Human autoantibodies against a nucleolar protein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 163, No. 2, 1989

Pages 1119-1127

September 15, 1989

HUMAN

AUTOANTIBODIES

AGAINST

Ingeta

Institute

A NUCLEOLAR

PROTEIN

Kind~s-MOgge

of T u m o r b i o i o g y and Cancer Research U n i v e r s i t y oF Vienna Vienna, Austria, B o r s c h k e g a s s e 8a

A-f090 Received June 6, 1989

SUMMARY: A u t o i m m u n e sera showing p r o m i n e n t i m m u n o f l u o r e s c e n c e in nucleolus were selected and analysed by immunoblotting techniques. Immunoblots using a nucleolac extract as antigen source revealed seva r e c o g n i z i n g a ]8 kDa n u c l e o l a v protein. Low c o n c e n t r a t i o n of A c t i n o m y c i n D, which inhibits the ribosomal RNA synthesis, caused a loss of f l u o r e s c e n c e . This suggests that the nucleolar antigen may be associated with the assembly or p a c k a g i n g of the ribosomes. The present nucteolar antigen has p r o p e r t i e s similar to the p r e v i o u s l y d e s c r i b e d n u c l e o l a c phosphoprotein B2] of cat cells and the recently d e s c r i b e d n u c l e o l a c protein N038 of mouse and Xenopus cells, oigsgAc~ae~icP..... Znc.

Antibodies served

specific

in human

gressive

systemic

patients

are

nucleolav

7-2

mevase to be

65,

and

In

of

bodies by

present

which

has

a

]4

nueteolus

The

kD

sera

ace

are

frequently

sera

ob-

in pro-

of

sclevodetma

particles

containing

protein

in the n u c t e o l u s

rheumatic

which

with

bind

several

of mouse

B2] and

[

(4).

RNA

has been

autoimmune

directed

against

attempt

to

are

sera

of

recognized

autoimmune

prominently

properties

phosphopcotein NO]8

located

and

with

immunofluorescence

which

(2).

the most

precipitate

(})

study

antigens patients

revealed

of

(1),

poly-

described

diseases

(5).

The

proteins

of

120,

classify

the

24 kDa.

the

nucleolac

these

(PSS)

to

UsRNA

I, e x c l u s i v e l y

42,

seca

observed RNA,

in

components diseases

sclerosis

immunoveactive

antibodies

for

autoimmune

cat

to

cells cells

]8

(6,

1119

material

kDa

were

to

in

anti-

selected

experiments

nucleolar

ascribed

7) and

found

containing

Immunoblotting a

those

(8,

antibodies Sera

to n u c l e o l a c

microscopy.

Xenopus

by

diseases.

recognizing similar

further

the

protein, nucleolar

the n u c l e o l a r

protein

9). 0006-291X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 163, No. 2, 1989

