0022·1767/86/ 1 371 2·3802$()2.00/0
T'HE JOURNAL 0 ' IMWUNOLOGY
Copynght C 1986 by Thc Amcncan AssoctaUon of JmmunoJoglsts
Vol. 137. 3802- 3808. No. 12. Dttember 15. 1986
Prlnled In U.S.A.
IMMUNOLOCALIZATION AND PARTIAL CHARACTERIZATION OF A
NUCLEOLAR AUTOANTIGEN (PM-SeI) ASSOCIATED WITH
POLYMYOSITIS/SCLERODERMA OVERLAP SYNDROMES 1
GEORG REIMER,2 ULRICH SCHEER,* JAN-MICHAEL PETERS.* ANO ENG M. TAN3
From the W. M. Keck Autotmmune Dtsease Center, Scrtpps Cltntc and Research Foundatlon, 10666 North Torrey Ptnes Rd ..
La Jolla, CA 92037; and the -Institute 01 Cell and Tumor Btol09Y, German Cancer Research Center. D·6900 Heidelberg,
Federal Republtc 01 Germany
Precipitating anti-PM-Sel antibo<lies are present
in sera from patients with polymyositis. sclero-
derma. and polymyositis/scleroderma overlap syn-
dromes. By indirect immunofluorescence micros-
copy. anti-PM-Scl antibodies stained the nucleolus
in cells of different tissues and species. suggesting
that the antigen is highly conserved. By electron
microscopy, anti-PM-Scl antibodies reacted primar-
ily with the granular component of the nuc1eolus.
Drugs that inhibit rRNA synthesis had a marked
effect on the expression of PM-Scl antigen. In acti-
nomycin D-treated cells, immunofluorescence
staining by anti-PM-Scl was signüicantly reduced
with residual staining restricted to the granular
regions of nuc1eoli. Treatment with 5,6-dichloro-ß-
o-ribofuranosylbenzimidazole (DRB) also selec-
tively reduced nuc1eolar staining. On a molecular
level, anti-PM-Sel antibodies precipitated 11 poly-
peptides with molecular weights (Mr) ranging from
110,000 to 20,000. The Mr 80,000 and 20.000 poly-
peptides were phosphorylated. Evidence suggests
that the PM-SeI antigen complex may be related to
a prerlbosomal particle.
AutoanUbodles agalnst nucleic acids and a variety of
cellular proteins and RNA protein complexes are pro-
duced by paUents with systemic autoimmune disease (1).
These serum antibodies not only are of diagnostic signif-
icance, e.g., anti-native DNA and anti-Sm anUbodies for
systemic lupus erythematosus (2, 3), but also have proved
to be valuable immunologic tools in molecular and cellu-
lar biology. For example, anUbodles against U1 ribonu-
cleoproteln were shown to effecUvely inhlblt splicing of
heterogeneous nuclear RNA, thus ImplicaUng this nu-
clear ribonuc1eoprotein particle in the processing of
Recelve<! for publlcatlon May 24,1986.
Accepted for publlcatlon September 2. 1986.
The costs of publlcatlon of thls artlcle were defrayed In part by the
payment of page charges. Thls artlcle must therefore be hereby marked
advertfsement In accordance wlth 18 U.S.C. SecUon 1734 solely to Indl-
cate thls fact.
1 Thls Is Publlcatton No. 4340BCR from the Department of Basic and
Cllnlcal Research. ScrippS Cllnlc and Research Foundatlon. La Jolla. CA.
Thls work was supporte<! by Natlonal Institutes of Health Grants
AM32063 and A1l0386. G. Reimer was supporte<! by Grant Re 585/1-1
from the Deutsche Forschungsgemeinschaft.
• Current address: Or. Georg Reimer. Dermatologtsche Unlversltatskll·
nlk. Hartmannstr. 14. 0·8520 Erlangen. Federal Republlc of Germany.
3 Reprint requests to: Eng M. Tan. M.O .. W. M. Keck Autoimmune
DlseaseCenter. Department of Basic and Cltnlcal Research. Scrlpps Cltnlc
and Research FoundaUon. 10666 North Torrey Plnes Rd .. La Jolla. CA
mRNA (4). Most recently, antibodtes against the Sm ri-
bonueIeoprotein partieIe that is complexed with uridine-
rieh smaU nueIear RNA, named UI. U2, U4, U5, and U6
(5), have facilltated cloning of cDNA that contains se-
quences complementary to mRNA for the E protein (Mr
11,000) of the Sm partieIe (6).
In arecent paper, a new preclpitatinganUgen-antibody
system termed PM-SeI was described In patients with
polymyosiUs, scleroderma. and polymyoslUsjsclero-
derma overlap syndromes (7), These antlbodtes have
identlcal specificities as antl-PM-1 antlbodies, which had
been prevtously reported In patients with polymyositis
(8). The immunofluorescence patterns obtained with
anti-PM-Scl sera were initially described as being both
nueIeolar and nucleoplasmic in nature. Recently, Targoff
and Reichlin (9) provlded evldence for a nucleolar locali-
zation of the PM-Scl antigen. Although some Mr data on
the PM-SeI antigen were recently reported by Bernstein
et aL (10), the macromolecular nature and origin of the
PM-SeI antigen remain unknown. To understand the
significance of autoanUgens in the context of diseases
with which they are assoctated, it may be important to
elueidate their structure and funetion. In this study, we
have shown that the PM-Scl antigen is a complex partlcle
composed of at least 11 polypeptides. We further provlde
evidence that the PM-Scl antigen complex may be of
MATERIALS AND METHODS
Patient sera. Antl-PM-Sel sera were obtalned from patlents with
polymyositIs, sclerodenna, or polymyoslUs/sclerodenna ovedap syn-
dromes and were selected from the serum bank of thls laboratory.
