Biochim~caet BiophysicaAcl"a.1077(1991) 119-126 © 1991 ElsevierSciencePublishersB.V.0167-4838/91/$03.50 ADONIS 016748389100131N
119
BBAPRO33856
Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E Ba! R a m S i n g h *, J u a n A . G i m ~ n e z a n d B i b h u t i R . D a s G u p t a FoodResearchInstitute, Universityof Wisconsin,Madison, WI (U.S.A.)
(Received21 May 1990) (Revisedmanuscriptreceived12 October 1990)
Key words: Botulinumneurotoxin:Circulardichroism:Derivativespectroscopy-(C. bmulinum); (C butyricurn) Production of ~ l ~ k e nemotox3n by a non-CIostridium bomllnmn organism has prdoumi implications in the epideminlo~ of the disease botulism. Molecular topography of the = 150 kDa neterotoxie protein produced by Closuidium Imtyrienm (strain 5839) and its activation kinetics were examined and compared with a s e r o l o ~ y related botulinmn nemotoxln p~dluced by ¢~ botu/~um type E to further characterize the butyrlcum nemotoxin. Bo~l|num mmrotox~ was fully activated within 30 min of i n c u h a t ~ with trypsin, whereas b~.rlcum ~ r o t o x i n achieved ~ acfivafiou within 5 rain of incubafiou. Molecular topography of the two neurotoxins was analyzed in terms of secondary s m ~ w e s and the surface accessH~fities of the p o l y ~ domains containing aromatic amino ~ The secondary stmctm-e parameters of the but}xicmn nemotoxin (a-halix 22%, ~ s h e e t 41% and random coil 37%), as estimated from the far ullra~io]et circular dichroic spectra, appeared siraHar to that of botulinum neurotoxin. (Singk, B.IL and ~ B.R., (1989) M o t Ceil Biochem. 86, 87). Second derivative ultravin~t spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricmn and botulinum neurotoxius, respectivdy, suggesting a differential surface ~ of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the b o h d | m u type E and Ibutyrlenm nem'otoxins were in a relatively hydrophobic environment as indicated by the blue-shlfted emisslou maxima (334 ran). About half of rite fluorescent Trip residues of both proteins were aecesslble to acrylamide, a neutral ~ queneh~, and appeared to be in a similar molecular environment. The ionic surface probe, ! - , quendaed the Tip fluorescence of bot~ilmnn significantly, but not that of butyricmn neurotoxin. Thus, a consideraCie n m n l ~ of fluorescent Trp residues were apparently located on the surface of the botulinmn, bet not on of m e ~ - r i c u m neurotoxin. Boudinum and butyricum neurotoxias, i n d ~ b ~ S s ~ t e by pobacryhnicte g d in rite presence of sodium dedecyl s~lfate, migrated differently in the absence of sodium dedecyl stdfate suggesting ~ f e r e n c ~ s ) in their surface charge distn'lmtinn. These results provide the first report of the secondary and tertiary stmebJ-e parameters of tha nemmoxiq In'O~ ~x~d by a nou-botulinum species and eomparlson of tha molecular t e p o g r a ~ of the nemetoxln with the antlgenlcally related bogdlnum neurotoxln type E.
Introduction Botulinum ncurotoxin causes the disease botulism. Seven distinct serotypes (A through G) of the neurotoxin, produced by different strains of Clostridium botulinum, have similar pharmacological action on neuromuscular junctions [1,2]. Some strains of C bu~ricum were recently found responsible for infant botulism * Present address: Department of Chemistry, Southeastern MassachusettsUniversity,North Dartmouth, MA. U.S.A. Corresp~dence: B.R. DasGupta,Food ResearchInstitute, Uni~'sity of Wisconsin,1925WillowDrive. Madison.Wl 53706. U.S-A.
cases. The neurotoxin produced by C butyricum was neutralized by the antiserum raised against the type E neurotoxin produced by C. bomlinum [3,4]. Immunodiffusion tests of the neurotcxlns from these two sources with antiserum raised against C. botulinum type E neurotoxin showed that at least one epitope present in ¢2. botulinum neurotoxin was absent in C. butyricum [5]. Another difference was in their specific toxicities; based on intravenous mouse assay the neurotoxins from C. botulinum and C. butyricum were 6-107 vs. 20-107 LDs0/mg protein, respectively [5]. Expression of the botulinum neurotoxin gene by a non-C, botulinum organism could have profound impfications in the epidemiology of botulism. Therefore, it is important to
120 compare the neurotoxins produced by C botulinum and C bu~ricum, at the molecular level. Out of the total = 1300 amino acid residues expected in each of the = 150 kDa proteins, 68 and 71 residues in the two neurotoxins have been identified [6]. A comparison of the partial amino acid sequences shows only three non-identical residues in their primary structures. No other structural comparison has been made for these two neurotoxins. In this study, we analyzed the secondary structures of the neurotoxins from C. botulinum type E strains 43 and 5545 and C. butyr/cum (strain 5839) u . ~ g eir~alar d/chroic (CD) spectroscopy. In order to examine the polypeptide foldings, (i) degrees of tyrosine exposure to the aqueous solvent were determined using second derivative ultraviolet spectroscopy and (ii) microenviruuments of tryptophan residues were probed using fluorescence quenching techrdque. The results suggest that while the secondary sUructures of the neurotoxins from C. botulinum and C. butyricum remained the same, the polypeptide foldings appear to differ s/gnificantly. Mmerials and Me~mds Neurotoxin preparations. C botulinum type E strain 43 was kindly supplied by Dr. Lynn S. Siegel, Fort Derrick, Frederic, MD. C. botulinwn type E strain 554"5 and C. butyricum strain 5839 were kindly provided by Dr. Charles L. Hatheway, Centers for Disease Control, Adanta, GA. The organisms were grown and the neurotoxin purified from the bacterial culture as previously described [7]. The pure neurotoxin preparations (single band in polyaerylam/de gel electrophoresis in the presenee of sodium dodecyl sulfate (SDS)), stored at 4 ° C as precipitated proteins (39 g ammonium sulfate/100 n'd), were recovered by centrifugation. After dissolving in 10 m M sodium phosphate buffer (NaPB) (pH 6.0) the neurotoxin was dialyzed extensively against the same buffer and filtered through a 0.2 /am Acrodisc fdter (Gelman Sciences) before optical measurements. Protein concentrations were determined as described before
tions of pure neurotoxin detoxified with 0.5% formaldehyde [10]. Polyactylamide gel electrophoresis without a denaturant (nati~-PAGE). The electrophoretic mobilities of neuromxins in the absence of SDS or urea were determined using the PhastSystem (Pharmacia, Piscataway, N-D. Protein samples (---0.5 m g / m l in 0.05 M Tris-HC! buffer, pH 8.5) were loaded on the gel with a Sample Applicator 8 / 1 (8 samples, 1 ~! each). Phastgel Gradient 8-25% and Phastgel Native Buffer strips were used. Circular ~chroisr~ Far UV-CD spectra were recorded be~,een 190 and 240 n m on a JASCO J2OA C D / O R D spectropdlarimeter at room temperature ( 2 3 - 2 5 ° Q . The chart speed used was 2 c m / m i n with a wavelength expansion of 10 n m / c m . The slit-width and time constants were fixed at 1 n m and 4 s, respectively. The spectra were recorded with 1 nun pathlength quartz cuvette using protein concentrations of 0.14-0.16 m g / m L Spectra] recordings of each protein were made in tr/plicate. Mean residue weight ell/ptieities were calculated at every n m between 190 and 240 nm. Mean residue weight of 114.49, derived from the published amino acid composition of type E botulinum neurotoxin [11], was used for the neurotoxins isolated from the three sources. Secondary structure parameters were caleu|ated according to Chang et at. [12] using a program provided by Drs. C-SC. Wn and J.T. Yang, University of California, San FrancLseo. In this procedure secondary structures are derived from a linear leastsquare analysis of the reference C D spectra of 15 proteins of known X-ray crystaUographic structure [12]. Absorption and second derivative ultraviolet spectroscopy. Absorption and second derivative spectra were recorded on an Uvikon 860 (Kontron Instruments) at room temperature ( 2 3 - 2 5 0 0 . The degree of the exposure of tyrosine residues to solvent ( a ) was estimated according to Ragone et al. [13] using the equation: a=
¥°-¥~ "r.Y~×tO0
[81.
