Synthesis and invitro activity of a hormone-diphtheria toxin fragment a hybrid

Vol.

133,

No. 2, 1985

December

17,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

SYNTHESIS AND --IN VITRO HORMONE-DIPHTHERIA TOXIN Thomas

N.

ACTIVITY FRAGMENT

430-435

OF A A HYBRID

Oeltmann

Department of Medicine Vanderbilt School of Medicine Nashville, TN 37232 Received

October

25,

1985

The synthesis and in vitro biological activity of intact human chorionicgonadotropin and fragment disulfide conjugate is reported. This hybrid retained of uncoupled hCG and was shown to be specifically tumor which binds hCG while being non-toxic towards for hCG. 0 1985 Academic Press, Inc.

During

the

toxins”. or

past

The

term

polyclonal, on

amodel

system

tropin-diphtheria activity

Since

the

hCG

currently

thus

as

have many fail

to

into

the

cytoplasm

Abbreviations NaCl, pH 7.2); pyridyldithiopropionate; trichloroacetic

hCG

we

the

will

on be

able

intracellular with cell

the

many

where

to

tumor

this cell

to

it

can

and exert

430

continuing

we

have

developed

chorionic

gonado-

investigated

its

Leydig

cell

tumor

which

internalized,

that our

-in

tumor.

antigens is

surfaces

are efforts

such

as

acidification

the

escape

of

its

toxic

effect.

used: PBS, phosphate buffered saline SPDP, N-succinimidyl-3-(2-pyridyldithiol)-propionate; SDS, sodium dodecyl sulfate; acid; hCG, human chorionic gonadotropin.

0006-291X/85 $1.50 CopVright 0 I985 by Academic Press, Inc. All rights of reproduction in any form resewed.

our

receptor

concentrate

events lysosome,

and

unlike bound

In

mono

usingmonoclonal

a human

defined

“isxnuno-

antibody,

encountered

a well

function,

in

binding

proteins,

(hCG-DTA)

using

interest

toxin.

synthesised

toxin

and

in

a cell

wehave

protein

complexes

fusion of

problems

the

a known

subsequent

vesicle,

of

function,

internalize, of

hybrid

these

We have

antigen-antibody

investigation endocytotic

no known

to

of

A hybrid

has

refers

a hybrid protein composed A of diphtheria toxin in a >90% of the binding ability toxic to a mouse Leydig cell cells which lack receptors

increase

uptake

specific

receptor

an

bacterial

moiety.

a cell

been

a plant,or

some

fragment

vitro

to

mediated

binding

has

usually

bound

to overcome the

there

“immunotoxin”

receptor

as

unlike

years

covalently

studies

antibody

five

of

the

toxic

(10

ml4 phosphate,

CON A,

Conconavalin

shed

and

on

the

of

the

moiety

0.15 M PDP, 2A; TCA,

Vol.