MATERIAL

AND

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MEIHODS

Autoantibodies: All sera used in this study gave prominent n u c l e o l a r s t a i n i n g in i n d i r e c t i m m u n o f l u o r e s c e n c e tests. The sera were obtained from patients with s y s t e m i c lupus e r y t h e m a t o s u s (SLE), p o t y m y o s i t i s and one p a t i e n t had a u t o i m m u n e c h r o n i c a c t i v e h e p a t i t i s . The c l i n i c a l d i a g n o s i s was b a s e d on g e n e r a l l y a c c e p t e d criteria. Sera containing antibodies against ribosomal ribonucleoprotein particles (anti-rRNP) were o b t a i n e d from p a t i e n t s with s y s t e m i c lupus e r y t h e m a t o s u s . C o n t r o l sera w e r e c o l l e c t e d from 42 h e a l t h y i n d i v i d u a l s . Antibody screeninq procedure: L o c a l i z a t i o n of a n t i g e n was d e t e r m i n e d by i n d i r e c t i m m u n o F l u o r e s c e n c e tests and p e r f o r m e d on Hep-2 c e l l s a c c o r d i n g to the i n s t r u c t i o n s of the m a n u f a c t u r a ( K a l l e s t a d Inc. A u s t i n , Texas). TiLers > 40 were r e c o r d e d as p o s i t i v e . P r e p a r a t i o n of HeLa ceil n u c t e i ~ n u c l e o t i and r i b o s o m e s : HeLa cell n u c l e i w e r e p r e p a r e d as d e s c r i b e d (IO). R i b o s o m e s were obt a i n e d from the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t by c e n t r i f u g a t i o n for three hours at 120.000 g, the nucleoli were isolated essentially by the p r o c e d u r e of P e t e r s and C o m m i n g s (It). Thus, sonicated nuclei were centrifuged through a layer of 0.9 M sucrose, I0 mH T r i s - H C I (pH 7,4) for 20 min at 3 . 0 0 0 g. The pellet containing the n u c l e o l i was r e s u s p e n d e d and r e s e d i m e n t e d t h r o u g h 0.9 M s u c r o s e . P r e p a r a t i o n of n u c t e o l a r p r o t e i n B23: The p u r i f i c a t i o n of p r o t e i n B23 was c a r r i e d out as d e s c r i b e d by M i c h a t i k et at. (12). However, a Mono Q c o l u m n (5x50 mm, P h a r m a c i a ) was used i n s t e a d of a DEAE-cellulose c o l u m n . B23 was e t u t e d at tow salt c o n c e n t r a t i o n . For p r e a d s o r b t i o n s t u d i e s of a n t i s e r u m with B2J, B23 was d i l u t e d with five v o l u m e s of a c e t o n and the p r e c i p i t a t e was s o l v e d in PB5. Potyacrylamide gel e t e c t r o p h o r e s i s and i m m u n o d e t e c t i o n or anttqens on n i t r o c e l l u l o s e : R i b o s o m e s , n u c l e o l i and n u c l e o l a r p r o t e i n B23 w e r e d i s s o l v e d in 2% s o d i u m d o d e c y [ s u l f a t e , 10% g l y c e r o l , 5% mercaptoethanol in i00 mM T r i s - H C l (pH 6,8) and s u b j e c t e d to etectrophoresis on 13% or 10% gels (I]). The p r o t e i n s were then transferred onto n i t r o c e l l u l o s e (14) and the r e s u l t i n g r e p l i c a was cut into 5 mm s t r i p s for the a n t i b o d y b i n d i n g assay. Each strip was incubated with d i l u t e d h u m a n sera (l:lO0, 1:50) in saturation buffer (PBS, I% BSA, 0.2% Tween). After extensive washing, immunocomple_xes w e r e detected by i n c u b a t i n g the blots for 2 h o u r s w i t h ( I Z S I ) - p r o t e i n A (OlpCi/ml saturation buffer). Autoradiography was p e r f o r m e d u s i n g Kodak X - 0 m a t Film. In some blotting experiments, (125 I) l a b e l l e d a n t i - h u m a n IgG (0.5 p C i / m l saturation buffer: P8S, 10% fetal c a l f serum, 0 . 5 % Tween) or alkaline phosphatase conjugated anti-human IgG was u s e d i n s t e a d of p r o t e i n A. P r e a d s o r b t i o n of a n t i s e r u m w i t h B23: 50 pt s e r u m was incubated for 6 h o u r s at 4°C with 6 Pg of i s o l a t e d B23 in a total v o l u m e of 150 pl. The s e r u m + B23 was d i l u t e d to 2.5 mi w i t h s a t u r a t i o n b u f f e r and used f u r t h e r as d e s c r i b e d above. A c t i n o m v c i n D t r e a t m e n t of HeLa cells: To i n h i b i t r i b o s o m a l ribon u c l e i c a c i d s y n t h e s i s , HeLa c e l l s w e r e e x p o s e d to a c t i n o m y c i n D (0.04 #g/ml) for 2-3 hours (15, 16). The nuclei were then isolated, Fixed for 20 min in 3% f o r m a l d e h y d e and i m m e d i a t e l y used in i n d i r e c t i m m u n o f l u o r e s c e n c e tests.

1120

Vol. 163, No. 2, 1989

RESULTS

AND

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION

Autolmmune

sera

were

staining.

Eight

sera

reaction

(fig.

la).

reacting

to

staining

in

the

sera

have

also

the

meeting patients using of

shown

with

selected

sera

to

stain,

not

these

sera

extract

had as

small

the

~ere

staining

a protein

of

quantities

ribosomal

(17).

Anti-rRNP (fig.

determined. the

38

kDa a

region,

study

70

The

the

were

(fig. kDa

2).

weight

eight

four In

All

containing

the

protein

sera

performed

molecular

nueleoli,

but

lb).

sera Of

ribo-

Some a n t i - r R N P

Immunoblots

of

active

the

by

of

chronic

cytoplasmic

source.

weak

patients

cell

in

SLE.

for

very

three

collected.

the

recognized

antibodies prominent

were

antigen

showed

SLE,

antibodies

used

autoantibodies

autoimmune

only

of

were

sera had

immuno-

nucleolar

contained

five

had

nucleolar

strong

patients

with

region

criterion

recognize

detected

while

patient

autoantigens

for

sera

(anti-rRNP)

nucleolar this

anti-nucleolar

shown

to

giving

the

Four

sera

particle

a nucleolar the

one

Additional

nucleoprotein

of

cytoplasma.

and

hepatitis.