Toxici~ determination. The neurotoxins (20/~g/60 pl 50 m M NaPB, pH 6.0) were mixed with trypsin (10 pl, neurotoxin:trypsin was 15:1, w/w). After 5, 15, 30 and 60 rain of incubation at 35°C, 15/xi aliquots were withdrawn and mixed with 15 /~I of soybean trypsin inhibitor (3 × more than trypsin, w/w). These samples were immediately diluted 10-fold with 60 m M NaPB (pH 6.2) containing 0.2% gelatin and injected into the tall vein of mice. Lethality (LDso/ral) was determined from the time to death method [9]. lmmunodiffusion test. Preparation and composition of the gels and development of immunoprecipitate fines were previously described [5]. The antineurotoxin sel~ were raised in rabbits by multiple subcutaneous injec-
where 3'. is the a / b ratio (see Fig. 6 for the notations) of the second derivative peaks of the native protein, 3", is the a / b ratio for the unfolded protein (in 6 M guanidine hydrochloride), Ya is a correction factor calculated on the basis of Tyr to Trp ratio for a given protein (see Table ! in Ref. 13). The T y r / T r p ratie determined by the procedure of Edelhoch [14] for butyricum and botulinum neurotoxins were 4.706 and 4.772, respectively. Mean of these two values, 4.739, was used for further calculations (see Table II). The number of Trp residues in the two neurotoxins were estimated by the spectroscopic procedures of Edelhoch [14] and also that of Servillo et at. [15]. Botulinum and butyricum neurotoxins were each found
121 to have 15 residues based on Edelhoeh's method. Earlier, using this method 16 residues were found in botulinum type F neurotoxin [11]. The number of Trp estimated by the method of Servillo et aL [15] were 20 (4-17%) and 18 (4-15%) for botullnum and butyrienm neurotoxins, respectively. The important poml that comes out by utiliTJng the two methods is that the Trp content in the two neurotoxins is virtually identical. Fluorescence spectroscopy. Fluorescence spectra were recorded on an SLM 8000 "Smart' spectrofluorometer at room temperature (23-25°C). The excitation wavelength of 295 nm was used to preferentially excite the Tip residues. To w ; n i m i T e t.he inner filter effect, the protein concentrations were used such that A295 remained ~ 0.05. For quenching experiments, stock solution (1-5 FI) of a quencher (KI or acrylamide) was added to a 700 tal protein solution in several steps (total --- 35 /d) and fluorescence recorded at the emission maximum. The fluorescence data recorded in triplicate were averaged and analyzed for Stern-Volmer plots [16]: -~= 1 + K~IQ] where Fo and F are the fluorescence intensities in the absence and presence of a given quencher concentration [Q], K~ is the Stcrn-Volmer quenching constanL The data were also analyzed using modified Stern-Volmer plots I171: F0
t
t
where F0 is the fluorescence intensity in the absence of a quencher, A F the difference in the fluorescence intensities due to the presence of a quencher, fa the fraction of the maximum accessible fluorescence and K o is the effective quenching constant. Most of the experiments were repeated with neurotoxin isolated from a separate batch of bacterial culture. Ultrapure guanidin~HC! (Schwarz/l~ann: Cleveland, OH), potassium iodide (Sj~rna; St. Louis, MO), Trypsin (L-l-tosylamide-2-phenylethyl chloromethyl ketone treated, activity of 268 U / r a g from Worthington, Freehold, NJ), soybean trypsin inhibitor (SBTI) (Sigma) and other chemicals were of highest quality available commercially. Buffers and solutions were prepared with deionized distilled water.
20"
El n c ~ t a ~/1~E o
q
~o
qx
0
-+ 0
5
lS
30
time
(~)
60
Fig. 1. Kinetics o f activation o f b o r u l i n u m type E a n d butyricum neurotoxins incubated with trypsin ( 1 5 : 1 , w / w ratio in 50 m M
NaPB, pH 6, 35°C). The lethality valu~ for botulinum type E and butyricum neorotoxins at time zero wen: 0.03-107 and 0.0"/.10~ LD~o/mg.respectively.
retained about 70% of the maximum toxicity for an additional 30 rain of incubation. In.contrast, butyricum neurotoxin was activated more rapidly; the maximum lethality was attained within 5 rain, but only 9~ of the activity remained after a total of 60 rain incubation with trypsin. lmmunodiffusion test. The antigenic relatedness betwecn botulinum and butyricum neurotoxins was evident from the spur formation in the immunoprecipitation lines (Fig. 2A and B). Similar observation was made earlier [5] using only the antibotulinum neurotoxin serum. Here we ha~e also used antibutyrieum neurotoxin serum to demonstrate that each neurotoxin has at least one epitope that is not present in the other protein. Native-PAGE. Electrophuresis of neurotoxins from C. botulinum and C. butyricum on a native (Le., without SDS) polyacrylamide gradient gel (8-25~) showed that
O
O
O
O
O
0
Results
Kinetics of activation. Fig. 1 shows changes in toxicities (LDs0/mg protein) of botulinum and butyricum neurotoxins as a function of incubation time in the presence of trypsin. Botulinum neurotoxin was activated to its highest lethality after 30 rain of digestion and
Fig. 2. Immunodiffusionassay of botulinum and butyricumneurotoxins. The central wells were charged with about 10 international units (1 unit neutralizes 1000 LD~) of type E botulinum(A) and butyricom(13)antitoxins. Wells, 1, 2 and 4 contained 6/tg of type E botulinumneurotoxinand wells3 and 5 contained6 Fg of butyricum neuroroxin.
122
f
o 0
J c: o
1[
18o
2oo
2~o
~;o
Wave'~ngth, nm
Fig. 4. Far ultraviok,t CD spectra of neurotoxins from C botulinma strain 5545 and from C but)r/cam strain 5839, dissolved in 10 mM NaPB (pH 6.0). The spectra were recorded at room temperature (23-25°C) using a l-ram pathlength quartz cuvene. Spectra are plotted afte~ respective baselines for 10 mM NaPB. pH 6.0. were subtracted. Un/ls of mean residue weight elfipficity on the y axis are degrees cm2/dmoL
123456 Fig. 3. Native polyacryhmidegradient (8-25%) gel electrophoresis of q,pe E botulinum (lanes 4, 5 and 6 containing 0.25, 0.5 and 0.75/Lg proteins, respectively) and buV2T/cum( h n ~ 1, 2 and 3 containing 0.25, 0.5 and 0.75 pg proteins, respectively) neurotoxins. The bands are sta/ned with Coomassie brilliant blue R-250.
at p H 8.5 the b o t u l i n u m n e u r o t o x i n m o v e d faster t h a n the b u t y r i c u m n e u r o t o x i n (Fig. 3), w h i c h indicates difference in their surface charges. T h e s e p r o t e i n p r e p a r a tions, t h a t m i g r a t e as single s h a r p b a n d s in S D S - P A G E [5,10,11] a n d a p p e a r h o m o g e n o u s b a s e d o n sequences o f N - t e r m i n a l residues [6,11], exhibited a s e c o n d a r y b a n d in the absence o f SDS. Circular dichroism- F a r ultraviolet C D s p e c t r a o f the n e u r o t o x i n s f r o m C. lvatyricum (Strain 5839) a n d f r o m C. botulinum (Strain 5545) a r e s h o w n in Fig. 4. E a c h s p e c t r u m s h o w s a shallow d o u b l e well s t r u c t u r e w i t h negative m a x i m a a t 217 + 1 n m a n d 207 + 1 n m , a n d negative m i n i m a at 212 + I n m . T h e m e a n residue elllptic/ty values were -:ery simAlar for n e u r o t o x i n s f r o m
C. botulinum strain 5545 and C. bu(yricum strain 5839. F o r e x a m p l e , a t 207 4-1 n m extremaL, the respective m e a n residue elliptic/ties were: - 1 1 805 a n d - 1 1 791 deg. c m 2 / d c e i m o L S e c o n d a r y s t r u c t u r e analysis o f the f a r ultraviolet C D s p e c t r a revealed the following: 24.2% a-helix, 40.0% fl-sheets, 1.0% f l - m r u s a n d 34.7% r a n d o m coils f o r the n e u r o t o x J n f r o m C. botulinum t y p e E strain 5545; a n d 22.2% a-helix, 40.7% fl-shects, n o fl-turns a n d 37.0% r a n d o m coils f o r t h e n e u r o t o x i n f r o m C. butyricum (Table I). T h e d i f f e r e n c e in the s e c o n d a r y structures o f b o t u l i n u m a n d b u t y r i c u m n e u r o t o x i n s c o u l d b e signific a n t ; t o verify this o b s e r v e d difference, b o t u l i n u m n e w r o t o x i n f r o m a n o t h e r source, C. botulinum t y p e E strain 43, w a s also e x a m i n e d . T h e e s t i m a t e d p a r a m e t e r s were 20_5% a-helix, 43.7% fl-shects, n o fl-turns a n d 35.7% r a n d o m coils. T h e analysis s h o w e d the s a m e extent o f v a r i a t i o n s in the s e c o n d a r y structures b e t w e e n n e u r o toxins f r o m C botulinum strains 43 a n d 5545, as between n e u r o t o x / n s f r o m C butyricum and C botulinum strain 5545. T h e r e f o r e , c o m p a r i s o n o f tertiary s t r u c t u r a l features, in t e r m s o f T y r e x p o s u r e a n d m o l e c u l a r t o p o g -
TABLE ! Secondary structure parameters of neurotoxins from C botulinum (strain 43), C. botulinura (Mrain 5545) and C. b~ayric~,n (strain 5839) in !0 raM soc~um phosphat~ pH 6.0, estimated from the circular dichraic spectra between 190 and 240 n,-n Ncoroloxin source C botu/iwan (strain 43) C. botulinum (strain 5545) C. bmryicum (strain 5839)
a-helix (%)
fl-sheets (~)
fl-turns (~)
Random coils (~)
20.5 ( + 0.50) +
43.75 ( =[:1.75)
0.00
35.75 ( + 2.25)
24.25 ( + 0.25)
40.00 ( + 5.00)
1.00 ( + 1.0)
34.75 ( + 4.25)
22.25 ( 4-1.75)
zh3.75( + 4.25)
0.00
37.00 ( 4-2.5)
* Standard error indicated within parenthesis.
