Role of arginine residues in the structure and biological activity of botulinum neurotoxin types A and E

Vol. March

93, No.

2, 1980

28,

BIOCHEMICAL

AND

EIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 369-375

‘1980

ROLE OF ARGININE RESIDUES IN THE STRUCTURE AND BIOLOGICAL

ACTIVITY

OF BOTULINUM NEUROTOXIN TYPES A AND E

B. R. DasGupta Food Research University

Received

and H. Sugiyama

Institute and Department of Bacteriology of Wisconsin, Madison, Wisconsin 53706

January

30,

1980

Summaryz When nicked types A and E as well as the unnicked (i.e., single chain) type E botulinum neurotoxins were treated with 1,2-cyclohexanedione, which specifically modifies the arginine residues in 0.2 M borate buffer, pH 8.0 i) both the nicked and unnicked neurotoxins were detoxified, ii) the unnicked single chain neurotoxin became resistant to nicking with trypsin, and iii) the serological reactivity of type A (type E was not tested) was altered. Reversal of the arginine modification partially restored toxicity. In the electroimmunodiffusion test the modified type A neurotoxin appeared as 2 cones; the height of one cone increased and the other decreased as the modification reaction progressed. These results indicate that i) at least one arginine residue is involved in maintaining the toxigenic structure of types A and E neurotoxins; ii) the site of nicking in type E is an arginyl bond; and iii) arginine residue is critical for at least one antigenic determinant of type A neurotoxin.

-Clostridium logically

distinct

F neurotoxin molecule

a single

composed

chain

respectively)

ratio

of single

toxin

depends

of the

of some (e.g.,

Trypsinization

of the

(i.e.,

activation);

in its

native

state,

held

type,

type

single also type is

in

mol.

wt.

by an interchain molecules

and conditions

molecule

not

in all

neurotoxin

the increase

invariably

more than

369

(e.g.,

The of neuro-

is nicked

of endogenously

type

of any type in its

-S-S-

(1,2,3).

physiology

A) but

(nicked) ~100,000

preparation

E neurotoxin, activated

of

immuno-

A, B, C, D, E or

in a purified

chain

chain

but

or a dichain

together

and dichain

The single

type

(unnicked),

molecules

the organism.

and generally

are

similar

the

(H- and L-chains

single

on the neurotoxin

in cultures

nicking

Of these

protein

that

to dichain

pharmacologically

types.

of two chains

or a mixture

culturing

produces

neurotoxin

is

and ~50,000, bond(s)

botulinum

E) types.

results

specific

toxicity

a single

chain

any other

type

in its

protein

(1,2,3).

0006-291X/80/060369-07$01.00/0 Copvright 0 1980 by Academic Press, Inc. All rights of reproducfion in an-vform reserved.

Vol.

93, No.

BIOCHEMICAL

2, 1980

Types A, B and F neurotoxins

AND

undergo

BIOPHYSICAL

endogenous

RESEARCH

COMMUNICATIONS

activation

as well

the mechanism

of nicking

as

nicking. Although

speculations

activation

(1,3),

and if

nicking

ficity

of trypsin

or a lysyl

is

this

E; type

because

pH 8.0,

without from

(see

Co.), were

CHD served

to stop

the reaction

0.2 M arginine

for

hourly

of the

changes

two portions. ketone

volume protein

the excess

to the other

of the enzyme solution solution).

(final

The trypsinized

were

containing

of mice

and analyzed

into chloromethyl

to one portion

of trypsin, that

30 Ug/ml,

of the dialyzed samples

for

toxicity

by polyacrylamide

gel

370

assayed

with

divided

was added

l/10

acid)

mixture.

