Dissociation of tetanus neurotoxin into two polypeptide fragments

Vol. 57, No. 4, 1974

BIOCHEMICAL

DISSOCIATION

OF TETANUS NEUROTOXIN INTO TWO POLYPEPTIDE Morihiro

Matsuda

Research

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February

and Masahiko

Yoneda

Institute for Microbial Osaka University, Osaka

21,

FRAGMENTS

Diseases

1974

SUMMARY. Analyses of neurotoxin protein of Clostridium tetani by polyacrylamide gel electrophoresis showed that the toxin as purified from culture filtrates ("extracellular" toxin, molecular weight 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment o) and 107,000 (Fragment f3) by treatment with dithiothreitol and sodium dodecyl sulfate. The toxin as purified from bacterial extracts ("intracellular" toxin) was found to consist of a single 160,000 dalton polypeptide chain, which is undissociable by such treatment but, when pretreated with trypsin, becomes dissociable into two fragments apparently identical with ~1 and S . In recent its

physical

been well

years,

tetanus

and chemical characterized

(1~5).

highly

neurotoxic

having

six

simple

little

has been known

free

information biologically

about

investigation chains.

that

paper

of the finding.

MATERIALS

AND METHODS

Clostridium tetanus pared

toxin from

tetani

culture

"Intracellular"

extracts

about

gation

42 hours.

was concentrated

of ammonium

sulfate

at 100,000

of the

and purified

fractionation x g for

were

for

at 35 C for

1257

insoluble

on

in the

fraction), materials

of

was pre-

4%5 days.

of the

cells

at 0 C with

The toxin

saturation

structure

the production

at O-4 C by subsequent

(20~40%

two polypeptide

toxin

extracts

of the

("extracellular"

molecular

prepared

(4).

120 min to remove

Copyright G 1974 by Academic Press, Inc. A 11 rights of reproduction in any form reverved.

grown

the

to isolate

into

"Extracellular"

organisms

very

molecule,

a course

of two types

(6).

as a

of structure-

be dissociated

et al.

daltons

However,

attempting

A47 was employed

The extracts

150,000 toxin

and during

from bacterial

of Raynaud

form

5).

their

have

in pure

elucidation

and discuss

medium

was obtained

to the method

could

Harvard

filtrates toxin

at 35 C for according

toxin

Mueller

about of the

the

and some of

composition

(4,

Thus we have been

toxin

strain

in modified

for

the dissociation

of tetanus

the basis

weight bridges

of the neurotoxin the

acid

obtained

structure

a clue

we report

and "intracellular")

has been

"subunit"

toxin.

fragments

we found

In this

the

extensively

amino

of molecular

provide

of the

active

including

and two disulfide

will

relationship

has been purified

The toxin

protein

SH-groups

of which

function

toxin

properties

grown

agitation

filtrates

steps

or

comprising

ultracentrifuand gel

filtra-

Vol. 57, No. 4, 1974

tion

on Sephadex

toxin

preparations

x

lo4 minimum

G-200

column

lethal

ratio

of 2.0~2.1. antitoxin

Davis

(7).

carried

They each formed

dodecyl

employing

Pharmaceut. brilliant

blue

pepsin

at 550 nm with

recorder

equipped

relative

mobilities

from

the

ences.

top

which

trypsin

were

determined

from

to the maximum peak height, and soy bean

scans

Worthington

Biochem.