123 030"
0005
030 i
0006
/
/ oo
015
i
015
L/v
,j/ !
O0
I
%% oo
- 3005 3OO 32O
Fig- 5. Absorption (broken line) and second derivative (solid line) spectra of the neurotoxin from C bozulinum strain 5545, dissolved in 10 r a m N a P B p H 6.0.
, ~-0.006 i:~oO 280 300 320 V~velength nm Fig. 6. Absorption (broken llne) and second derivative (solid line) spectra of the neurotoxin from C. botulinum strain 5545, dissolved in 6 M guanidine-HCI.
raphy of Trp residues were confined to botulinum neurotoxin from only one source (strain 5545) and the butyricum neurotoxin. Degree of Tyr exposure. The degree of Tyr exposure was determined usiz~g second derivative ultraviolet spectroscopy (between 280-300 rim). The absorption and second derivative spectra of the neurotoxin from C. bmulinum (strain 5545) are shown in Fig. 5. The second derivative spectrum shows two positive peaks at 288.5 and 295.5 nm, and two negative peaks at 284.5 and 291.5 nm. The notation "a" refers to the arithmetic sum of the negative d2A/dA2 of the peak at 284.5 n m and the positive d2A/dX2 of the peak at 288.5 n m of the derivative spectrum. The notation "b' refers to the arithmetic sum of the negative d2A/dA2 of the peak at 291.5 n m and the positive d2A/dA2 of the peak at 295.5 n m of the derivative spectrum. The a / b ratio (Fig. 5) for one batch of the neurotoxin in 10 m M NaPB (pH 6.0) was 2.147 (Table II). Treatment of the neurotoxin with 6 M guanidine-HCl increased the intensities of the derivative peaks, showing two negative peaks at 283.5 and 291 n m a:ld two positive peaks at 287.0 and 294.5 n m (Fig. 6).
The a / b ratio for the botulinum neurotoxin in 6 M guanidine-HC! was 3.794 (Table II). The absorption and second derivative spectra of the butyricum neurotoxin (in 10 m M NaPB, pH 6.0) are shown in Fig. 7. The second derivative spectra shows two positive and two negative peaks at wavelengths that are similar to the corresponding peaks of the botulinum neurotoxin. However, there is a notable difference in the ratio of the two positive peaks (i.e., 295.5 nm/288.5 nm) which is 0.99 and 1.15 for the neurotoxins from C. botulinum and C bu~,ricum, respectively. The a / b ratio of one batch of the bntyricum neurotoxin (in 10 m M NaPB, p H 6.0) was 1.900 (Table II). Treatment with 6 M gaanidine-HCl increased the intensities of the positive (287.5 and 295 rim) and negative (283 and 291 nm) peaks as in the case of neurotoxin from C botulinum (spectrum not shown). The a / b ratio was 3.730 (Table Il). The average degree of the Tyr exposure in botulinum and butyricum neurotoxins was 58.2 + 1.2~ and 5 3 . 4 + 0 . 9 ~ respectively (Table II). Fluorescence spectroscopy. The fluorescence spectra of both the neurotoxins, excitated at 295 nm, showed
240
260
280
O0
240
T A B L E It
Degree of t.vrosine exposure in the botulinura neurotoxins from C. botulinum (strain 5545) and C bulyricum as determined by second deriontiv¢ ultraviolet spectroscopy Neurotoxin soulx~
"In *
7,
x
Ya
a
a **
C. botulinura
2.147 ZISI
3.794 3.969
4.739 4.739
- 0.263 - 0.263
59.4% 57.0%
58.2 + 1.2%
C buo'r/ct,n
1.900 1.911
3.730 3.877
4.739 4.739
- 0.263 - 0.263
54.2% 52.5~
53.4 4- 0.9~
* For the notations see Materials and Methods. ** "D-e degree of tyro~ne exposure, a, is the average of two independent determinations for neurotoxin samples isolated from two different batches of bacterial cultures.
124 T A B L E !11
Quenching Forameters for ! - a n d acrylamide ~zenching of Tip fluorescence in botulintml nem-otoxins from C bolulinum strain 5545 a n d C butyricum strain 5839
Neurozoxin
I-
source
K~ (M - I )
K 0 (M -1)
f~
Acz'ylarr~de K~. ( M - ' )
Ko (M-')
]'~
C botulinum C./gayr/cum
1.48 - *
5.38 -
0.57 -
2.88 2.20
9.23 1024
0.49 0.44
• Quenchingwas not observed.
/'\
0 30
-omo
1~ i 1.4!
u o L
1.1 015
O0 ~
1.0 0.9 -u.02
\ 280
300
|
! 0.18
[Aery~mide], M
,010 260
i 0.08
Fig. 9. Stern-Volmer plots for aucrylamidc quenching of tryptophan
OC 240
|
fluorescencein neurotoxinsfromC botu//nu~,and C butyr/cmn.See legend m Fig. 8 for other details.
320
Wavelen91h . nm
Fig. 7. Absorption(broken line) and second derivative (sohd line) spectra of the neurotoxinfromC butyr/cumstrain 5839, dissolved in 10 mM NaPB(pH 6.0). emission maxima at 334 nm (spectra not shown). The fluorescence quenching with the anionic I - and neutral acrylamide quenchers revealed that I - quenched the Trp fluorescence of the botulinum neurotoxin, but even 0.2 M KI did not quench the Trp fluorescence of the butyricum neurotoxin (Fig. 8). Above 0.2 M KI, quenching increased for both the neurotoxins showing upward curvatures. Since such upward curvatures appear mainly due to the static quenching [18], the data derived from > 0.2 M KI were not used for Stern-
Volmer or modified Stern-Volmer kinetic analysis. The Ksv, KQ and fa for I - quenching derived from SternVolmer and modified Stern-Volmer plots (not shown) were 1.48 M - , 5.38 M - and 0.57, respectively (Table III). Acrylamide was almost equally effective in quenching both botulinum and butyricum neurotoxins. The quenching parameters K~, derived from Stern-Volmer plots (Fig. 9) and KQ and f= derived from modified Stern-Volmer analysis (plots not shown) for the botulinum neurotoxin were 2.88 M -i, 9.23 M - t and 0.49, respectively. The corresponding values fcr the butyricum neurotoxin were 2.20 M -I, 10.24 M -1 ~J,d 0.44, respectively (Table III). Discussion
C. ~ulinum
1.0~ 09 -u.02
~ 0.08 (Kq, M
'
' 0.18
Fig. 8. Stern-Volmerplots for I- quenchingof tryptophan fluorescence in neurotoxinsfromC botulinum and C bulyricum. Fo and F are the fluorescenceintensitiesat the emissionmaximain the absence and presence of quencher, 1 - , respectively.