133,

No. 2, 1985

BIOCHEMICAL

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

Reagents and cell culture: Human chorionic gonadotropin (hCG) was obtained as a gift from the National Pituitary Agency, Baltimore, Maryland. N-Succiminidyl-3(Z-pyridyldithiol-propionate (SPDP) was obtained from Sigma. The M548OP Leydig tumor was obtained from the PapanicolauCancer Research Institute inMiami, Florida and was originally a gift of Dr. William Neaves of the University of Texas Health Science Center at Dallas to Dr. David Puett when he was at this institution. The tumors are maintained by serial transplantation every 2 weeks into 7-9 week old male C57 B1/6 mice by the method of Neaves (1). Single cell suspensions of tumor are prepared by the method of Ascoli and Puett (2). K562, a human erythroid precursor cell, and Molt-4, a human T cell line, were used as control cell lines. Anti-diphtheria toxin was obtained from Connaught Laboratories, Swiftwater, PA and Anti-hCG was obtained from Miles Laboratories, Inc., Elkhart, IN. All other chemicals and reagents were of the highest quality available commercially. Methods: hCG was labeled with lz51 by the Chloramine-T method of Greenwood et al (3) asmodifiedby Bellisarioand Bahl (4) for the retention of biological actigtr Protein was estimated by the ultraviolet spectrophotometric method of Waddel (5). Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate was carried out by the method of Laemmil (6) as modified by O'Farrell Crude diphtheria --et al (7). toxin was obtained from Connaught Laboratories, Toronto, Canada. After purification, Fragment A was prepared as described by Chung and Collier (8). Synthesis of hCG-diphtheria toxin fragment A: hCG was coupled to fragment A by methods we have reported (9) and was originally described by Martinez St (10). Briefly, to 5mg hCG in 1 ml O.lM sodium phosphate, pH 7.2 containing 0.15 M NaCl (PBS) was added SPDP in the amounts indicated in 50 pl of absolute ethanol. The solution was allowed to stand at room temperature for 30 minutes followed by dialysis at 4°C against PBS for 18 hours. Diphtheria toxin fragment A (3-fold molar excess) was reduced with 50 mM dithiothreitol overnight, passed over a G25 Sephadex column and immediately mixed with the Z-pyridyldithiol-propionateThis mixture was incubated at room temperature for 1 hour and the hCG (PDP-hCG). resulting coupled product was isolated as described in the results section. Cellular Protein Synthesis: Protein synthesis was estimated by measuring L(14C)amino acid incorporation in trichloroacetic acid insoluble cell protein as previously described (11). Binding Experiments: Binding of 1251-labeled hCG and 1251-labeled PDP-hCG was carried out using freshly prepared Leydig cells by the method of Chang --. et al (12). Prior to binding, control and PDP-hCG samples were reduced with 20 mM dithiothreitol to release the reactive pyridine 2-thiol to determine the extent of derivitization (13) and remove the highly reactive group which reacts with cell surface proteins and produces artifactuallyhigh binding. Following the reduction, all hCG samples were reannealed by dialysis against PBS at 4°C for 24 hours.

RESULTS AND DISCUSSION The inactivation of free the

fragment

SPSP method

modification that

of

A into

in cells

the cytosol.

by diphtheria Fragment

we have previously

used (14).

free

groups

and it

to four

or more

hormone

to its

modification

the binding

of EF-2

amino

of three

ability

of this

radiolabeled

hCG with

be used which

would

SPDP in order

is

431

requires

A was therefore However, known

of the specific

to determine

give maximum derivatixation

toxin

this

from

coupled method

the

lysines

work

ratio

of hCGwith

to hCG by involves

the

of Puett

(15)

of hCG greatly

receptor. what

the release

Thus,

reduce

we titrated

of SPDP to hCG could minimal

loss

of binding

Vol.

133,

activity.

The results

not result with

BIOCHEMICAL

No. 2. 1985

in loss

of these

of hCG and these Once the

ratio

derivatization

not

are

of

hCG using mixture

bound

was first

while

the

retained

by virture

A with

the Sepharose

hCG-DTA of the

its

was then

mannose

fragment retained

containing

The free

with

Thus,

only

column.

diphtheria

Ratio

toxin

*Binding derivatized

A) was not

bound

to

the

the bound hCG-diphtheria

toxin

TO LEYDIG

40

1.0

97

a3

5.0

40

>I00

10.0

10

>I00

20.0

9

>lOO

50.0

10

>lOO

at 4°C for 1 hour.

54,074

dpm with

non-specific

"Based on release of pyridine 2-thiol (13). there was more than 1 mole of PDP incorporated incorporated for the 5O:l ratio.

432

fragment coupled

of toxin

to hCG

Control

affinity A chain

column. hybrid.

CELL

binding (nonbinding subtracted.

With ratios per mole

of of

is

of the protein

Molar % hCG derivatized**

96

was

were

The resulting

A bound

0.5

hCG)

of the

diphtheria

1, the majority

(X control)

was conducted

A chain,

hCG, because

fragment

Binding*

(SPDP:hCG)

column.

to

hCG was

as the A chain

to ConA whereas

Table 1 EFFECT OF SPDP MODIFICATION OF hCG ON BINDING Molar

free

10 mM adenine.