None

prominent

foc

collected

nucleoplasma

the

polymyositis

screened

were

sera

sera were

addition, and

two

two other

Fig. i: a Indirect immunofluorescence staining on HEp-2 cells with an autoimmune serum containing anti-nucleolar antibodies. b Anti-rRNP, which in addition to cytoplasmic staining also shows nucleolar staining.

1121

Vol. 163, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

92-

i ~ ~;~

68-

92es-

,

..... ....

43-

43g.....

~

W

q

31--

31-

21-"

21-

Q

1

2

3

.....

---

4 5

~---

iii!

123

Q

Fig. 2: N u e l e o l a r proteins recognized by a n t i - n u c l e o l a r a u t o i m m u n e sera. The total proteins of isolated nucleoli v J e r e separated by SDS-PAGE and transferred to n i t r o c e l l u l o s e sheets~ incubated with a n t i - n u c l e o l a r a u t o L m m u n e sera and detection of the antigens ~as p e r f o r m e d as described in the M a t e r i a l s and Methods. (1) control serum; (2-5) sofa recognizing nucleolar antigen. The arro~ indicates the position of a 38 kDa protein. Fig. 3: N u c l e o l a r proteins recognized by a n t i - n u c l e o l a r positive serum and by a n t i - r R N P serum e x h i b i t i n g nucleolar staining in indirect immunofluorescenee. I m m u n o b l o t t i n g e x p e r i m e n t with a solution of nucleolar proteins as antigen. (1) control serum; (2) a n t i - n u c l e o l a r a u t o i m m u n e serum; (3) anti-rRNP serum. The arro~ shows the p o s i t i o n of a 38 kDa protein.

sera

a

nizing

72 a

kDa 69

protein.

kDa

An

protein

antinucleolar

has

been

autoimmune

described

in

serum

a patient

recog-

with

PSS

(18). Anti-rRNP

sera,

showed

prominent

action

with

recognized

or

stained 19).

that

the

Our

antibodies reacting

the

serum

(fig.

which with

a

In

autoantigen that

identify 38

kDa

38

a

of

1122

for

nucleolar

lane is

2

and

the

their

rewas

lane

de-

same

3 for

addition,

r-RNP

16

kDa

kDa

the

protein sera

serum

(lane

autoantigen

3)

antigen. were

the serum

sometimes

nucleolar

also

protein

autoimmune

nucleolar

analyzed.

staining

anti-nucleolar

anti-rRNP 38

tested

kDa

distance

the

3).

cytoplasmic

were

A

Comparison

either

indicate

to

staining

migration

by

ribosomal

data

addition

extract.

sera.

the

recognized

anti-rRNP

in

nucleolar

nucleolar

by

monstrates proteins

a

which

also (17,

include Sera further

Vol. 163, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

92--

b m

68-43-m 31--

21-

I

14-

Q

1

2

3

4

5

Q

Fig. 4: Lack of reaction of anti-nucleolar autoimmune sera with ribosomal proteins. Immunoblotting experiments with total proteins of isolated ribosomes as antigens. (l) control serum; (2) anti-rRNP serum; (]-5) anti-nucleolar autoimmune sera. Arrows show the position of ]8, 16 and 15 kDa proteins. Fig. 5: Nucleolar segregation celts to Actinomycin immunostained with immuno-staining on a) to Actinomycin D; b) Actinomycin D for two exposed to Actinomycin

Antibodies ribosomal (17,

19).

nucleolar which formed

by Actinomycin D. After exposure of HeLa D (0.04 pg/ml), nuclei were isolated and anti-nucleotar positive serum. Indirect HeLa nuclei isolated from cells not exposed HeLa nuclei isolated from cells exposed to hours; c) HeLa nuclei isolated from cells D for three hours.

specific

for

r-RNP

phosphoproteins To

antibodies

recognize using

antibodies

of

investigate

a of

a 38

representative

bands off the

at

the

identical ribosomal extract

anti-rRNP

contrast,

none

kDa

approximately

whether

were

ribosomal

predominantly

15,

to

16,

and

1123

the

kDa

positive

with and

three

15

described

immunoblots source

indeed 38

]6,

anti-rRNP

antigen

serum

anti-nucleolar

38,

presently

protein, as

react

kDa

anti-

antibodies ~ere

(fig.

per-

4).