The neurotoxin produced by C. butyr/cum strains isolated from infant botulism cases, causes the same symptoms in mice as botulinum neurotoxins and can be neutralized by the antiserum for type E botulinum neurotoxin [3,4]. Type E botulinum and butyrlcum nenrotoxins are synthesized as ---150 kDa single chain proteins. Limited digestion with trypsin nicks the single chain molecule to a two chain structure [5]. Trypsinization of the neurotoxins also increases their specific toxicities by = 200-fold. However, as shown in Fig. 1, the kinetics of their activation differ substantially. There m ; ~so differences in the serological properties of these neurotoxins. Although both proteins can be neutralized with the antiserum raised against the heter-
125 ologous protein, the immunodiffusion test shows that type E botulinum and butyricum neurotoxins differ, at least, in one epitope (Fig. 2A and B). The similarities and differences in the toxic and serological properties of the two neurotoxins are likely to be rooted to their primary, secondary a n d / o r tertiary structural features. Secondary structural features of the two neurotoxins are virtually identical (Table I). Are the small differenees between the two proteins (2.00~ in a-helix, 0.75~ in fl-sheet, 1.00~ in /~-turus and 2.25~ in random coil) significant? To judge this issue we analyzed type E neurotoxin produced by another strain of C botulinum type E (strain 43). The neurotoxins produced by the two strains (i.e., 43 and 5545) of C. botulinum type E also differ in their secondary 'structure parameters (Table I) to a similar extent (3.65~ in a-helix, 3.75~ in fl-sheet and 1% in random coil). Absence of //-turns in the secondary structure may not be unequivocal because the analysis method of Chang et al. [12] is particularly weak in deriving the/~-turns from far ultraviolet CD spectra. Similarity in the secondary structural features of the neurotoxin from C. butyricum and C botulinum type E indicate that their core structures are similar. This is consistent with the limited sequence information available for the two proteins. Within the 5~ of the known sequences (68 and 71 residues out of 1300 have been identified) that can be compared, 96% of the amino acids are identical [6]. The solvent exposure of Tyr residues in the botufinum and butyricum neurotoxins suggests only a small difference (58.2~ vs. 53.4~, Table II). This amounts to 41 and 37 Tyr residues exposed to the surface of botulinum and butyricum neurotoxins, respectively, based on 70 Tyr residues present in botnlinum [11] and assuming the same number of Tyr residues in butyricum neurotoxin. This conclusion may be tentative because the number of Tyr residues assigned to the butyricum neurotoxin was taken from the type E botulinum neurotoxin [11] as the amino acid composition of the butyricum neurotoxin is not yet known. However, the assumption has validity from the a / b ratios of botulinum and butyricum neurotoxins in 6 M gnanidine-HCl where the a / b ratio is only a function of T y r / T r p ratio and not the folding of protein [13]. Since the mean a / b (¥~) ratio of the two neurotoxins 3.881 and 3.803 in 6 M gnanidine-HCl are within 2~ of each other (Table II), their T y r / T r p ratio should be essentially the same. This provides further evidence to the significant difference in the degree of Tyr exposure in the native neurotoxins as evident from a more than 11~ difference in the a / b ('in) ratios of the botulinum and bntyricum neurotoxins (Table II). Topography of Tyr residues is apparently important for the toxicity of the type E botulinum neurotoxin because modification of Tyr residues with tetranitromethane abolishes the toxicity while retaining the immunological properties of the protein [19].
Fluoreseence spectra of both the neurotoxins showed emission maxima at 334 nm compared to 348 nm for free L-tryptophan indicating that the fluorescent Trp residues are in a relatively hydrophobic environment within the protein matrix. Trp residues in polar environmcnts show red-shifted emission maximum [20]. About half of the fluorescent Trp residues of both the neurotoxins were accessible to the neutral quencher, acrylamide. The fluorescence quenching parameters K~v, KQ and f~ derived from acrylamide quenching, were very similar for the two neurotoxins (Table III), indicating that about half of the Trp residues in these two proteins are in similar molecular environments. Acrylamide, being a neutral molecule, can penetrate into the protein matrix. Therefore, most of the fluoreseent Trp residues of a protein are generally accessible to this quencher [21]. Thus, about half of the fluorescent Trp residues that were not quenched by acrylamide can be considered deeply buried in the protein matrix where acrylamide could not reach, possibly owing to steric hindrance. Tiffs interpretation is consistent with the blue-shifted emission maximum (334 rim) relative to Trp residues (vide supra). The acrylamide quenching appears to be mostly 'dynamic" rather than "static" in nature as no upward curvatures in the Stern-Volmer plots were observed (Figs. 8 and 9). At higher coneentrations of acrylamide ( > 0.2 M) and downward curvature was observed (data not shown) indicating beterogenous populations of the fluorescent Trp residues. The tryptophan fluorescence quenching by the anionic probe l - (a surface quencher) was significantly discriminatory between the neurotoxins from C. botulinum and C. butyricum (Table Ill). I - quenched the Trp fluorescence of the botulinum neurotoxin significantly suggesting a considerable exposure of Trp residues on the surface of the protein. Virtual absence of any quenching by I - in the butyricum neurotoxin indicates that the fluorescent Trp residues are not located on the surface of this protein. Since the StemVolmer quenching plots showed straight fines up to 0.2 M I - concentration, it is considered that 'static" quenching contribution is very little [20], especially when Ksv is low (Table IIl, Ref. 18). However, at higher concentrations ( > 0.2 M) of I-, sharp upward curvatures were observed in the Stern-Volmer quenching plots of the neurotoxins from both C botulinum and C butyricum, indicating significant static quenching beyond 0.2 M I - concentrations. For Ksv, KQ and fa calculations, I - concentrations below 0.2 M were used. In the botulinum neurotoxin the fraction of maxim u m fluorescence accessible to both I - and acrylamide were comparable (Table Ill) suggesting that both quenchers interact with the same population of fluorescent Trp residues. The Ksv and KQ values for acrylamide quenching were about 2-fold more than in
126 the case of I - quenching, which is understandable because of their neutral and ionic properties. Anionic l may not be as effective a probe as acrylamide because of the repulsive charges in the protein. A comparison of I - and acrylamide quenching is valid as both probes have a fluorescence quenching efficiency of a unity [18]. Botufinum and butyricum neurctoxins, that migrated identically in SDS-PAGE indicating their similarity in molecular size [5], have differences in their surface charges as suggested by a differential electrophoretic migration in native conditions (Fig. 3). Our data indicate that while the neurotoxins produced by C. botulinum and C. butyricum have similar secondary structures, differences in their tertiary structures (polypeptide folding) are detectable. This s u m m a r y is based on differences in (i) the degree of solvent exposed Tyr residues, (ii) mobilities of the two native neurotoxins on the native polyacrylamide gel and (iii) quenching of the Trp fluorescence b y 1-. The inner cores of the two neurotoxins are apparently similar as is evident from the inaccessibility of about half of the Trp fluorescence to acrylamide, whereas most of the differences are reflected at the surface of the two neurotoxins. Comparative examination of the C. botulinum types A, B and E neurotoxins has indicated some similarities and range of differences in their secondary and tertiary (exposed Tyr residues and state of Trp residues) structure parameters [23]. The similarities and differences between the type E botulinum neurotoxin and the type E-like neurotoxin produced by C. butyricum, reported here, are in agreeu,eat with what is k n o w n between types A, B and E botalinum neurotoxins. Whether these variations are responsible for the differences in the toxic potencies and serological properties of the b o t u l i n u m type E and butyricum neurotoxins remains to be investigated. Acknowledgments This study was supported in part by N I H grants NS17742, NS24545 and NS25063, D e p a r t m e n t of Defense-University Research Instrumentation Program
award DAAG-29-83-GO063 and DAAG-03-87-G-0089, the Food Research Institute and the College of Agricultural a n d Life Sciences of the University of Wisconsin-Madison.