Sephadex

Coomassie

determined

by

distance

markers

were G-200

(Armour

Biochem.

using

as refer-

obtained

chloromethyl

Corp.,

using

multiple absorbance was estimated from

standard

L-1-toluenesulfonylamido-2-phenylethyl

from

was

albumin

were

of gels

inhibitor

by

(8),

using

bands weight

employing

trypsin

serum

stained

of protein

spectrophotometer-Gilford Molecular transport.

a linear

described

(Worthington

Gels were

electro-

electrophoresis

bovine

and trypsin

markers.

crude

gel

by Weber and Osborn Co.),

intensities

Beckman

with

Dithiothreitol

Chem. Co.,

weight

against

originally gel

as described

(Schwarz/Mann

and the relative

scanning

band

by the methods

(2x crystallized)

molecular

Purified 2~4

polyacrylamide

sulfate(SDS)-polyacrylamide

ovalbumin

Co.),

as standard

(pH 7.5).

precipitation Analytical

7.5% gel

5% or 10% gel

human gamma-globulin, Corp.)

a single plates.

using

buffer

units(Lf)/Absorbance(28Onm),

and Absorbance(280nm)/Absorbance(260nm)

in Ouchterlony's

Sodium

out

flocculating

doses(MLD)/Lf

was performed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in 0.1 M NaK phosphate

have 360%385

horse phoresis

BIOCHEMICAL

from

Sigma

ketone-treated

from Pharmacia

Fine

Chem. Co.. RESULTS AND DISCUSSIONS Dissociation

of "extracellular"

the purified

"extracellular"

acrylamide

gel

in the

The molecular

weight

of molecular agreement method

weight with

(9).

that

on the

of SDS (Fig. ated

into (Fig.

The faster B, for were: ratio

Similar

(Fig.

the

reduced

(Fig.

weights

f 3,000,

of Fragment

the

entire

Fragment

60 min gave no inin the absence

was subjected

to gel

preparation

dissoci-

Fragment

1258

the slower

B = 107,000

protein toxin.

one Fragment

and band

+ 4,000

10% gel with

of Fragment

a faint

of the untreated n,

in close

by different

of the two fragments

done using B to that

that

is

a plot

in 50 mM Tris-HCl,

gel

toxin

on polyfrom

value

by others (1 mg/ml

that

of SDS(O.l%).

2b, CL and B), remaining

corresponding

was designated

were

2a) This

on polyacrylamide

thiol

of SDS, almost

The molecular experiments

component

as determined

reported toxin

behavior when

2a illustrate

(100 mM) at 25 C for

components

a = 53,000

+ 5,000

on SDS gel.

150,000)

dithiothreitol

component

of the amount

or presence

"extracellular"

2b, T) at the position

Fragment

la)

mobility

in the presence

moving

protein

relative

of the

However,

two protein

as a single

was 160,000

electrophoretic

convenience.

f 5,000.

(Fig.

la and Fig.

runs

toxin

(approximately with

lb).

electrophoresis band

absence of the

Reduction

1 mM EDTA, pH 8.2) fluence

toxin

versus

Fig.

toxin:

similar

o determined

T

and T= 160,000 results.

The

by densito-

Vol. 57, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(b) Fig. 1. Polyacrylamide gel electrophoresis in the absence of SDS of "extracellular" toxin. (a) untreated and (b) reduced with dithiothreitol. Approximately 10 pg protein was applied on each gel. The electrode buffer was 0.01 M Tris, 0.054 M glycine (pH 8.3). Electrophoresis was at a constant current of Stained with Coomassie brilliant blue. 2.0 ma per gel at 4 C for 3.5 hours. Migration was from top to bottom.

metric

analyses

scan curves with

that

ments

by amount

(S/d) predicted

are

(2.02)

complementary

T band varied

of stain

= 2.0 ? 0.16.

from

for

estimated

1:l

ratio

for

the

suggesting

the whole

to preparation

areas

is in reasonable

stoichiometry,

each other

preparation

from weighing

The observed

toxin

that

the

the two frag-

molecule.

and usually

under agreement

The amount

occupied

about

of

5% of

the preparation. Dissociation

of "intracellular"

"intracellular" protein

toxin,

component

to the

latter,

molecular trophoresis

(Fig.

two types

rence

of at least

cysteine

residues

former 3b).

of toxin

weight

remains

Since after

one cleavage forming

Fig.