As shown in Figure

eluted

Uncoupled

site

as well

as maximum

The resultant

(8).

column.

A-Sepharose binds

conducted

A was coupled

as unreacted

A chain

diphtheria

Fragment

ofo-methylmannoside

fragment

reported

PBS containing

oligosaccharide,

not.

as well

of the NAD+ binding

to a Conconavalin

on the affinity

(unincorporated Addition

applied

A does

interaction

eluted

toxin

as well

did

80% derivatization

maximum binding

previously

hybrid,

reactions

in over

to a NAD-Sepharose

bound NAD+.

to hCG was subsequently eluent

applied

ratio

molar

1.

diphtheria

we have

COMMUNICATIONS

a I:1

In addition,

in Table

SPDP to hCG to give

methods

that

resulted

more

summarized

RESEARCH

indicated of hCG.

or

had been established,

derivatized reaction

binding

at 10 mg/ml

results

BIOPHYSICAL

experiments

of significant

hCG concentrations

AND

5:l or greater, hCG up to 5 moles

Vol.

133,

No.

2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

.5

.4

x a

I!IO 50

83

20

,2

-i

1 70

so

30

I

I

I

80

90

loo

FRACTION

of

63,

65,

mercaptoethanol

there

is

upon

no

free

A chain

recution

hCG u and

FIGURE 2. (a) 20 ug 70; (e) 20 ul fraction e were not phoresis.

the

A chain

(10

ug)

b

the is

does

c

present

well

d

It

under (lane

can

our

is

presence be

seen

and in

fractions

Figure (b,c,d)

interesting

conditions

absence

(lane

to

note

a),

but

f).

f

e

the

eluted

(g,h,i).

stain

in As

gel.

or-methylmannoside

not

stain

column (3ml) was directly with PBS. The column was was then eluted with 0.5 M

examined

SDS-polyacrylamide

in

I3 subunits

a

1) were

67 (Figure

on a 14%

reduction,

non-reduced

and

I3

NUMBER

The fraction eluted from the NAD-Sepharose FIGURE 1. applied to the Con-A Sepharose column equilibrated washed with PBS until A 280 = 0 (insert). The hybrid a-methylmannoside in PBS.

Frac:tions

40

9

h

I

i

SDS polyacrylamide gel electrophoresis of samples from the Con-A column. hCG; (b) 40 pl fraction 58; (c) 40 ul fraction 60; (d) 40 ul fraction ug nicked diphtheria toxin; (f) 20 pg hCG; (g) 40 pl fraction 58; (h) 40 diphtheria toxin. Lanes a60; (i) 40 u1 fraction 70; (j) 20 pg nicked reduced, lanes E-j were reduced with 2-mercaptoethanol prior to electro-

433

2, but that upon

Vol.

133,

No. 2, 1985

BIOCHEMICAL

-+ -It-

AND

Dipbthcria KG-S-S-D14

1 IO-”

I lo-‘0

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Toxin

I 10-g

‘X-X

I 10-8

10-7

[TOXIN],

10-S

M

FIGURE 3. 1 ml of freshly isolated Leydig cells 105/ml in RPMI-1640 with the indicated concentrations of toxin or hybrid for 6 hours. acid mixture was added and the incubation continued for an additional incubations were stopped with the addition of 100 pl of 100% TCA and Controls incorporated 43,600 + 1540 cpm. content determined. average of three incubations.

Fractions results

shown

with

intact

are

resistant

is

a dose not

the

orders

10-6

effects

of

the

hybrid.

