proteins which defined synapsin I binding by blotting
Binding K affinity 5 nM
19 nM 180 nM
D b
Binding capacity 4.0
1.1 6.6
analyses A210, Q235. Recombinant fusion proteins were
a
purified by affinity chromatography inset, Fig. 4B and
Calculated by least squares analyses of binding isotherms using
125 125
ENZFITTER computer program.
labeled with I using the Bolton–Hunter reagent.
I-
b
Values reflect mg recombinant protein mg vesicle total protein.
labeled recombinant spectrin was incubated with small synaptic vesicles [14,28,29] and vesicle-associated spectrin
separated from unbound proteins by differential centrifuga- binding capacity of these two fusion proteins with synaptic
tion. The binding isotherms, for spectrin-vesicle binding, vesicles Table 1, indicating a difference in the binding
were calculated using an ENZFITTER computer program sites upon the vesicles for the recombinant Q235 and A210
R.J. Leutherburrow, Biosoft, Inc., Milltown, NJ. Full- spectrin fusion polypeptides.
length V-457 and truncated bSpIIS1 Q235 gave compar- able levels of saturable binding peptide K
of 5 nM and 3.4. Peptide specific antibodies to the 25 amino acid
D
19 nM, respectively. These values agree well with pub- bSpIIS1 synapsin I binding site block synaptic
lished values of synaptic vesicle–spectrin interaction 24 transmission in living cells
nM; [28] and synapsin I–spectrin interactions 9 nM;
27
[15]. This contrasts with the value of 180 nM 1.8310 Following localization of the synapsin I binding domain
M obtained with the bSpIIS1 peptide truncated at amino to amino acids 211 through 235 of the bSpIIS1 molecule
acid A210 Table 1. Thus, removal of 25 amino acids, Fig. 3 and the demonstration that these amino acids
between L211 to Q235, from the bacterial bSpIIS1 comprised the attachment site for synaptic vesicles Fig.
peptides causes a 10-fold reduction in synaptic vesicle 4, we next examined the function of this region in vivo.
binding, indicating that the major binding domain of We generated a peptide specific antibody to residues 207 to
bSpIIS1 for synapsin I synaptic vesicles lies between 221 which spans the actual synapsin I attachment site. This
these residues. Further, there is a change in the calculated antibody, referred to as Ab 921, was used to confirm the
Fig. 4. Quantitative analysis of bSpII-GST fusion peptides with synaptic vesicles. Binding of the bSpII-GST fusion proteins with isolated small synaptic vesicles was analyzed by a differential centrifugation assay. The fusion proteins were purified by chromatography upon a glutathione agarose affinity
125 125
column and the purified proteins labeled with I using the Bolton–Hunter reaction. Various amounts of the
I labeled proteins were incubated with the synaptic vesicles and then the reaction mixtures centrifuged at 200,0003g for 30 min. The supernatant and pellet were analyzed for labeled peptide and the
Bound pellet versus Free supernatant data plotted. From these data a K binding affinity and binding capacity were derived using the ENZFITTER
D
computer program. Panel A shows the plot of demonstrated binding with the full length bSpII-V457 protein filled circles and GST alone controls open circles, while panel B illustrated the data obtained with the bSpII Q235 and bSpII A210 peptides. The inset shows an SDS–PAGE of the purified proteins
used in this analysis.
W .E. Zimmer et al. Brain Research 881 2000 18 –27
25
expression of the amino terminal domain in the baculo- were averaged and normalized to the initial maximal
virus system Fig. 1 thus demonstrating the antibody response. The decline of ESPCs developed rapidly and
specificity. We reasoned that injection of Ab 921 should maximal suppression was observed after approximately 45
occlude the brain spectrin–synapsin I binding site, thus min. Injection of Ab 921 at 70 mg ml caused a more rapid
reducing neurotransmission, if this bSpIIS1 segment was suppression of ESPCs amplitude which reached maximal
functionally important for this process. Therefore we effect in about 25 min Fig. 5C. Therefore, these data
examined the effects of Ab 921 in cultured hippocampal illustrate that occlusion of the brain spectrin–synapsin I
neurons in which the presynaptic neuron of synaptically interaction causes a specific reduction of synaptic transmis-
paired cultured rat hippocampal neurons was injected with sion in coupled hippocampal neurons. The inhibition of
different concentrations of Ab 921 IgG, whereas control synaptic transmission is site specific on bSpIIS1 because
cells were injected with a similar concentration of rabbit peptide specific antibodies against residues 8–24 Ab 43
pre-immune IgG. The consequence upon synaptic trans- and 581–597 Ab 49 have no effect on synaptic transmis-
mission of infusing Ab 921 was measured in the amplitude sion [30].
of excitatory postsynaptic currents ESPCs recorded from a synaptically-coupled postsynaptic neuron. Fig. 5A dia-
grammatically summarizes our experimental protocol. As
4. Discussion