3 hours

were

to

dialyzed

the reaction

for

and untrypsinized

injection

incubated

M boric

PB buffer

samples

concentration

45 min and then

of

withdrawn

was immediately

in PB buffer,

at 35°C for

borate

concentration

tosylamido-2-phenylethyl

incubated

(5)

acid-sodium

6.0 + 0.005

PB buffer

and buffer

it.

and CHD

The neurotoxin

chilled

The dialyzed

dissolved

of type

activate

method)

CHD from

against

(EC 3.4.4.4.,

freshly

treated),

and PB buffer

buffer.

in

90 and 120 min and added

sample

against

selectively

to those

of 0.6 ml were

The diluted

first

E neurotoxin

does not

M).

pH

1 hr and then

Trypsin

or 0.116

0, 30, 60,

an arginyl

was included

at 35°C (final

NaH2P04-Na2HP04,

was done at ?"C,

either

(CHD) which

in 0.2 M boric

Aliquots

and remove

speci-

in type

purification

dissolved

and CHD, 0.058

(0.1

Substrate

are converse

1 for

after

nicking

A neurotoxin

and incubated

0.2 M arginine.

The dialysis

residue(s)

and

during

is

and trypsinization

ref.

mixed

0.1 ml of PB buffer

of nicking

properties

both

mixture

cleaved

activation.

The type

as a control.

the reaction

containing

(4).

molecule

A278 = 0,317

protein,

site

is

1,2-cyclohexanedione

two of its

T-ype E neurotoxin

buffer,

the

of arginine

residues

Chemical

for

The role with

bond

cause that

A is a dichain

(Aldrich

known what

suggests

examined

arginine

study

is not

made about

the primary

bond.

was therefore modifies

it

were

were

by the i.v. electrophoresis

Vol.

93, No.

2, 1980

BIOCHEMICAL

in presence

of sodium

samples

electrophoresis

for

The destained with

(c)

samples

and (d)

molecule

resulted mol.

was the

in

wt.

because

Fig.

CHD for

15,30,

147,000

mol.

the pattern

samples

generated

resistant

L-arginine, excess

The type (Table

I):

(control)

to 4.4

x lo3

product

from

not

from

band of nick

neurotoxin

The neurotoxin

to mol.

reaction

was activated corresponding

reaction

30 min of modification 1.9 x 10 to 1.8 x 10'

while

LD

371

/ml

did

not

147,000

in

(nick to the

more intense vs.

0.058

M,

Presence mixture,

resistant

of

in molecules.

Activation

the untreated increased

from

51 min survival

time)

was activated

to

by CHD in two other ii)

(toxicity

with

and 47,000

molecule.

of the nick

to 152 vs.

treated

was related

0.116

was detoxified.

by 44 fold

wt.

produced

resistant

e.g.,

here

samples

102,000

was affected

was quenched;

and

of the trypsinized

147,000;

formation

samples

corresponding

reduction

in the modification

chain

by trypsin

LD /ml

longer

the

102,000

presented

of these

of CHD in the reaction,

CHD, inhibited

i)

band

to the H- and L chains,

L-lysine

wt.

(a),

to 147,000

The neurotoxin

wt.

of

trypsinized

is not

corresponding

reaction;

E single

the neurotoxin

toxicity

relative

over

the gels

-or reduction

of the band

cases

corresponding

as single

of mol.

as one

the case of (c)

of mol.

representative.

bands

more intense but

band

band in any gel;

concentration

produced

toxin

three

of modification Higher

ways

of the

molecule)

band.

molar

7 is

treated

of these

in the

of the

of the H- and L chains

or 120 min appeared

Intensity

and in

single

(7).

mercaptoethanol)

Reduction

of

of

min appeared

The band

molecule,

of the

(a) not

zero

(with

147,000.

trypsinization

change

duration

wt.

1A of ref.

wt.;

CHD for

reduction

chain

The preparation

the neurotoxin

with

A photograph

60,

(6).

COMMUNICATIONS

has been described

of the band.

the disappearance

respectively.

also

single

and appearance

gel.

(d)

pattern

of mol.

47,000,

each

treated

or

change

RESEARCH

(PAGE-SDS)

as follows:

and (b)

not

BIOPHYSICAL

and electrophoresis

trypsinization

did

dichain

dodecylsulfate

appeared

CF!D (control)

band;

(b)

gels

AND

9.4

fold

(increase

or 240 min vs.

119 min

of neuro1 x 10' the in

Vol.

93, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

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TABLE I EFFECT OF C 11 C ON TYPE E NEUROTROXIN (SURVIVAL TIME)

ToxIcIn

NOT TRYPSINIZED CONTROL

152 MIN.

KEACTION 30 MIN. 60

TRYPSINIZED

~240

"

51 MIN.

"

>16 HRS.

