Vol. 169, No. 2, 1990 June 15, 1990

BINDING

BIOCHEMICAL

AND INTERNALIZATION

AND BJOPHYSICAL RESEARCH COMMUNICATIONS Pages 602-609

OF RICIN LABBLLED ISOTHIOCYANATE

WITH FLUORESCEIN

A. Bellelli! R. Ippoliti! M. Brunori!* Z. Kam? H. Benv niste? F&toy s F. Emmanuel? E. Turpin 3 A. Alfsen3and J.P. ' Dipartinento Holecolare

di de1

*Polymer

Scienae C.N.R.,

Department,

3Equipe

de

Biochimiche Univsrsita' Roma,Italy

The Weizmann Israel

e

Centro Roma "La

di

Institute

of Science,

Recherche n. 64 du C.N.R.S., U.E.R. Saints Pe'res, Paris, France

Received April

di Biologia Sapienza", Rehovot,

Biomedioale

des

12, 1990

lectin ricin has been covalently labelled with The toxic active A chain. isothiocyanate on the enzymatically fluorescein The fluorescein reacted toxin maintains its biological activity. The lateral diffusion coefficient of cell surface bound ricin, studied in two cell lines by fluorescence photobleaching Fluorescence microscopy recovery, is D = 1 - 2 x lo-lo cm*/,. evidence for secondary endosomes in the provides preliminary cytoplasm. a 1990 Academic Press, Inc. the

Ricin, communis, named

cytotoxic

A

and

B

The

(HW

chain

B

N-acetyl

galactosamine

triggers

the

inhibits

protein to

rRNA

reaches

A (105the

is

uptake

said

*Address for M. Brunori, Universita'

of

30

kDa

binds

of

each)

the

toxin

large

number

lo6

sites/cell), under

to be sufficient

of ricin but limiting to kill

seeds

and by

602

chains,

the

cell;

binds

A chain

of to

number

conditions, one cell

and

the

4324

a small

or

inactivation,

at position molecules

bridge

glycolipids,

the the

of

(4).

Roma,

28s cell

toxins

one molecule

di Soienze Biochimiche Piazza Aldo Horo 5, 00185

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Ricinus

of galactose

ribosome

only

of

by a disulfide residues

by irreversible adenine

the

two polypeptide

linked

glycoproteins

a single

correspondence: Dipartimento La Sapienza,

of

to terminal

on

cytoplasm;

from

consisting

synthesis

removal

(3).

membrane

ricin

isolated

is a heterodimer,

(1,2).

due

lectin

Italy.

of

Vol.

169, No. 2, 1990

Ricin

gold

receptor

mediated

not

with

labelled

colloidal

is

BIOCHEMICAL

yet

active

A

ribosome;

has

ferritin,

been

chain

escape(s)

moreover

the

the

5 for

enter

ricin

by selecting

different

via

however,

molecule

macromolecules,

or

cell

the

compartment

with

interfere

peroxidase

a review);

complete

vesicular

labelling

may

to

(see

how and where

RESEARCH COMMUNICATIONS

horseradish

demonstrated

endocytosis

clear

endocytosis,

AND BIOPHYSICAL

to

it

or the

attack

which

the affect

intracellular

routings. These

problems

living

cells.

ricin

labelled

chain microscopy receptors, preliminary

may As

be

a first

with

(called we

attacked step

fluorescein

F-ricin),

is

determined

the

its evidence

in

lateral for

this

microscopy

direction,

we report

isothiocyanate, fully

active.

binding

mobility

of and

secondary

MATERIALS

by quantitative

bound Employing F-ricin

endocytosis,

endosomes

containing

on that

to

the

A

optical to membrane providing F-ricin.

AND METHODS

A homogeneous preparation of ricin was obtained from Ricinus communis (var. Vilmorin-Andrieux, Paris, seeds sanguineus, France) following the method of Nicolson and Blaustein (6); its concentration was determined by absorbance at 280 nm, at pH 7.0, using the extintion coefficient 1.4 l/g.cm (7) and 0.765 and 1.49 l/g s cm for the A and B chains respectively (6). Reaction of ricin with fluorescein isothiocyanate (Sigma, USA), carried out at pH 8.1 as described by Rinderknecht (9) for serum proteins was monitored by alkaline electrophoresis. Fluorescein concentration was estimated using E=72/mM.cm at 495 nm (10). F-ricin was purified by means of ion exchange chromatography a 2x22 cm column of DEAE cellulose (DE52, Whatman), in 0.025 M Giis-HCl buffer PH 6.8 using a linear gradient of NaCl (0 to 0.5 M) in 400 ml of buffer; flow rate was 8 ml/h. Page electrophoresis and isoelectric focusing were carried out following standard procedures. Hemoagglutination assays were carried out in Coke microplates as previously described (11) with human O-erythrocytes 2% final (CNTS, Orsay, France). Protein synthesis concentration experiments were performed on Zajdela hepatoma cells following M. Decastel (personal communication); IC50 in cell free assay was rabbit reticulocyte lysate according to Emmanuel et measured in al. (8). a Cary 210 speotrophotometer or Spectra were recorded with with a Jobin et Yvon JY3 spectrofluorimeter coupled to a Hewlett Packard 9815 A desk computer and 9862 A plotter. reoovery experiments were carried Fluorescence photobleaching and ohinese hamster ovary cells with an apparatus out on A 431 603