References I Hahermann. E. and Dreyer. F. (1986) Curr. Topics Microbiol. Immunol. 129. 93-179. 2 Simpson. L L (1981) Pharmacol. Rev. 33. 155-188. 3 Aureli, P~ Fenicia. L., Pasolini. B., Gianfranceschi, M., McCroskey, LM. and Hatheway, C.L. (1986) J. Infect. Dis. 154. 207-21 I. 4 McCroskey, LM_ Hatheway, C.L. Fenicia, L., Pasolini. B. and AurelL P. (1986) J. Cfin. Microbiol. 23. 201-202. 5 Gimtnez, J.A. and Sugiyama. H. (1988) Infect. Immun. 56. 926929. 6 Gimtnez. J.A., Foley. J. and DasGupta, B.IL (1988) FASEB J. A1750 (abstr.). 7 Gim,~neT..J.A. and Sugiyama. H. (1987) Appl. Environ. Microbiol. 53. 2827-2830. 8 Singh, B.IL and DasGupta, B.IL (1989) Mol. Cell. Biochem. 85, 67-73. 9 Boroff. D.A. and Rock. U. (1966) J. Bacteriol. 92. 1580-1581. 10 DasGupta, B.IL and Rasmussen, S. (1984) Arch. Biochem. Biophys. 232, 172-178. 11 Sathyamoorthy, V. and DasGupta, B.IL (1985) J. Biol. Chem. 260, 10461-10466. 12 Chang. T.C., Wu, C.-S.C. and Yang. J.T. (1978) Anal. Biochem. 91, 13-31. 13 Ragon¢, R.. Colonna, G. Balestrieri. C., Servillo, L. and lrace. G. (1984) Biochemistry 23. 1871-1875. 14 Edelhoch, H. (1967} Biochemistry 6. 1948-1954. 15 Servillo, L., Colonna, G, Balestrieri, C. Ragooc, IL and lrace. G. (1982) Anal. Biochern. 126, 251-257. 16 Stern, O. and Volmer, M. (1919) Phys. 7.. 20, 183-188. 17 Lehrer, S.S. (1971) Biochemistry 10, 3254-3267. 18 Eftink. M.IL and Ghiron. C.A. (1981) Anal. Biochem. 114, 199227. 19 Woody. M.A. and DasGupta. B.IL (1989) Mol. Cell. Biochem. 85, 159-169. 20 Lakowicz, J.IL (1983) Principles of Ruoresceoce Spectroscopy, Plenum Press, New York. 21 Eftink, M.IL and Ghiron. C.A. (1984) Biochemistry23, 3891-3899. 22 Lehrer, S.S. and Leavis, PC. (1978) Methods Enzymol. 49, 222236. 23 Singh, B.IL and DasGupta, B.R. (1989) Mol. Cell. Biochem. 86, 87-95.
119
BBAPRO33856
Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E Ba! R a m S i n g h *, J u a n A . G i m ~ n e z a n d B i b h u t i R . D a s G u p t a FoodResearchInstitute, Universityof Wisconsin,Madison, WI (U.S.A.)
(Received21 May 1990) (Revisedmanuscriptreceived12 October 1990)
Key words: Botulinumneurotoxin:Circulardichroism:Derivativespectroscopy-(C. bmulinum); (C butyricurn) Production of ~ l ~ k e nemotox3n by a non-CIostridium bomllnmn organism has prdoumi implications in the epideminlo~ of the disease botulism. Molecular topography of the = 150 kDa neterotoxie protein produced by Closuidium Imtyrienm (strain 5839) and its activation kinetics were examined and compared with a s e r o l o ~ y related botulinmn nemotoxln p~dluced by ¢~ botu/~um type E to further characterize the butyrlcum nemotoxin. Bo~l|num mmrotox~ was fully activated within 30 min of i n c u h a t ~ with trypsin, whereas b~.rlcum ~ r o t o x i n achieved ~ acfivafiou within 5 rain of incubafiou. Molecular topography of the two neurotoxins was analyzed in terms of secondary s m ~ w e s and the surface accessH~fities of the p o l y ~ domains containing aromatic amino ~ The secondary stmctm-e parameters of the but}xicmn nemotoxin (a-halix 22%, ~ s h e e t 41% and random coil 37%), as estimated from the far ullra~io]et circular dichroic spectra, appeared siraHar to that of botulinum neurotoxin. (Singk, B.IL and ~ B.R., (1989) M o t Ceil Biochem. 86, 87). Second derivative ultravin~t spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricmn and botulinum neurotoxius, respectivdy, suggesting a differential surface ~ of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the b o h d | m u type E and Ibutyrlenm nem'otoxins were in a relatively hydrophobic environment as indicated by the blue-shlfted emisslou maxima (334 ran). About half of rite fluorescent Trip residues of both proteins were aecesslble to acrylamide, a neutral ~ queneh~, and appeared to be in a similar molecular environment. The ionic surface probe, ! - , quendaed the Tip fluorescence of bot~ilmnn significantly, but not that of butyricmn neurotoxin. Thus, a consideraCie n m n l ~ of fluorescent Trp residues were apparently located on the surface of the botulinmn, bet not on of m e ~ - r i c u m neurotoxin. Boudinum and butyricum neurotoxias, i n d ~ b ~ S s ~ t e by pobacryhnicte g d in rite presence of sodium dedecyl s~lfate, migrated differently in the absence of sodium dedecyl stdfate suggesting ~ f e r e n c ~ s ) in their surface charge distn'lmtinn. These results provide the first report of the secondary and tertiary stmebJ-e parameters of tha nemmoxiq In'O~ ~x~d by a nou-botulinum species and eomparlson of tha molecular t e p o g r a ~ of the nemetoxln with the antlgenlcally related bogdlnum neurotoxln type E.
Introduction Botulinum ncurotoxin causes the disease botulism. Seven distinct serotypes (A through G) of the neurotoxin, produced by different strains of Clostridium botulinum, have similar pharmacological action on neuromuscular junctions [1,2]. Some strains of C bu~ricum were recently found responsible for infant botulism * Present address: Department of Chemistry, Southeastern MassachusettsUniversity,North Dartmouth, MA. U.S.A. Corresp~dence: B.R. DasGupta,Food ResearchInstitute, Uni~'sity of Wisconsin,1925WillowDrive. Madison.Wl 53706. U.S-A.
cases. The neurotoxin produced by C butyricum was neutralized by the antiserum raised against the type E neurotoxin produced by C. bomlinum [3,4]. Immunodiffusion tests of the neurotcxlns from these two sources with antiserum raised against C. botulinum type E neurotoxin showed that at least one epitope present in ¢2. botulinum neurotoxin was absent in C. butyricum [5]. Another difference was in their specific toxicities; based on intravenous mouse assay the neurotoxins from C. botulinum and C. butyricum were 6-107 vs. 20-107 LDs0/mg protein, respectively [5]. Expression of the botulinum neurotoxin gene by a non-C, botulinum organism could have profound impfications in the epidemiology of botulism. Therefore, it is important to
120 compare the neurotoxins produced by C botulinum and C bu~ricum, at the molecular level. Out of the total = 1300 amino acid residues expected in each of the = 150 kDa proteins, 68 and 71 residues in the two neurotoxins have been identified [6]. A comparison of the partial amino acid sequences shows only three non-identical residues in their primary structures. No other structural comparison has been made for these two neurotoxins. In this study, we analyzed the secondary structures of the neurotoxins from C. botulinum type E strains 43 and 5545 and C. butyr/cum (strain 5839) u . ~ g eir~alar d/chroic (CD) spectroscopy. In order to examine the polypeptide foldings, (i) degrees of tyrosine exposure to the aqueous solvent were determined using second derivative ultraviolet spectroscopy and (ii) microenviruuments of tryptophan residues were probed using fluorescence quenching techrdque. The results suggest that while the secondary sUructures of the neurotoxins from C. botulinum and C. butyricum remained the same, the polypeptide foldings appear to differ s/gnificantly. Mmerials and Me~mds Neurotoxin preparations. C botulinum type E strain 43 was kindly supplied by Dr. Lynn S. Siegel, Fort Derrick, Frederic, MD. C. botulinwn type E strain 554"5 and C. butyricum strain 5839 were kindly provided by Dr. Charles L. Hatheway, Centers for Disease Control, Adanta, GA. The organisms were grown and the neurotoxin purified from the bacterial culture as previously described [7]. The pure neurotoxin preparations (single band in polyaerylam/de gel electrophoresis in the presenee of sodium dodecyl sulfate (SDS)), stored at 4 ° C as precipitated proteins (39 g ammonium sulfate/100 n'd), were recovered by centrifugation. After dissolving in 10 m M sodium phosphate buffer (NaPB) (pH 6.0) the neurotoxin was dialyzed extensively against the same buffer and filtered through a 0.2 /am Acrodisc fdter (Gelman Sciences) before optical measurements. Protein concentrations were determined as described before
tions of pure neurotoxin detoxified with 0.5% formaldehyde [10]. Polyactylamide gel electrophoresis without a denaturant (nati~-PAGE). The electrophoretic mobilities of neuromxins in the absence of SDS or urea were determined using the PhastSystem (Pharmacia, Piscataway, N-D. Protein samples (---0.5 m g / m l in 0.05 M Tris-HC! buffer, pH 8.5) were loaded on the gel with a Sample Applicator 8 / 1 (8 samples, 1 ~! each). Phastgel Gradient 8-25% and Phastgel Native Buffer strips were used. Circular ~chroisr~ Far UV-CD spectra were recorded be~,een 190 and 240 n m on a JASCO J2OA C D / O R D spectropdlarimeter at room temperature ( 2 3 - 2 5 ° Q . The chart speed used was 2 c m / m i n with a wavelength expansion of 10 n m / c m . The slit-width and time constants were fixed at 1 n m and 4 s, respectively. The spectra were recorded with 1 nun pathlength quartz cuvette using protein concentrations of 0.14-0.16 m g / m L Spectra] recordings of each protein were made in tr/plicate. Mean residue weight ell/ptieities were calculated at every n m between 190 and 240 nm. Mean residue weight of 114.49, derived from the published amino acid composition of type E botulinum neurotoxin [11], was used for the neurotoxins isolated from the three sources. Secondary structure parameters were caleu|ated according to Chang et at. [12] using a program provided by Drs. C-SC. Wn and J.T. Yang, University of California, San FrancLseo. In this procedure secondary structures are derived from a linear leastsquare analysis of the reference C D spectra of 15 proteins of known X-ray crystaUographic structure [12]. Absorption and second derivative ultraviolet spectroscopy. Absorption and second derivative spectra were recorded on an Uvikon 860 (Kontron Instruments) at room temperature ( 2 3 - 2 5 0 0 . The degree of the exposure of tyrosine residues to solvent ( a ) was estimated according to Ragone et al. [13] using the equation: a=
¥°-¥~ "r.Y~×tO0
[81.