recognized

(fig. sera

4,

lane

tested

The the

2). con-

In

Vol. 163, No. 2, 1989

rained

antibodies

lack

of

shows

present To

in the

tration

of

sulting

seva

a

cells

D

not

autoimmune treatment,

causing

nucleoli

These

associated

particle. (20),

The

in

synthesis. from

the

B23

B23

Novikoff

ribosomal

precursor

to c o r r e s p o n d mouse

and

the

to

of

its

by

assembly

those

of

of

(22).

the

was

The

pve-rRNP

recently

38

kDa

with

been

as

shown

and

its

from

antigen

B23/N038

particles

the

a member

nucleolar

the

in the

isolated

identified

resembles

isolated

proteins

N038,

al.

ribosomal

associated

has

et

nucleolar

and

to be

B23

Yung the

kDa

may

ribosomal

of

37

hours

antigen

by

major

5b).

three

the

of

of

observed

(fig.

after

a

with

hours

was

of

protein,

N038

paper

two

inhibition

the

believed

9).

stained

of

as

nuclei,

exhibited

nucleolar

observed

weight

(21).

family

present

the

packaging

one

D

re-

known

irregular

completely

HeLa

concen-

5a,

when

translocation

is

(8,

fig.

after

and

that

nueleolar

cells

in

fluorescence

small

molecular

particles

the

of

Low

by

synthesis

Actinomycin

selective

is

RNA

nucleolus

ov

identified

components,

However,

the

cells

main

nucleoplasmin in

the

suggest

protein

a

Xenopus

identified terms

The

ribosomal antibodies

pg/ml).

shown

with

loss

on

a

The

biosynthesis,

(0.04

ribosomal

As

assembly

hepatoma

(6).

D

nucleolar

resembled

with

ribosomal

serum.

induced

nucleolus

of

of

become

studies

phosphoprotein

protein

disappeared

effect

their

nucleolac

16).

a

results

with

of

in

in

to

fluorescence

5c).

with

treated

D

(fig.

protein.

sera

kDa

(15,

anti-nucleolar

Nucleolav

ribosomal

groups

inhibit

immunofluorescence

the

kDa

different

Actinomycin

Actinomycin

be

38

redistribution

from

strong

two

involved

with

segregation

isolated

38

seva.

Actinomycin

in

nucleolar

the

anti-nucleolar

of

the

is

treated

to

the

respective whether

selected were

of

evidence

clarify

cells

reacting

reactivity

proteins

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

both

in

molecular

weight. A

recent

identifled found were

in

by

anti-rRNP antibodies obtained kDa

human

patients

detected

recognized

38

publication

a

with

37

kDa

PSS

was

sera

SLE.

autoantigen,

the

patients

nucleolar

a

in

with

antigen

SLE.

no

70%

of

the

identified

To

(12)

1124

of

as

were

kDa

protein.

investigate

and

autoantigens the

to

sera

B23.

Some

shown

to

if

These the

B23/N038,

immunoblotting

were

antibodies

characterized

study, 38

corresponds

isolated

nucleolar

anti-nucleolar

One

this

nucleolar

few (18).

whereas

with

analyzed

against

phosphoprotein

autoimmune

in p a t i e n t s

seva,

from

describes

of

seva the

contain

sera

were

recognized

the

nucleolav

experiments

Vol. 163, No. 2, 1989

were

performed.

serum, is

in

against

Lhe

Lhe

When

of

the

58

LhaL

Lhe

the

kDa

addition

to

5,

be

Lhe

wiLh

seva 58

a

thaL

as

58

B25

inLerpveLed band

nucleolar

B23.

summary,

Lhis

someLimes

kDa

more

nucleolar

be

found

This

This

when

a protein

of

lane

This

form

6b, in

Lhe

As when

pcesenLs

These

(23),

can

be

the

Lo

Lhe

anti-nucleolar

70

70

thaL

kDa

was

could

However,

kDa fig.

proLein 6,

lane

preincubated

human

antibodies,

idenLify

protein

was

Lhat

How-

menLioned

in

Two wiLh

SLE.

abouL

seen

serum

a

SLE.

preparation.

evidence

anLi-nucleolar

anLigen.