3a shows

"extracellular"

even when reduced

tween

trypsinization

like

of molecular

the

weight

toxin:

just

this

160,000

to run with

bridge

1259

as a single

However,

in

contrast

of the

prior

to SDS-gel

elec-

in electrophoretic

behavior

be-

bond between

same

to be due to the occurthe two particular

in "extracellular"

behavior

the purified

component

was considered

of peptide

disulfide

on SDS gel.

as a single

difference

that migrates

dithiothreitol

reduction

on the electrophoretic

toxin,

of "intracellular"

toxin,

effect toxin

was

of

BIOCHEMICAL

Vol. 57, No. 4, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(b)

(4

Fig. 2. SDS-polyacrylamide gel electrophoresis (5% gel) of "extracellular" toxin. (a) not reduced (10 ug sample) and (b) reduced with dithiothreitol (20 pg sample). Mercaptoethanol was omitted from the sample buffer (10) the unreduced toxin. Electrophoresis was performed at a constant current per gel for 5.5 hours. Migration was from top to bottom.

examined. pH 8.2)

Thus

the

above

and then

trypsin

trypsinized

illustrates

that

under

two components. with

those

conditions

The molecular

of the

ratio.

consists

with

weights

of these

Incidentally, identical

At the end of the and after

dithiothreitol

the preparation

weights

60 min.

was added

as described

dissociates

behavior indicate

were

two components

as shown

3c

completely

two components

and the

several

of SDS. Fig.

in Fig.

identiwere

3d, about

toxin preparation dissociated into with those of Fragment C( and S

with trypsin and subsequent electrophoresis contrast to the case of "extracellular" toxin,

of a single

1 mM FDTA, chloro-

in the presence

"intracellular"

in the electrophoretic The data presented above

the cells

rig/ml)

~1 and 6 respectively

of molecular

by merely treating This is in sharp change

was reduced

in equimolar

fraction

two components

the

in 50 mM Tris-HCl,

at 25 C for (1.5

to electrophoresis

of Fragment

to exist

two-thirds

(1 ug/ml) inhibitor

toxin

subjected

(1 mg/ml

L-1-toluenesulfonylamido-2-phenylethyl

soy bean trypsin

minutes

found

with

ketone-treated

incubation

into

toxin

was preincubated

methyl

cal

"intracellular"

for 8 ma

on SDS gel. where no such

was observable upon such treatment. that tetanus toxin synthesized within

polypeptide

1260

chain

of molecular

weight

160,000,

BIOCHEMICAL

Vol. 57, No. 4, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SDS-polyacrylamide gel electrophoresis (5% gel) of "intracellular" Fig. 3. toxin. (a) not trypsinized, not reduced (10 ng sample), (b) not trypsinized but reduced (10 ug sample), (c) trypsinized and reduced (20 ug sample) and (d) trypsinized, not reduced (20 ug sample). Other experimental conditions were the same as described in the legend for Fig. 2.

while

most

composed

of the

linked

tetanus

of diphtheria

toxin

being

ment with

SDS for

ther

these

that

dissociable

are held

53,000

into

(13),

together

firmly

also

respecand

and "nicked"

the

fact

requires

two polypeptide

each

and 107,000,

"intact"

However,

toxin

medium are

Thus "intracellular"

to correspond

diphtheria

the dissociation

conversion

with

"extracellular"

by the action

Incidentally,

toxin

of "intracellular"

identical

naturally

a year)

formation.

weight

bridge.

11, 12) respectively. from

chains

apparently

bacilli. half

appear

the culture

that

tetanus

addtional

chains

forms

treat-

indicates

by non-covalent

fur-

bonds

by SDS.

Artificial can occur

toxins

into

(molecular

one disulfide

(10,

different

released

chains

by at least

"extracellular"

form

molecules

of two polypeptide

tively)

toxin,

toxin

we observed

of a purified

toxin

of proteolytic that

prolonged

"intracellular"

toxin

in the

This is possibly due to the presence preparation. Reduction was necessary for

lular"

toxin.