Toxici~ determination. The neurotoxins (20/~g/60 pl 50 m M NaPB, pH 6.0) were mixed with trypsin (10 pl, neurotoxin:trypsin was 15:1, w/w). After 5, 15, 30 and 60 rain of incubation at 35°C, 15/xi aliquots were withdrawn and mixed with 15 /~I of soybean trypsin inhibitor (3 × more than trypsin, w/w). These samples were immediately diluted 10-fold with 60 m M NaPB (pH 6.2) containing 0.2% gelatin and injected into the tall vein of mice. Lethality (LDso/ral) was determined from the time to death method [9]. lmmunodiffusion test. Preparation and composition of the gels and development of immunoprecipitate fines were previously described [5]. The antineurotoxin sel~ were raised in rabbits by multiple subcutaneous injec-
where 3'. is the a / b ratio (see Fig. 6 for the notations) of the second derivative peaks of the native protein, 3", is the a / b ratio for the unfolded protein (in 6 M guanidine hydrochloride), Ya is a correction factor calculated on the basis of Tyr to Trp ratio for a given protein (see Table ! in Ref. 13). The T y r / T r p ratie determined by the procedure of Edelhoch [14] for butyricum and botulinum neurotoxins were 4.706 and 4.772, respectively. Mean of these two values, 4.739, was used for further calculations (see Table II). The number of Trp residues in the two neurotoxins were estimated by the spectroscopic procedures of Edelhoch [14] and also that of Servillo et at. [15]. Botulinum and butyricum neurotoxins were each found
121 to have 15 residues based on Edelhoeh's method. Earlier, using this method 16 residues were found in botulinum type F neurotoxin [11]. The number of Trp estimated by the method of Servillo et aL [15] were 20 (4-17%) and 18 (4-15%) for botullnum and butyrienm neurotoxins, respectively. The important poml that comes out by utiliTJng the two methods is that the Trp content in the two neurotoxins is virtually identical. Fluorescence spectroscopy. Fluorescence spectra were recorded on an SLM 8000 "Smart' spectrofluorometer at room temperature (23-25°C). The excitation wavelength of 295 nm was used to preferentially excite the Tip residues. To w ; n i m i T e t.he inner filter effect, the protein concentrations were used such that A295 remained ~ 0.05. For quenching experiments, stock solution (1-5 FI) of a quencher (KI or acrylamide) was added to a 700 tal protein solution in several steps (total --- 35 /d) and fluorescence recorded at the emission maximum. The fluorescence data recorded in triplicate were averaged and analyzed for Stern-Volmer plots [16]: -~= 1 + K~IQ] where Fo and F are the fluorescence intensities in the absence and presence of a given quencher concentration [Q], K~ is the Stcrn-Volmer quenching constanL The data were also analyzed using modified Stern-Volmer plots I171: F0
t
t
where F0 is the fluorescence intensity in the absence of a quencher, A F the difference in the fluorescence intensities due to the presence of a quencher, fa the fraction of the maximum accessible fluorescence and K o is the effective quenching constant. Most of the experiments were repeated with neurotoxin isolated from a separate batch of bacterial culture. Ultrapure guanidin~HC! (Schwarz/l~ann: Cleveland, OH), potassium iodide (Sj~rna; St. Louis, MO), Trypsin (L-l-tosylamide-2-phenylethyl chloromethyl ketone treated, activity of 268 U / r a g from Worthington, Freehold, NJ), soybean trypsin inhibitor (SBTI) (Sigma) and other chemicals were of highest quality available commercially. Buffers and solutions were prepared with deionized distilled water.
20"
El n c ~ t a ~/1~E o
q
~o
qx
0
-+ 0
5
lS
30
time
(~)
60
Fig. 1. Kinetics o f activation o f b o r u l i n u m type E a n d butyricum neurotoxins incubated with trypsin ( 1 5 : 1 , w / w ratio in 50 m M
NaPB, pH 6, 35°C). The lethality valu~ for botulinum type E and butyricum neorotoxins at time zero wen: 0.03-107 and 0.0"/.10~ LD~o/mg.respectively.
retained about 70% of the maximum toxicity for an additional 30 rain of incubation. In.contrast, butyricum neurotoxin was activated more rapidly; the maximum lethality was attained within 5 rain, but only 9~ of the activity remained after a total of 60 rain incubation with trypsin. lmmunodiffusion test. The antigenic relatedness betwecn botulinum and butyricum neurotoxins was evident from the spur formation in the immunoprecipitation lines (Fig. 2A and B). Similar observation was made earlier [5] using only the antibotulinum neurotoxin serum. Here we ha~e also used antibutyrieum neurotoxin serum to demonstrate that each neurotoxin has at least one epitope that is not present in the other protein. Native-PAGE. Electrophuresis of neurotoxins from C. botulinum and C. butyricum on a native (Le., without SDS) polyacrylamide gradient gel (8-25~) showed that
O
O
O
O
O
0
Results
Kinetics of activation. Fig. 1 shows changes in toxicities (LDs0/mg protein) of botulinum and butyricum neurotoxins as a function of incubation time in the presence of trypsin. Botulinum neurotoxin was activated to its highest lethality after 30 rain of digestion and
Fig. 2. Immunodiffusionassay of botulinum and butyricumneurotoxins. The central wells were charged with about 10 international units (1 unit neutralizes 1000 LD~) of type E botulinum(A) and butyricom(13)antitoxins. Wells, 1, 2 and 4 contained 6/tg of type E botulinumneurotoxinand wells3 and 5 contained6 Fg of butyricum neuroroxin.
122
f
o 0
J c: o
1[
18o
2oo
2~o
~;o
Wave'~ngth, nm
Fig. 4. Far ultraviok,t CD spectra of neurotoxins from C botulinma strain 5545 and from C but)r/cam strain 5839, dissolved in 10 mM NaPB (pH 6.0). The spectra were recorded at room temperature (23-25°C) using a l-ram pathlength quartz cuvene. Spectra are plotted afte~ respective baselines for 10 mM NaPB. pH 6.0. were subtracted. Un/ls of mean residue weight elfipficity on the y axis are degrees cm2/dmoL
123456 Fig. 3. Native polyacryhmidegradient (8-25%) gel electrophoresis of q,pe E botulinum (lanes 4, 5 and 6 containing 0.25, 0.5 and 0.75/Lg proteins, respectively) and buV2T/cum( h n ~ 1, 2 and 3 containing 0.25, 0.5 and 0.75 pg proteins, respectively) neurotoxins. The bands are sta/ned with Coomassie brilliant blue R-250.