B25

oligomers

dimev.

i).

be

Lhat

reacting wiLh

I wanLed to

antibodies

with

sera,

patients

de-

implies

patienL

positive from

antigen,

B25/N058,

clearly

contains

antigen,

disappeared

contain

in

anLi-rRNP

wiLh

It n e e d s

a

study

kDa

B25/N058.

available

(fig.

can

nucleolar

58

an

preadsorbed

B25/N058.

kDa

as

a

sometimes

obtained

conLamination

kDa

In

70

no

visualized to

considering could

Lhe

sera

anti-nucleolar

were

with

appeared.

protein also

were

autoantigen

occasionally correspond

can

B25/N058

was

protein

anti-rRNP

proLein, sera

against

kDa

B25/N058

of

antiserum

nucleolar

collected

kDa these

58

main to

four

58

ever,

in

6.

the

anLibodies

anLibodies

fig.

staining

monstrates

of

Immunodetection

conLaining

shown

no

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

auLoimmune

recognizing

anLibodies

are

b

~'i ~~i I ~i

92-

i;!!,i~i ~

~,

i!i i!!ii l

iill ' i

,

) 43-

21-

});):

!

i~i ¸ i"

1

2

3

123

Fig. 6: Nucleolar pvoLein B25 idenLified by anti-rRNP serum recognizing a 38 kDa nucleolar protein (serum A). Immunodetection of isolated nucleolar pvoLein B23. a/(1) Serva Blue R staining of isolated B25; 2/ serum A; 3/ co~nLro[ serum, b/(1) serum A~ (2) control serum; (5) serum A preincubaLed with B25.

1125

a not

VoI. 163, No. 2, 1989

identical 38

kDa

to

be

with

from

were

of

snRNPs,

probes

an

integral

autoantibodies For examining

to the

to

precursor

particles

part

B23/NB38.

with and

SLE. one

a 38 kDa

RNP

the

complexes

structure of

specific

recognizing

antigen

polymyositis

determining

sera

kDa n u c l e o l a r

patients

recognized

autoantibodies in

38

phosphoprotein in

with also

The

in anti-rRNP

ribosomal

major

recognized

patient

tools

way,

with

the

hepatitis

Human Ful

as

present

protein.

associated

one

active

antibodies

ribosomal

identified bodies

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

organization

Two

patient

have

and

was

obtained

with

chronic

autoantigen.

shown

to be power-

Function

of a variety

"spliceosome" protein

of the

shown

autoanti-

sera,

nucleolar

and

nucleolar

These

was

a

In a similar may

be

useful

nucleolus.

ACKNOWLEDGMENTS I wish to thank Dr.G.Sauermann For laboratory space and support. I am indepted to Dr. Penner for helpful d i s c u s s i o n s and For p r o v i d i n g the human sera. Some anti-rRNP sera were generous gifts from Dr. Smolen, Dr. H a s s f e l d and Dr. Tan. I am grateful to Mr. Paukovits for the valuable help with the column c h r o m a t o g r a p h y and for many stimulating discussions. The d e d i c a t e d technical assistance of E.Hitchmann, H.Bauer and l.Fr~hlich is gratefully acknowledged.

REFERENCES 1. Pinnas, [.L., North~ay, I.D. and Tan, E.M. (1973) J. Immunol. iii, 996-1004. 2. 8ernstein, R.M., Steiger~ald, J.O. and Tan, E.M. (1982) Olin. Exp. Immunol. 48, 43-51. 3. Reddy, R., Tan, E.M., Henning, D., Nohga, K. and Busch, H. (1983) J. Biol. Chem. 258, 1383-1386. 4. Lisehwe, M.A., Oehs, R.L., Reddy, R., Cook, R.G., Yeoman, L.C., Tan, E.M., Reichlin, M. and Busch, H. (1985) J. Biol. Chem. 260, 26, 14304-14310. 5. Stetler, D.A., Rose, K.M., Wenger, M.E., Berlin, C.M. and Jacob, S.T. (1982) Proc. Natl. Acad. Sci. USA, 79, 7499-7503. 6. Lischwe, M.A., Smetana, K., Olson, M.O.J. and Busch, H. (1979) Life Sci, 25, 701-708. 7. Michalik, J., Yeoman, L.C. and Busch, H. (1981) Life Sci. 28, 1371-1379. 8. S c h m i d t - Z a c h m a n n , M.S., HOgle-D6rr, B. and Franke, W.W. (1987) EMBO J. vol. 6, 7, 1881-189B. 9. S c h m i d t - Z a c h m a n n , M.S. and Franke, W.W. (1988) Chromosoma (Berl.) 96, 417-426. 10. Penner, E., K i n d ~ s - M O g g e , I., Hitchman, E. and Sauermann, G. (1986) Clin. Exp. Immunol. 63, 428-433. 11. Peters, K.E. and Comings, D.E. (1980) J. Cell Biol. 86, 135-155. 12. Michalik, J., Yeoman, L.C. and Busch, H. (1981) Life Sciences, 28, 1371-1379. 13. Laemmli, U.K. (1970) Nature, 227, 680-685. 14. Towbin, H., Staelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354.

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