W .E. Zimmer et al. Brain Research 881 2000 18 –27
19
the presynaptic terminal consistent with a key role in by DNA sequencing, this shuttle plasmid was used to
neurotransmission. Biochemical and molecular studies also make a viable baculovirus encoding the 6His-bSpIIS1
support a role for spectrin in regulating neurotransmitter fusion protein using the Bac-to-Bac
E kit as indicated by release. Brain spectrin binds end-on to small synaptic
the supplier Gibco BRL, Gaithersburg, MD. The pres- vesicles via synapsin I [28,29], a major phosphoprotein of
ence of the fusion protein in Sf 21 cells infected with the synaptic vesicle membrane which regulates the availa-
bSpII 1–457 virus was confirmed in all lysates by Western bility of synaptic vesicles during synaptic transmission
analysis using both a His-tag antibody Pharmacia Biotech, [7,10]. That this binding occurs close to the brain spectrin
Chicago, IL and one of several bSpIIS1 peptide specific actin binding domain was demonstrated by low angle
antibodies. rotary shadowing electron microscopy [20].
For protein purification, Sf 21 cells were infected with The interaction of brain spectrin with synapsin I appears
bSpII 1–457 virus 15, T 150 flasks and the cells similar to that observed between protein 4.1 and erythroid
incubated at 288C for 72–96 h. The cells were collected by spectrin in red blood cells. For example, protein 4.1 and
centrifugation and then lysed in buffer containing 50 mM synapsin I bind directly to the beta subunit of the spectrin
Tris–HCl pH 8.5, 5 mM 2-mercaptoethanol, 100 mM protein in red blood cells and neural tissues, respectively
KCl, 1 mM PMSF, 1 Nonidet P-40 at 48C by vortexing. [5,15]. Moreover, protein 4.1 can competitively inhibit the
Cell debris was removed by centrifugation 10,0003g for binding of synapsin to brain spectrin and synapsin I can
10 min and the supernatant loaded onto a Ni-NTA column. inhibit protein 4.1 binding to the red blood cell spectrin
The column was washed with 2 to 3 column volumes of aSpISI bSpISI [2,20]. Comparison of the predicted amino
buffer containing 50 mM Tris–HCl pH 8.0, 5 mM acid sequence of bSpIIS1with its erythroid counterpart
2-mercaptoethanol, 100 mM KCl, 10 glycerol and 3 mM detected a region adjacent to the actin binding domain that
Imidazole, after which bound proteins were eluted in the was 87 identical between these isoforms. We predicted
same buffer containing 100, 200, and 300 mM Imidazole. that this region spanning amino acid residues 207–445 of
The eluted fractions were examined by SDS–PAGE and the b molecule maybe a potential synapsin I binding
Western blotting as described previously [4,23,34]. domain of brain b spectrin bSpIIS1 [23]. In this report, we
A bacterial vector containing the glutathione-S-transfer- have used recombinant peptides to identify the exact site of
ase GST gene was used for making recombinant GST- synapsin I–bSpIISI interaction. Our results demonstrate
fusion proteins. The bSpIIS1 cDNA served as a template that amino acids residues 211 through 235 of the b-
to clone the segment of DNA encoding amino acids 1–457 molecule are essential for synapsin I binding. Further, we
into the pGEX-5 GST-fusion protein vector Pharmacia demonstrate that a peptide specific antibody against this
Biotech, Chicago, IL. This placed the GST coding se- region of bSpIISI inhibits synaptic transmission in patch
quence at the amino terminus of the bSpIIS1 sequence. clamp studies of paired hippocampal neurons. These
The plasmid was transferred into BL 21 bacteria and results indicate that interaction of synaptic vesicles with
grown at 378C in Luria Broth LB supplemented with 75 these 25 amino acids within brain spectrin bSpIISI is
mg ml ampicillin and 0.2 glucose. To make truncated essential for neurotransmission.
proteins, PCR fragments with a common 59 end, beginning
431
at the ATG initiation codon, and ending with codons A ,
406 381
355 331
306 285
259 235
210
R , Y
, 12 , A
, K , V
, D , Q
, and A were cloned into the same GST vector. Each vector was
2. Materials and methods confirmed by DNA sequence analysis and then transferred
into BL 21 bacteria for generation of recombinant proteins. 2.1. Recombinant protein expression and purification
One hundred microliters of LB media containing 75 mg ml ampicillin was inoculated with bacteria housing
The initial recombinant protein used in this study was plasmids for the GST-fusion proteins and the bacteria
generated using the Bac-to-Bac E Baculovirus System
grown in a 378C shaker-incubator until an A of 0.4–0.6
600
Gibco BRL, Gaithersburg, MD. bSpIIS1 cDNA clone was obtained. IPTG was then added final concentration of
14T3-1 containing the 5 UTR and |2 Kb of coding 0.5 mM and the cultures incubated for an additional
sequence [23] was used as a template for PCR amplifying 24–48 h. The cells were collected by centrifugation,
the coding segment beginning with the translation initia- resuspended in 13PBS phosphate-buffered saline con-
1 457
tion codon Met and extending through the GTT, Val taining Triton X-100 2. Cells were lysed by sonication
codon. The amplified product contained synthetic Eco RI and cell debris removed by centrifugation. GST-fusion
and Hind III restriction sites at the 59 and 39 ends of the proteins were then enriched from the lysates using a spin
fragment, respectively. This fragment was cloned into like column of Gluthionine Sepharose 4B as indicated by the
cut pFAST BAC HTa Gibco BRL, Gaithersburg, MD supplier Gibco BRL, Gaithersburg, MD. Some proteins
which placed the bSpIIS1 sequence in frame with a 63 were further purified by again loading the enriched fraction
histidine amino acid tag. The 63His tag was placed at the onto a Gluthionine-Sepharose 4B column and elution with
amino terminus of the bSpIIS1 segment. After verification reduced glutathione 10 mM final concentration. Protein
20 W
purity was estimated by SDS–PAGE followed by staining studies was previously characterized [20], and was used in
with Coomassie Blue as previously described [4,23]. the present study at dilution of 1:1000.