at p H 8.5 the b o t u l i n u m n e u r o t o x i n m o v e d faster t h a n the b u t y r i c u m n e u r o t o x i n (Fig. 3), w h i c h indicates difference in their surface charges. T h e s e p r o t e i n p r e p a r a tions, t h a t m i g r a t e as single s h a r p b a n d s in S D S - P A G E [5,10,11] a n d a p p e a r h o m o g e n o u s b a s e d o n sequences o f N - t e r m i n a l residues [6,11], exhibited a s e c o n d a r y b a n d in the absence o f SDS. Circular dichroism- F a r ultraviolet C D s p e c t r a o f the n e u r o t o x i n s f r o m C. lvatyricum (Strain 5839) a n d f r o m C. botulinum (Strain 5545) a r e s h o w n in Fig. 4. E a c h s p e c t r u m s h o w s a shallow d o u b l e well s t r u c t u r e w i t h negative m a x i m a a t 217 + 1 n m a n d 207 + 1 n m , a n d negative m i n i m a at 212 + I n m . T h e m e a n residue elllptic/ty values were -:ery simAlar for n e u r o t o x i n s f r o m
C. botulinum strain 5545 and C. bu(yricum strain 5839. F o r e x a m p l e , a t 207 4-1 n m extremaL, the respective m e a n residue elliptic/ties were: - 1 1 805 a n d - 1 1 791 deg. c m 2 / d c e i m o L S e c o n d a r y s t r u c t u r e analysis o f the f a r ultraviolet C D s p e c t r a revealed the following: 24.2% a-helix, 40.0% fl-sheets, 1.0% f l - m r u s a n d 34.7% r a n d o m coils f o r the n e u r o t o x J n f r o m C. botulinum t y p e E strain 5545; a n d 22.2% a-helix, 40.7% fl-shects, n o fl-turns a n d 37.0% r a n d o m coils f o r t h e n e u r o t o x i n f r o m C. butyricum (Table I). T h e d i f f e r e n c e in the s e c o n d a r y structures o f b o t u l i n u m a n d b u t y r i c u m n e u r o t o x i n s c o u l d b e signific a n t ; t o verify this o b s e r v e d difference, b o t u l i n u m n e w r o t o x i n f r o m a n o t h e r source, C. botulinum t y p e E strain 43, w a s also e x a m i n e d . T h e e s t i m a t e d p a r a m e t e r s were 20_5% a-helix, 43.7% fl-shects, n o fl-turns a n d 35.7% r a n d o m coils. T h e analysis s h o w e d the s a m e extent o f v a r i a t i o n s in the s e c o n d a r y structures b e t w e e n n e u r o toxins f r o m C botulinum strains 43 a n d 5545, as between n e u r o t o x / n s f r o m C butyricum and C botulinum strain 5545. T h e r e f o r e , c o m p a r i s o n o f tertiary s t r u c t u r a l features, in t e r m s o f T y r e x p o s u r e a n d m o l e c u l a r t o p o g -
TABLE ! Secondary structure parameters of neurotoxins from C botulinum (strain 43), C. botulinura (Mrain 5545) and C. b~ayric~,n (strain 5839) in !0 raM soc~um phosphat~ pH 6.0, estimated from the circular dichraic spectra between 190 and 240 n,-n Ncoroloxin source C botu/iwan (strain 43) C. botulinum (strain 5545) C. bmryicum (strain 5839)
a-helix (%)
fl-sheets (~)
fl-turns (~)
Random coils (~)
20.5 ( + 0.50) +
43.75 ( =[:1.75)
0.00
35.75 ( + 2.25)
24.25 ( + 0.25)
40.00 ( + 5.00)
1.00 ( + 1.0)
34.75 ( + 4.25)
22.25 ( 4-1.75)
zh3.75( + 4.25)
0.00
37.00 ( 4-2.5)
* Standard error indicated within parenthesis.
123 030"
0005
030 i
0006
/
/ oo
015
i
015
L/v
,j/ !
O0
I
%% oo
- 3005 3OO 32O
Fig- 5. Absorption (broken line) and second derivative (solid line) spectra of the neurotoxin from C bozulinum strain 5545, dissolved in 10 r a m N a P B p H 6.0.
, ~-0.006 i:~oO 280 300 320 V~velength nm Fig. 6. Absorption (broken llne) and second derivative (solid line) spectra of the neurotoxin from C. botulinum strain 5545, dissolved in 6 M guanidine-HCI.
raphy of Trp residues were confined to botulinum neurotoxin from only one source (strain 5545) and the butyricum neurotoxin. Degree of Tyr exposure. The degree of Tyr exposure was determined usiz~g second derivative ultraviolet spectroscopy (between 280-300 rim). The absorption and second derivative spectra of the neurotoxin from C. bmulinum (strain 5545) are shown in Fig. 5. The second derivative spectrum shows two positive peaks at 288.5 and 295.5 nm, and two negative peaks at 284.5 and 291.5 nm. The notation "a" refers to the arithmetic sum of the negative d2A/dA2 of the peak at 284.5 n m and the positive d2A/dX2 of the peak at 288.5 n m of the derivative spectrum. The notation "b' refers to the arithmetic sum of the negative d2A/dA2 of the peak at 291.5 n m and the positive d2A/dA2 of the peak at 295.5 n m of the derivative spectrum. The a / b ratio (Fig. 5) for one batch of the neurotoxin in 10 m M NaPB (pH 6.0) was 2.147 (Table II). Treatment of the neurotoxin with 6 M guanidine-HCl increased the intensities of the derivative peaks, showing two negative peaks at 283.5 and 291 n m a:ld two positive peaks at 287.0 and 294.5 n m (Fig. 6).
The a / b ratio for the botulinum neurotoxin in 6 M guanidine-HC! was 3.794 (Table II). The absorption and second derivative spectra of the butyricum neurotoxin (in 10 m M NaPB, pH 6.0) are shown in Fig. 7. The second derivative spectra shows two positive and two negative peaks at wavelengths that are similar to the corresponding peaks of the botulinum neurotoxin. However, there is a notable difference in the ratio of the two positive peaks (i.e., 295.5 nm/288.5 nm) which is 0.99 and 1.15 for the neurotoxins from C. botulinum and C bu~,ricum, respectively. The a / b ratio of one batch of the bntyricum neurotoxin (in 10 m M NaPB, p H 6.0) was 1.900 (Table II). Treatment with 6 M gaanidine-HCl increased the intensities of the positive (287.5 and 295 rim) and negative (283 and 291 nm) peaks as in the case of neurotoxin from C botulinum (spectrum not shown). The a / b ratio was 3.730 (Table Il). The average degree of the Tyr exposure in botulinum and butyricum neurotoxins was 58.2 + 1.2~ and 5 3 . 4 + 0 . 9 ~ respectively (Table II). Fluorescence spectroscopy. The fluorescence spectra of both the neurotoxins, excitated at 295 nm, showed
240
260
280
O0
240
T A B L E It
Degree of t.vrosine exposure in the botulinura neurotoxins from C. botulinum (strain 5545) and C bulyricum as determined by second deriontiv¢ ultraviolet spectroscopy Neurotoxin soulx~
"In *
7,
x
Ya
a
a **
C. botulinura
2.147 ZISI
3.794 3.969
4.739 4.739
- 0.263 - 0.263
59.4% 57.0%
58.2 + 1.2%
C buo'r/ct,n
1.900 1.911
3.730 3.877
4.739 4.739
- 0.263 - 0.263
54.2% 52.5~
53.4 4- 0.9~
* For the notations see Materials and Methods. ** "D-e degree of tyro~ne exposure, a, is the average of two independent determinations for neurotoxin samples isolated from two different batches of bacterial cultures.
124 T A B L E !11
Quenching Forameters for ! - a n d acrylamide ~zenching of Tip fluorescence in botulintml nem-otoxins from C bolulinum strain 5545 a n d C butyricum strain 5839
Neurozoxin
I-
source
K~ (M - I )
K 0 (M -1)
f~
Acz'ylarr~de K~. ( M - ' )
Ko (M-')
]'~
C botulinum C./gayr/cum
1.48 - *
5.38 -
0.57 -
2.88 2.20
9.23 1024
0.49 0.44
• Quenchingwas not observed.
/'\
0 30
-omo
1~ i 1.4!
u o L
1.1 015
O0 ~
1.0 0.9 -u.02
\ 280
300
|
! 0.18
[Aery~mide], M
,010 260
i 0.08
Fig. 9. Stern-Volmer plots for aucrylamidc quenching of tryptophan
OC 240
|
fluorescencein neurotoxinsfromC botu//nu~,and C butyr/cmn.See legend m Fig. 8 for other details.
320
Wavelen91h . nm
Fig. 7. Absorption(broken line) and second derivative (sohd line) spectra of the neurotoxinfromC butyr/cumstrain 5839, dissolved in 10 mM NaPB(pH 6.0). emission maxima at 334 nm (spectra not shown). The fluorescence quenching with the anionic I - and neutral acrylamide quenchers revealed that I - quenched the Trp fluorescence of the botulinum neurotoxin, but even 0.2 M KI did not quench the Trp fluorescence of the butyricum neurotoxin (Fig. 8). Above 0.2 M KI, quenching increased for both the neurotoxins showing upward curvatures. Since such upward curvatures appear mainly due to the static quenching [18], the data derived from > 0.2 M KI were not used for Stern-
Volmer or modified Stern-Volmer kinetic analysis. The Ksv, KQ and fa for I - quenching derived from SternVolmer and modified Stern-Volmer plots (not shown) were 1.48 M - , 5.38 M - and 0.57, respectively (Table III). Acrylamide was almost equally effective in quenching both botulinum and butyricum neurotoxins. The quenching parameters K~, derived from Stern-Volmer plots (Fig. 9) and KQ and f= derived from modified Stern-Volmer analysis (plots not shown) for the botulinum neurotoxin were 2.88 M -i, 9.23 M - t and 0.49, respectively. The corresponding values fcr the butyricum neurotoxin were 2.20 M -I, 10.24 M -1 ~J,d 0.44, respectively (Table III). Discussion
C. ~ulinum
1.0~ 09 -u.02
~ 0.08 (Kq, M
'
' 0.18
Fig. 8. Stern-Volmerplots for I- quenchingof tryptophan fluorescence in neurotoxinsfromC botulinum and C bulyricum. Fo and F are the fluorescenceintensitiesat the emissionmaximain the absence and presence of quencher, 1 - , respectively.