Vol.

169, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

similar to that described by Livneh et al. (12), and analyzed as described by Koppel et al. (13). Quantitative nicro%copy experiments were carried out with a Zeiss Axiomat microscope connected with a Venus image intensified TVZM video camera; digitation and image processing were achieved using a Gould De Anza FD 5000 interfaced with a Digital Vax 11/780 computer. RESULTS AND DISCUSSION Preparation

and

of

activity

fluorescein

labelled

ricin

(F-ricin). The

reaction

sluggish.

of

After

isothiooyanatt protein

focusing.

of

F-ricin

estimated is

homogeneous

of

nm upon F-ricin It

retains

ie

to

haemagglutinating pg/ml

cells

for from

binding

is

either

native

partially

prevented ricin

displaced

to

to per

since

F-ricin

indicate

that

F-ricin

mole.

Emission

indicate

spectra

a detectable

residues

intensity

a

was

1.1;

tryptophanyl

biological

activitiee

galactosides

to

bound

of emission

native (as

0'

of

at 521

from

25 jWm1

to

lactose; the

shown

(to binds

the

moreover cell

604

membrane

toxin.

by

affinity

P-150)

with

by fluorescence

addition

native

BioGel

F-ricin

observed

or

(as

trythrocytes

ricin).

by

of the

- aminoethyl

oonctntration

mouse

in

290 nm.

group

the

protein

combination

290 or 495 nm)

lactosaminyl

human

detected.

fluorescein

this

enhanced

also

labelled

FPLC and isoelectric

bound

the

of

by PAGE,

to be 0.8

we take

bind

on

agglutinate

of

of the

the

able

chromatography

18

initial

is

nH fluorescein

fraction

"20% of the

at

at

the

yields

from

excitation

C,

0.25

were

fluorescein

because

with

components

(excitation

fluorescein,

8'

isothiocyanate

50%; minor

above)

of

transfer

energy

and

spectroscopy

(set

one mole

F-ricin

incubation

stoichiometry

by optical

contain%

fluorescein

a% controlled

form,

The

of

8.1

PH

exceed

purified

with

days

not

Purification highly

two at

did

ricin

a

and to minimum

be compared

with

to B18 melanoma microscopy)

incubation bound

F-ricin

by incubation

and

medium

of

can be for

1

Vol.

169, No. 2, 1990

hour

with

(added

10 to

l/2

in

F-rioin

rat

experiments), of

After

fig.2,

essentially

chain,

while

2.2

x lo-'M

indicating

that

of

the

by

ricin

or lactose

(fig. for

inhibition

1A). native

The IC5Cis ricfn

toxicity

is

only

chain

interchain

by all

of

protein

5.1

x lo-'ll

(average halved

of three by

ricin)

covalent

reacted

=

is

is eluted

amount

the

chromatography.

fluorescein

after

(IC50

disfulfide,

affinity

the

a small

fluorescein A

native

of F-ricin),

cells

separated

non splitted

native

either

estimated

hepatona

reduction were

The

of

RESEARCH COMMUNICATIONS

fluorescein.

chains

the

excess

was

and

AND BIOPHYSICAL

addition

toxicity

sinthesis

binding

50 fold after

hour

F-ricin

for

BIOCHEMICAL

addition

of

A

retains

chain

1 x 10'M),

0.1

As shown in

eluted

with

A and B

with

the

the

B chain

(and

toxicity

of

M lactose. all

the

as estimated

in

the

rabbit

B

Molar

-10 -9 Concentration

-8 (log)

-7

-13 Molar

A

-11 Concentration

-9

-7 (log)