So the site(s)

of cleavage

by trypsinization that

conversion

produced

storage

(at

preparation

bond(s)

by tetanus

4 C for resulted

of such enzyme(s) the dissociation

of the peptide

1261

suggests

enzyme(s)

to "nicked"

more than in "nick"

contaminated of "extracelinvolved

in the

Vol. 57, No. 4, 1974

conversion

must be between

1ar"toxin

corresponding

Fragment tion

BIOCHEMICAL

c1 and 8 by forming

of the

sinization

"intracellular"

toxin

pair(s) have

are

concerned,

that both

above,

of its

ible

reported

definite

of toxicity

by trypsinization, association

Elucidation

on type

increase

the particular

Recently

Bizzini

properties (5).

fine

they

et

examined

However,

molecular

alteration

tryp-

of the "intra-

between

identical

(more in

the "nick"

as pre-

structure,

of the

toxin

during

a and 8 is

studies

now in progress

nature (Fig. with

of Clostridium physical employing

3~).

of the This

botulinum purified

reproduc-

of "intracellular" toxin.

The

suggests

the structural

and chemical

of young

observed

increase)

progenitor

of toxicity toxin

of "autolysates"

We have also

three-fold formation

biological, further

(14).

than

of enhancement E progenitor await

in toxicity

of trypsin

also

of detailed

ment a and 6 must of Fragment

are

frac-

upon

in majority

formed

in their

structural

suggesting

resulted

as reported

toxin

two-thirds

dissociated

that

and chemical

each other

to link

production.

enhancement

possible

employed is

of "intracelluserve

About

in the dissociation.

of definite

by the action

trypsinization

bridge.

bridge

as physical

from

residues toxin

suggests

involved

bacilli

toxin

This

of tetanus

differ

the course

et al.

preparation

no disulfide

types

they

the occurrence

Seki

disulfide

as far

reflecting

tetanus

interchain

residues

reported

cysteine

in "extracellular"

reduction.

molecules,

of cysteine

al.

two particular which

toxin

even without

cellular"

sented

the

those

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

alteration, (15).

properties fragments.

of FragIsolation

in our laboratory.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Murphy, S. G., and Miller, K. D. (1967) J. Bacterial. 94, 580-585. Dawson, D. J., and Mauritzen, C. M. (1968) Aust. J. Biol. Sci. 21, 559568. Holmes, M. J., and Ryan, W. L. (1971) Infect. Immun. 3, 133-140. Bizzini, B., Turpin, A., and Raynaud, M. (1969) Ann. Inst. Pasteur 116, 686-712. Bizzini, B., Blass, J., Turpin, A., and Raynaud, M. (1970) Eur. J. Biochem. 17, 100-105. Latham, W., Bent, D., and Levine, L. (1962) Appl. Microbial. lo, 146-152. B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. Davis, Chem. 244, 4406-4412. Weber, K., and Osborn, M. (1969) J. Biol. Mangalo, R., Bizzini, B., Turpin, A., and Raynaud, M. (1968) Biochim. Biophys. Acta 168, 583-584. Gill, D. M., and Dinius, L. L. (1971) J. Biol. Chem. 246, 1485-1491. Collier, R. J., and Kandel, J. (1971) J. Biol. Chem. 246, 1496-1503. Drazin, R., Kandel, J., and Collier, R. J. (1971) J. Biol. Chem. 246, 1504-1510. Matsuda, M., Kanei, C., and Yoneda, M. (1972) Biken J. 15, 199-206. H., and Yamaguchi, H. (1954) Med. J. M., Hirabayashi, Seki, T., Takaki, Osaka Univ. 2, 271-289. DasGupta, B. R., and Sugiyama, H. (1972) Biochem. Biophys. Res. Commun. 48, lb8-112.

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