119

"

193

"

TABLE II EFFECT OF C H D ON TYPE A NEUROTOXIN

T REACTION TIME

TOXICITY SURVIVAL TIME

LD50/ml

38 MIN.

0 MIN.

4.8

SURVIVAL TIME I

LOSS %

GAIN

LD50/ml

--

x lo5

REVERSAL EXPT.

I 30

"

60 60

”

1.0 x lo5 1.0 lo5

79.0

60

"

117

It

8.9 X lo3

98.0

90

"

68 MIN.

99.3

CONTROL 90 MIN.

--

6.8

x lo4

7.6 X

x lo4

3.9 x

105

"

1.3

39

"

4.5 x lo5

--

To reversethe modification (type A neurotoxin was modified with CHD as in case of type E) neurotoxin samples, after appropriate length of modification reaction, were divided into two 0.3 ml portions; to one portion 0.6 ml of 0.1 M NaH2P04-Na2HP0,, buffer, pH 7.2 + 0.005 M boric acid was added, and it was dialyzed against 100 ml of this buffer at 7°C; to another portion 0.6 ml of 0.2 M TRIS-HCl buffer, pH 7.2 was added and it was dialyzed against 100 ml TRIS buffer at 3O'C. Each buffer was changed four times during the 24 hours of dialysis in dark. The dialyzed samples were assayed for toxicity. survival

time).

dichain

form

detoxified That Table

II.

(see ref.

In separate (produced

by trypsinizing

when treated this

detoxification

Availablility for

experiments

purification

with

the activated the single

neurotoxin

chain

in

neurotoxin)

was

CHD. is not unique

of a steady method)

but

with

small

and rabbit

372

type supply

E is

shown in

of type

antiserum

A neurotoxin

(prepared

Vol.

93, No.

2, 1980

Fig.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1. Ouchterlony gel diffusion analysis of type A neurotoxin not treated with CHD (well 1); treated with CHD for zero min (about 20-30 set) (well 2), 30 min (well 3). 60 min (well 5) and 90 min (wells 4 and 6). Rabbit antitype A neurotoxin serum (8133) 1:2 diluted in central well. Each of the peripheral wells had 32.7 pg protein in 20 ~1. Duplicate plates were made with 1.5% agar in 0.05 M borate buffer, pH 7.4 + 0.85%

NaCl. against

i)

the

detoxified

neurotoxin

the restoration

reversing

(Table

of toxicity

and ii)

of the neurotoxin. II);

e.g.,

modification

the

gained

conditions

(4,

or complete

sample

7.6X

in PAGE-SDS

The neurotoxin zero

min

(actually

in Ouchterlony

analysis imposable from

not

20-30

(Fig.

1).

cones.

that

cones

from

toxicity

subjecting

(a)

and (b)

Two clearly

a sample

progressed.

products; widened

373

difference

higher

yet. as a single

LB in ref.

treated

with

7).

CHD for

precipitin

increasingly

band diffused

Electroimmunodiffusion

each produced

distinguishable

for

appeared

one sharp

The band appeared

60 min of

optimum

see Fig.

and (b)

restored

to the reversal

been established or not

be

after

it

Conditions

produced

CHD) by

could

98% toxicity

picture

of the neurotoxin

with

of:

of CHD on serological

lost

modified

set)

30, 60 and 90 min reaction

the two tall

the

(control)

the study

modification

have not

treated

2) &owed

small

of

II).

whether

allowed

the effect

a representative

plate

(Fig.

after

after

of Table

about

as the modification

formalin)

had lost

of toxicity

(for

(a)

that

toxicity

and legend

recovery

Part

The type A neurotoxin band

(lost

the modification,

reactivity

with

two nearly taller

cones

superemerged

in the height

as the modification

reaction

between

Vol.

93, No.

BIOCHEMICAL

2, 1980

Fig.

one

as and

received

these

of

amounts

the

two

logical

RESEARCH

6 is

product in nearly

1 and after

migration found

the

COMMUNICATIONS

chemistry presence

2.