As

can

magnitude

reported

and

no

lack

that

up to

of

had

concentrated

3 indicate

toxic

hybrid

toxin

dependent

manner

and

while

assayed

there

is

M, remembering toxin,

for

little

the

cells

hybrid

has hCGB

than

the

K562,

Molt-4,

or

mouse

In

addition,

no

murine

Leydig

toxic

activity. or

that

the

be seen,

more

Typical toxicity

cells were an

seen

in

general

exquisitely

LD50

= 5 x 10-8~

chain-ricin

A chain

(11,141.

effect

on

hCG receptors.

diphtheria

was

able

which

to

inhibit

antibody the

paralleled

L cells

the ir

which

directed

toxic

activity

abi lity

to

were

towards of

the

inhibit

used either

hybrid

binding

in (data

shown). Based

-Ricin

on

these

A chain

previously hCG and the

pooled,

toxin

previously

controls or

the

several

The

hCG

figure

to to

we have

as

in

were

diphtheria

sensitive and

55-75

were treated [ l4C]-amino hour. The the radioactive Points are the

same

conjugate,

reported the

seed

results,

we using

(11,141, protein

gelonin

had

also

prepared

intact

hCG

an (16)

LD50

434

rather

= lo-9M.

exhibited

conditions.

two

other than The

an LD50

hCG the

hybrids.

13 subunit

conjugate = lo-gM

The alone

prepared when

assayed

hCG as

we

using under

Vol.

133,

The

above

of

the

of

monoclonal

for many

described

problems

its

Leydig

which

are

antibodies

receptor tumor

antigens

which

complexes

on

tumor

is cells

be

to

receptors

of

known

function.

deficient

in

binding

and/or

this

tumor

may

and

currently

For

this have

known

to

which

are

in

toxins.

cell

extended

here,

in

model

be

the

receptor no

and

model

to

include

Studies

for

overcoming hybrids

is

a known

of

In

unlike

addition,

and

other

study

the

antigen-antibody More

underway further

specific

function,

many

such

many composed

highly

internalize.

currently

internalization

of hCG

unlike to

in

function.

studies

are

study

has

known

fail

COMMUNICATIONS

be useful

instance,

internalized

shed

RESEARCH

of macromolecules,

reported

pathway

BIOPHYSICAL

encountered

on receptormediateduptake can

tumor

currently

Leydig

complex

AND

cell

coupled

on the

hCG-receptor

studies

BIOCHEMICAL

No. 2, 1985

the

generally,

hybrid

unique

proteins

cell

to

isolate

of

the

surface mutants

endocytotic

system.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Neaves, W. B. (1973) J. Natl. Cancer Inst., 0, 1069-1073. Ascoli, M. and Puett, D. (1978). Proc. Natl. Acad. Sci. USA, 75, 99-102. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J., E, 114-123. Bellisario, R. and Bahl, 0. P. (1975) J. Biol Chem. 250, 3837-3844. Waddell, W. J. (1956) J. Lab. Clin. Med., 48, 311-314. (1970) Nature, 227, 680-685. Laemmli, V. K. O’Farrell, P., Goodman, H., and O’Farrell, P. (1977) Cell, 12, 1133-1142. Chung, D. N. and Collier, R. J. (1977) Biochem. Biophys. Acta, 483, 248-257. Oeltmann, T. N. and Forbes, J. T. (1981) Arch. Biochem. Biophys., 209, 362-370. Wallace, E. F., and Wofsy, L. (19801, Fed. Proc. Fed. Amer. Martinez, O., Sot. Exp. Biol., 21, 719. Oeltmann, T.N. and Heath, E. C. (1979) J. Biol. Chem., 254, 1028-1032. Daxard, A. and Neville, D. M., Jr. (1977) J. xl. Chem., 252, Chang, T., 1515-1522. Stuchbury , T. , Shipton, M. and Norris, R. (1975) Biochem. .I., 151, 417-432. Oeltmann, T. N. and Heath, E. C. (1979) J. Biol. Chem., 254, 1022-1028. Puett, D. J., personal communication. Stirpe, F., Olsnes, S. and Pihl, A. (1980) J. Biol. Chem., 255, 6947-6955.

435