Fig.1. Panel A: Toxicity of fluorescein labeled and unlabeled ricr on Zajdela rat hepatoma cells. After 90 nin preincubation at 37°C with labelled (0) or unlabelled (0) rici,q, the cells (1.5 X 103) were incubated for 90 nin at 37°C with C-leucine in 0.5 ml of leucine free HEM; then the TCA precipitable radioactivity was determinqt. Results are expressed as percent of the toxin free control C-leucine incorporation into precipitable material. Panel B: Toxicity of the fluorescein labeled and unlabelled A chain on the reticulocyte lysate. Reaction mixtures (30 Pl) containing 10 ~1 reticulocyte lysate were incubated for 90 nin at 37 C in the presence of various concentrations of labelled (0) with 10 X and unlabelled (m) A chain. Proteins were precipitated trichloroacetic acid and harvested. Results are expressed as percent of the toxin free control 3H-leucine incorporation.

Vol. 169, No. 2, 1990

reticulocyte

lysate

F-ricin

and

rRNA.

a

(14)

Zajdela

this

alternative

these

cells.

The were

diffusive

Chinese

typical

trace

lateral

diffusion

fluorescence.

moderately

properties

hamster

not

in

for

the

of

that

the

binding

to the

cell

attack

to the

26 S

the

toxic

via

a nannose

effect

personal

toxicity

of the

of ricin

communication),

of F-ricin

tested

- F-ricin

photobleaching (CHO)

is

shown in

coefficients

may be fortuitous latter

recovery

and human

and

parameters ricin

the

receptors

ovary

recording

of

is concluded the

Decastel,

account

efficiency

pathway

part

(K.

properties

modified this

take

toxic

on

of F-rioin.

These

fluorescein

to

by fluorescence

lines:

it

but

cells

cannot

the

internalization

seems not

measured

ricin

secondary

dynamics

although

Since (fig.lA),

affects

hepatona

Cellular

(fig.lB>.

/ or internalization,

Since

receptor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

diminished

modification

surface

the

assay

is somewhat

covalent

on

BIOCHEMICAL

(15);

3; table

the

fractional reported

they in view

on two cell

A431 carcinoma.

fig.

were

complexes

are

I reports

before similar

which

leads

the

recovery

of

for to

of some uncertainty

preparation

A

a

ours, about

to a 120 KDa

derivative. 700.1

02

03

O.O 20.0

40.060.060.0 100.‘20~ time kec)

Fig.2. Separation of the polypeptide chains of reduced F-ricin affinity chromatography on lactosamynil BioGel P-150. Elution was monitored through fluorescence emission at 340 (A) and 530 wavelenght (Ah) nm; excitation was 290 and 480 nm respectively. The arrow indicates addition of 1 ml 0.1 Ii lactose. by

Fig.3. recovery for

Typical

experiment. 20 min at 20°C.

recording A 431

of a fluorescence cells were incubated 606

photobleaching with 150 nH FR

140.

Vol.

BIOCHEMICAL

169, No. 2, 1990

TABLE I.

Some diffusive

Cells

F-ricin

A431

150 nil

CHO

The

is

(16)

and

higher almost

of ricin

1.7

--10

x 10

(20.6)

receptors

X Mobile

x 10-l'

1.0 (+0.5)

RESEARCH COMMUNICATIONS

n

40 (t20)

60

50 (t20)

46

cells (2 x lo5 cells/plate) with 150 nh F-ricin in for 15'; measurements were taken within 1 hour from the incubation and the recovery of fluorescence ms laser pulse was followed for 2 to 3 min.; n number of measurements.

coefficient

diffusion

work

properties D (cm /s)

150 nM

Incubation of PBS at 20" C beginning of after a 100 represents the

AND BIOPHYSICAL

than

that

comparable

measured of

the

tetravalent

with

those

for

F-ricin

in

ltctin measured

the

present

Concanavalin for

many membrane

Fluorescence nioroscopy image of an A 431 cell Fig.4. with 150 nH F-ricin for 40 min at 2O“C. The sample was incubated epi illuminated with blue light and the image was recorded with a VAX RCA high resolution video camera oonnected with a Digital The large white spots (p) correspond to 11/760 computer. glycoprotein patches on the cell membrane, while the smaller spots (e) focus at a deeper level and are suggested to represent

secondary

endosomes.

607

A

Vol.