The

the of

6)

left

devoid

reacting of

in

the

and

of

an

The

of

formed (well

that

the

I)

that

neurotoxin

acquired

higher

electrophoretic

with

Compared

to

native

CHD.

neurotoxin

modification

towards plate

reaction. forms

the the

(Fig.

A CHD modified

a stable

complex

374

that

and

the

is

for

one

antigenic

the

serothe

in

1

Fig.

was

90

protein

in

present

in

mobility the

consistent

acquires

two

between

pole,

arginyl

formalin

least

neurotoxin

positive 3),

the

at

modified

determinant

in

The

damages

spar

well

gain

follows:

critical

neurotoxin

antigenic

the

arginine

indicated

immunoelectrophoresis

borate

as

is

critical

the

each

antibodies

residue(s)

plate)

CYD-modified

of

of

amounts

reaction

is

determinant.

(well

Since for

heights

the

increasing

modification

formation

of

this

antigen.

of

their

modification of

(particularly

only

between

with

explanation

min

Arginine

reactivity reaction

30

loaded

another one

after

induced

and

of

were

protein

specificities.

determinant

wells

of

cones

neurotoxin

different

wells

amounts

difference

treated

well

three

decreasing

progressive

the

if

equal

height

min

BIOPHYSICAL

2. Electroimmunodiffusion analysis of type A neurotoxin treated with and without CHD. Wells left to right: Neurotoxin treated with CHD for zero min (about 20-30 set), neurotoxin not treated with CHD (control), neurotoxin treated with CHD for 30, 60, 90 min, and control. Each well received 3.6 ug protein in 20 ~1. Plate (8 x 10 cm) is 0.6% agar in barbital-acetate buffer, pH 8.6 (see ref. 8 for composition of the buffer) containing antiserum (6133) 1:300 diluted. Current 10 mA (across 8 cm) for 5.5 hr at 7'C.

progressed, of

AND

faster

also with

residue a negative

the in

Vol.

93, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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Fig. 3. Immunoelectrophoresis of type A neurotoxin. Wells from left to right: Neurotoxin treated with CHD for zero (about 20-30 set), 30, 60 and 90 min. Each well received 16.3 ~8 protein in 10 ~1. After 4.5 hours of electrophoresis at 7°C at 20 mA current (across 10 cm) the parallel troughs were filled with antiserum (f/133) 1:4 diluted. Plate (8 x 10 cm) is 0.6% agar in barbital-acetate buffer, pH 8.6 (same as used in electroimmunodiffusion analysis). charge it

(4);

hence

undergoes

toxin

a protein

modification.

was modified

the heights

of arginyl

residues

reactivity

(the

to the modification

that

increasingly

3 also migrated.

therefore

are

and ii) of the

critical

of 150,000

negatively

shows

that

appear

the longer

to be due to:

resulting

and not mol.

wt.

critical

arginyl

from for

as

the neuroeffects i)

on

enhanced

the modification serological

has approximately

damage of one serologically critical

charged

The two opposing

of the neurotoxin

neurotoxin 1),

it

two cones

mobility

ref.

Fig.

the faster

of the

electrophoretic

residues,

becomes

reactive

37 arginine site

due

residue(s).

References 1.

2. 3. 4. 5. 6. 7.

a.

DasGupta, B.R., and Sugiyama, H. Biochemistry and Pharmacology of botulinum and tetanus neurotoxin in Perspectively in Toxinology, Bernheimer, A.W. (ea.). John Wiley & Sons. New York, pp. 87-119 (1977). & Environ. Microbial. 33, 400 (1977). Syuto, B. and Kubo, S. Aeel. & Immun. Miyazaki, S. and Iwasaki, M., and Sakaguchi, G. Infect. 17, 395 (1977). Pathy, L. and Smith, E.L. J. Biol. Chem. 250, 557 (1975). & Immun. 5, 587 (1972). H. Infect. DasGupta, B.R. and Sugiyama, Chem. 244, 4406 (1969). Weber, K. and Osborn, M. J. Biol. DasGupta, B.R., and Sugiyama, H. Biochem. Biophys. Res. Commun. 48, :t (1972). Appl. Microbial. 6, 801 (1972). Duncan, C.L. and Somers, E.B. 375