The neurotoxin produced by C. butyr/cum strains isolated from infant botulism cases, causes the same symptoms in mice as botulinum neurotoxins and can be neutralized by the antiserum for type E botulinum neurotoxin [3,4]. Type E botulinum and butyrlcum nenrotoxins are synthesized as ---150 kDa single chain proteins. Limited digestion with trypsin nicks the single chain molecule to a two chain structure [5]. Trypsinization of the neurotoxins also increases their specific toxicities by = 200-fold. However, as shown in Fig. 1, the kinetics of their activation differ substantially. There m ; ~so differences in the serological properties of these neurotoxins. Although both proteins can be neutralized with the antiserum raised against the heter-
125 ologous protein, the immunodiffusion test shows that type E botulinum and butyricum neurotoxins differ, at least, in one epitope (Fig. 2A and B). The similarities and differences in the toxic and serological properties of the two neurotoxins are likely to be rooted to their primary, secondary a n d / o r tertiary structural features. Secondary structural features of the two neurotoxins are virtually identical (Table I). Are the small differenees between the two proteins (2.00~ in a-helix, 0.75~ in fl-sheet, 1.00~ in /~-turus and 2.25~ in random coil) significant? To judge this issue we analyzed type E neurotoxin produced by another strain of C botulinum type E (strain 43). The neurotoxins produced by the two strains (i.e., 43 and 5545) of C. botulinum type E also differ in their secondary 'structure parameters (Table I) to a similar extent (3.65~ in a-helix, 3.75~ in fl-sheet and 1% in random coil). Absence of //-turns in the secondary structure may not be unequivocal because the analysis method of Chang et al. [12] is particularly weak in deriving the/~-turns from far ultraviolet CD spectra. Similarity in the secondary structural features of the neurotoxin from C. butyricum and C botulinum type E indicate that their core structures are similar. This is consistent with the limited sequence information available for the two proteins. Within the 5~ of the known sequences (68 and 71 residues out of 1300 have been identified) that can be compared, 96% of the amino acids are identical [6]. The solvent exposure of Tyr residues in the botufinum and butyricum neurotoxins suggests only a small difference (58.2~ vs. 53.4~, Table II). This amounts to 41 and 37 Tyr residues exposed to the surface of botulinum and butyricum neurotoxins, respectively, based on 70 Tyr residues present in botnlinum [11] and assuming the same number of Tyr residues in butyricum neurotoxin. This conclusion may be tentative because the number of Tyr residues assigned to the butyricum neurotoxin was taken from the type E botulinum neurotoxin [11] as the amino acid composition of the butyricum neurotoxin is not yet known. However, the assumption has validity from the a / b ratios of botulinum and butyricum neurotoxins in 6 M gnanidine-HCl where the a / b ratio is only a function of T y r / T r p ratio and not the folding of protein [13]. Since the mean a / b (¥~) ratio of the two neurotoxins 3.881 and 3.803 in 6 M gnanidine-HCl are within 2~ of each other (Table II), their T y r / T r p ratio should be essentially the same. This provides further evidence to the significant difference in the degree of Tyr exposure in the native neurotoxins as evident from a more than 11~ difference in the a / b ('in) ratios of the botulinum and bntyricum neurotoxins (Table II). Topography of Tyr residues is apparently important for the toxicity of the type E botulinum neurotoxin because modification of Tyr residues with tetranitromethane abolishes the toxicity while retaining the immunological properties of the protein [19].
Fluoreseence spectra of both the neurotoxins showed emission maxima at 334 nm compared to 348 nm for free L-tryptophan indicating that the fluorescent Trp residues are in a relatively hydrophobic environment within the protein matrix. Trp residues in polar environmcnts show red-shifted emission maximum [20]. About half of the fluorescent Trp residues of both the neurotoxins were accessible to the neutral quencher, acrylamide. The fluorescence quenching parameters K~v, KQ and f~ derived from acrylamide quenching, were very similar for the two neurotoxins (Table III), indicating that about half of the Trp residues in these two proteins are in similar molecular environments. Acrylamide, being a neutral molecule, can penetrate into the protein matrix. Therefore, most of the fluoreseent Trp residues of a protein are generally accessible to this quencher [21]. Thus, about half of the fluorescent Trp residues that were not quenched by acrylamide can be considered deeply buried in the protein matrix where acrylamide could not reach, possibly owing to steric hindrance. Tiffs interpretation is consistent with the blue-shifted emission maximum (334 rim) relative to Trp residues (vide supra). The acrylamide quenching appears to be mostly 'dynamic" rather than "static" in nature as no upward curvatures in the Stern-Volmer plots were observed (Figs. 8 and 9). At higher coneentrations of acrylamide ( > 0.2 M) and downward curvature was observed (data not shown) indicating beterogenous populations of the fluorescent Trp residues. The tryptophan fluorescence quenching by the anionic probe l - (a surface quencher) was significantly discriminatory between the neurotoxins from C. botulinum and C. butyricum (Table Ill). I - quenched the Trp fluorescence of the botulinum neurotoxin significantly suggesting a considerable exposure of Trp residues on the surface of the protein. Virtual absence of any quenching by I - in the butyricum neurotoxin indicates that the fluorescent Trp residues are not located on the surface of this protein. Since the StemVolmer quenching plots showed straight fines up to 0.2 M I - concentration, it is considered that 'static" quenching contribution is very little [20], especially when Ksv is low (Table IIl, Ref. 18). However, at higher concentrations ( > 0.2 M) of I-, sharp upward curvatures were observed in the Stern-Volmer quenching plots of the neurotoxins from both C botulinum and C butyricum, indicating significant static quenching beyond 0.2 M I - concentrations. For Ksv, KQ and fa calculations, I - concentrations below 0.2 M were used. In the botulinum neurotoxin the fraction of maxim u m fluorescence accessible to both I - and acrylamide were comparable (Table Ill) suggesting that both quenchers interact with the same population of fluorescent Trp residues. The Ksv and KQ values for acrylamide quenching were about 2-fold more than in
126 the case of I - quenching, which is understandable because of their neutral and ionic properties. Anionic l may not be as effective a probe as acrylamide because of the repulsive charges in the protein. A comparison of I - and acrylamide quenching is valid as both probes have a fluorescence quenching efficiency of a unity [18]. Botufinum and butyricum neurctoxins, that migrated identically in SDS-PAGE indicating their similarity in molecular size [5], have differences in their surface charges as suggested by a differential electrophoretic migration in native conditions (Fig. 3). Our data indicate that while the neurotoxins produced by C. botulinum and C. butyricum have similar secondary structures, differences in their tertiary structures (polypeptide folding) are detectable. This s u m m a r y is based on differences in (i) the degree of solvent exposed Tyr residues, (ii) mobilities of the two native neurotoxins on the native polyacrylamide gel and (iii) quenching of the Trp fluorescence b y 1-. The inner cores of the two neurotoxins are apparently similar as is evident from the inaccessibility of about half of the Trp fluorescence to acrylamide, whereas most of the differences are reflected at the surface of the two neurotoxins. Comparative examination of the C. botulinum types A, B and E neurotoxins has indicated some similarities and range of differences in their secondary and tertiary (exposed Tyr residues and state of Trp residues) structure parameters [23]. The similarities and differences between the type E botulinum neurotoxin and the type E-like neurotoxin produced by C. butyricum, reported here, are in agreeu,eat with what is k n o w n between types A, B and E botalinum neurotoxins. Whether these variations are responsible for the differences in the toxic potencies and serological properties of the b o t u l i n u m type E and butyricum neurotoxins remains to be investigated. Acknowledgments This study was supported in part by N I H grants NS17742, NS24545 and NS25063, D e p a r t m e n t of Defense-University Research Instrumentation Program
award DAAG-29-83-GO063 and DAAG-03-87-G-0089, the Food Research Institute and the College of Agricultural a n d Life Sciences of the University of Wisconsin-Madison.
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