169, No. 2, 1990

receptors

(like

recovery

of

table

I)

or

BIOCHEMICAL

EGF,

lies

higher

fluorescence

in well

correlates rioin

cm2/s,

after

12).

in

between

that

(40 of most

probes

mobility

and

F-ricin,

as compared,

the

The fractional

photobleaching

of non agglutinating

with

RESEARCH COMMUNICATIONS

greater

relatively

- 50 X, see

lectins

(80

more

X

to 90 X1. The

fractional for

(20

recovery

example

of

to Con A (12)

efficient

endocytosis

of

(4,5). Like

the

all

patching

of

spots

in

after

the

one hour

from

incubation of

cell

periphery

brilliant

spots

fluorescence analysis represent 0.3

17)

incubation

time

receptors,

toxicity

the

secondary labelling intracellular thereby intracellular

toxin.

endosonee,

contribute

already

incubation

areas

uneven

medium.

clearly Even

patching, detected

though

spots

of their

in

a refined

are

likely

diameter

frequency

The

within

of massive

cases

in view

to

microscopy does the

though A of

not

A chain,

(0.5

increases

to

impair

active elucidation

routing. 608

suitable cells,

to to

with

the

probe

for

since

binding

patching

to

indicates allow

to

subunit of

the

membrane

and internalization

internalized

preliminary, may

a

living

of

chain the

be

on

identification

the fate

is

4.

appreciably

smaller

appears

of The

of

fig.

to one hour).

dynamic modification

the

(fig.4).

their

F-ricin

chemical

in

these

because

(up

conclusion

quantitative

images

shown in

change

some

endosomes,

and

not

and the in

superficial

membrane

toxin

attempted,

secondary

pm,

In

not

free

were

microscopy was

cell

extensive

as large

image

and does

absence

the

the

on

induces

evident

microscopy

F-ricin

min

ricin

receptors,

fluorescence

in the

Far

lectins,

membrane

of 15-20

tiny

polyvalent

its

distribution

of

10

somewhat

and that

slightly

-10

x

fluorescence

also

lower)

2

AND BIOPHYSICAL

in the

F-ricin that

selective

investigate living dynamics

in

the cells of

and the

Vol.

169, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS The authors are deeply indebted to Dr. Il. Decastel for help with the protein synthesis inhibition on hepatoma cells and to Prof. L. Bellelli for some microscopy experiments, Support from the C.N.R.S. of France and the N.C.R.D. of Israel gratefully acknowledged. Grants were received from the iztituto Pa5teur - Fondazione Cenci Bolognetti, Rome, and the C.N.R., P.F. Biotecnologie e Biostrunentazioni. REFERENCES 1. 2. 3. 4. 5. 8. 7. 8. 9. 10. 11. 12. 13. 14. 15. 18. 17.

Olsnes S. and Pihl A. (1973) Biochemistry 12, 3121-3128. Stirpe F. and Barbieri (1988) FEBS Lett. 195, l-8. Endo Y. and Tsurugi K. (1987) J. Biol. Chen. 282, 8128-8130. Olsnes S. and Sandvig K. (1983) in Receptors and Recognition, Series B, vol 15, pp. 188-238, P. Cuatreoasas and T.F. Roth editors, Chapman and Hall, London. van Deurs B., Tonnessen T.I., Petersen O.W., Sandvig K. and Olsnes S. (1988) J. Cell Biol. 102, 37-47. Nicolson G. and Blaustein (1972) Biochin. Biophys. Acta 288, 543-557. Zentz C., Frenoy J.P. and Bourrillon R. (1978) Biochim. Biophys. Acta 538, 18-28. Emmanuel F., Turpin E., Alfsen A. and Frenoy J.P. (1988) Anal. Biochem. 173, 134-141. Rinderknecht H. (1982) Nature 193, 187-188. Chen R.F. (1989) Arch. Biochem. Biophys. 133, 283-278. Turpin E., Wantyghem J., Beaudry P., Neel D. and Goussault Y. (1984) Can. J. Biochem. Cell Biol. 82, 203-208. Livneh E., Benveniste M., Prywes R., Felder S., Kam Z. and Schlessinger J. (1988) J. Cell Biol. 103, 327-331. Koppel D.E., Axelrod D., Schlessinger J., Elson E.L. and Webb W.W. (1978) Biophys. 3. 18, 1315-1327. Skilleter D.N. and Foxwell B.M.J. (1986) FEBS Lett., 198, 344-348. Zagyansky Y.A. and Jard S. (1979) Nature 280, 591-593. Schlessinger J., Koppel D.E., Axelrod D., Jacobson K., Webb W.W. and Elson E.L. (1978) Proc. Natl. Acad. Sci. U.S.A. 73, 2409-2413. Alberts B., Eray D., Lewis J., Raff M., Roberts K. and Watson J.D. (1983) Molecular Biology of the Cell, Garland Publ., New York and London.

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