We developed an in vitro assay based upon the ability to remove synaptic vesicles from a mixture by differential
2.2. Brain spectrin and synapsin I isolation centrifugation. Differences in sedimentation properties
between synaptic vesicle and intact spectrin are small, thus Bovine brains were obtained from a local slaughterhouse
an assay based upon centrifugation was not feasible. and stored at 2708C until used to isolate spectrin. Brain
However, we reasoned that a peptide representing |15 of spectrin was purified from the frozen tissue as detailed
the beta subunit might allow separation, and in preliminary previously [29]. Synapsin I was isolated from frozen pig
experiments we found that .98 of vesicles were found brain tissue as detailed in previous studies from our
in the pellet of a 200,0003g centrifugation for 30 min laboratory [20,29]. Protein purity was assessed by SDS–
while 100 of the bSpIIV457 |80 KDa bacterial fusion PAGE and Coomassie Blue staining of the polyacrylamide
protein remained in solution under the same conditions. gels. The synapsin I was followed by analysis on Western
For binding analyses, the bacterially expressed bSpII blots. Red blood cell RBC membrane proteins were from
125
peptides were labeled with I using the Bolton–Hunter
isolated ghosts as described previously [27]. reagent. Typical binding assays were accomplished in a
200 ml volume of buffer containing 5 mM Tris–HCl pH 2.3. Synapsin I blotting assay
7.5, 65 mM NaCl, 1 mM EGTA, 0.2 mM DTT, 20 mg ml PMSF and 4 mg of synaptic vesicle protein. Binding was
125
To identify the attachment site of synapsin I on initiated by the addition of increasing quantities of
I- bSpIIS1, we modified the blotting technique of Iga et al.
labeled peptide and after a 1 h incubation at room [15]. Proteins to be analyzed were separated by SDS–
temperature 228C the reaction loaded onto a Ti 42.2 rotor PAGE and then electrophoretically transferred to a nitro-
Beckman Instruments
and spun
at 35,000
r.p.m. cellulose sheet as described previously [4,28,29]. The
200,0003g for 30 min at 48C. The pellet and supernatant
125
nitrocellulose membrane was then blocked by incubation were carefully separated and the amount of
I in each in buffer containing 5 BSA, 1 Triton X-100, 50 mM
determined using a gamma counter Packard Autogamma KCl, 1 mM EDTA and 20 mM HEPES pH 7.4 for 12–16
50DC; Packard Instruments Company, Meridan, CT. We h at 48C. The blot was washed 3–5 times with the same
initially determined that the kinetics of binding was rapid, buffer and then incubated in this buffer to which purified
reaching equilibrium in |1–5 min. We thus chose to synapsin I had been added. Early experiments contained
incubate our binding reactions for 1 h to ensure complete- 1.5 mg ml synapsin I [15], but we demonstrate that higher
ness of the binding reaction. All reactions were done in fidelity of the assay was obtained with lower concen-
triplicate and controls consisted of no added peptide or trations, 0.015 mg ml. The synapsin I incubation was
vesicles and using BSA instead of the bSpIIS1 peptides. allowed to continue for 1 h at room temperature, after
The amount of bound peptide pellet was plotted versus which the blot was washed with buffer without synapsin
the amount of free peptide supernatant and from these 5–7 times 30 min each wash with constant agitation. The
data the K binding affinity and maximal binding
D
nitrocellulose was then incubated in 4 paraformaldehyde capacity were determined using the ENZFITTER computer
for 30 min at room temperature and the excess paraformal- program as described previously [28,29].
dehyde removed by 3–5 washes in Western blotting buffer 0.9 NaCl, 0.05 Tween 20, and 10 mM Tris–HCl, pH
2.5. Whole-cell patch clamp recording 7.4. The blot was processed for Western blotting using
rabbit antisynapsin I antibody as described previously [29] Excitatory postsynaptic currents were followed in paired
125
and localization of the synapsin I antibody using I-
hippocampal neurons essentially as previously described
125
protein A New England Nuclear, Boston, MA. I- was
by our laboratory [30]. Briefly, hippocampal neurons localized by autoradiography using either X-ray film
cultured from neonatal rats postnatal day 2–3 were Kodak AR5 or by image analysis on a BioRad Phos-
bathed in extracellular recording solution 119 mM NaCl, phoimager.
5 mM KCl, 20 mM HEPES, 2 mM CaCl , 2 mM MgCl ,
2 2
30 mM glucose, 1 mM glycerine, 100 mM picrotoxin, pH 2.4. Isolation of small synaptic vesicles and binding
7.3, osmolality adjusted to 330 mOs with sucrose and analyses
communicating cells sealed with patch clamp recording electrodes and maintained at a holding potential of 270
Small synaptic vesicles were isolated essentially as mV. ESPCs were evoked in the presynaptic cell and
described previously [29]. Fractions from the controlled recordings were obtained from the postsynaptic cell using
pore glass bead column chromatography were examined the protocol previously described by our laboratory [30].
for synapsin I using a quantitative immunobinding assay as The presynaptic neuron pipette contained either bSpII
described by Jahn et al. [17]. The antibody used in these peptide IgG or control antibody pre-immune IgG which
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