Thelr Immunologie speclflclty was assessed by double ImmunodIf-
fusion agalnst a reference anU-PM-SeI serum (S.8.) as descrtbed (7).
Thls serum had also been the standard reference serum In the latter
lndtrect Immunofluorescence. Sera were dlluted startlng at 1/40
In phosphate-buffered saline (PBS). pH 7.4. and used for Indlrect
Immunofluorescence. Substrates Included commerclally avallable
Hep-2 cells (Blon, Park Rldge, IL) and KB cells (Electronucleonlcs.
Bethesda, MD). Other substrates were Vero and HeLa cells grown on
mlcroscoplc sildes and flxed In acetone/methanol at 3:1 for 3 mln.
unstlmulated and concanavaltn A (Con A)-stlmulated human pertph-
eral blood Iymphocytes, T cell lymphoma Molt 4 cells. rat RV cells.
rat kangaroo PTK2 cells. flsh FHM cells. turtle TH-l cells, duck
(Pe kin duck) embryonlc cells. and rat IIver cryostat sectlons. The
duck embryonie. FHM. and TH-l cells were obtained from the Amer-
Ican Type Culture Collectlon. Rockvllle, MD; the other cells. from
stocks In thls and Or. Scheer's 1aboratory. A fluoresceln goat antl-
human IgG conJugate (Tago, Burllngame, CA) was the detectlng
reagent. Intenslty of Immunofluorescence was graded from weak
(+), to moderate (++), to strong (+++). Double Immunofluorescence
stalnlng wlth the use of a mouse monoclonal antlnucleolar antlbody
of IgG2a Isotype and human anti· PM-SeI serum was perfonned as
descrlbcd (I I ). ResullS were read on a Lellz Ortholux II fIuorescence
f;/ecfroll mtcroscopic Immunocytochemlsfry. A representatlve
antl-PM-Scl serum (5125) was dlluled 1/ 100 In PBS and incubaled
for 2 hr w!th frozen sectlons (5-l'm lhickness) of regcneraling rat
livcr t hat had been flxed In acetone al -20°C for 10min. After
scvcral washcs In PBS. goal anti-human IgC coupled 10 colloldal gold
partlclcs of 5-nm diameter (Janssen Life Sclences. Beerse. Belglum)
was added at a dilution of 1/4. After overnlghl incubation at 4°C.
the speeirnens were f1xed and processed for electron mlcroscopy as
deserlbed In detail elsewhere (12).
Drug studtes. TIssue ulture Vero cells were Incubated wllh me-
dium contalnlng 0.1 I'g/ml actlnomycln D for 4 hr and wlth 50 I'g/
mI5.6-dlchloro-I-ß-o-ribofuranosylbenzlmldazole (DRB).· a halogen-
ated adenosine analogue. for 6 hr. Both drugs were purchased from
8ehring Dlagnoslics. La Jolla. CA. After drug treatment. cells were
f1xed and processed for indirecl immunofluorescence or double Im-
rnunofIuorescence stainlng as describcd.
Nuc/ease digestion experiments. For nuclease digestion experi-
ments. Vcro cells were grown on mlcroscopic slides and f1xed as
dcsrribcel above. The cells were then dlgested wlt.h 0.25 mg/mi
DNase I (MIllipore. Freehold. NJ) in PBS containing 5 mM MgCI2 and
2 mM phenylmethylsulfonylfluorlde (PMSF) or wlth 0.25 mg/mi
RNa e A (Milli pore) In PBS for 30 mln at 37"C. After digestion. cells
were washed in PBS and processed for Inellrecllmmunofluorescence.
ßurfers wlltlOut enzymes serveel as negative controls.
ImmuTlopreclpltation. HeLa cells were metabollcally labeled wlth
I35Sjmethlonlne and 132Pjorthophosphate (New England Nuclear. Bos-
ton. MA). Iyseelln buffer A (10 mM Trls-HCI. pH 7.4. ISO mM NaCI.
10 mM MgCh. 0.5% Nonldet P-40. and 2 mM PMSF). and further
proeessed as described (13) wlth mocllflcallons. All experIments were
performed In Eppenelorf mlcrocenlrlfuge tubes at O°C to 4°C. In brief.
sampies of 101'1 of patient serum were mixed for I hr wlth 1001'1 of
protein A-Sepharose CL-4B [Pharmacla. Uppsala. Sweden) sus-
peneled at 10% (v/v) In buffer B (10 mM Trls-HCI. pH 7.4.150 mM
NaCI. 10 mM EDTA. 0.1 % dcoxycholate. 0.1 % sOOlum dOOecyl sulfate
ISOSI 0.5°;' Nonldet 1'-40 23 U/ml Kalilkrein Inactlvator IBehrlng
Diagnosticsll supplemented wlth 2 mg/mi bovlne serum albumin.
After washing wlth buffer B. 100 1'11. of 3 ~ S _ 01' 32P-Iabeled HeLa cell
cxtract was added anel Incubateel for I hr with constanl mixing. The
beaels wert' then washed five ttmes In buffer B. boileel In Laemmll
sampie buffer (14) and processed by SDS-polyacrylamide gel electro-
phoresis (SDS-PACE). followed by aUloradlography at -70°C. Half of
the 32p pellet was processed by SDS-PAGE and the olher half was
phenol eXlracted: any RNA was preclpilated in ethanol. RNA analy-
sis was performed in 7 M urea. 8% polyacrylamide gels followed by
autoradlography as described (5).
ImmunoJluorescence stalning with anti-PM-Scl antl-
bodies with the use oJ dljJerent cell substrates_ Various
celllines and tlssues were used as substrates for stalning
wllh antl-PM-Sc1 sera from different patients. The stain-
Ing patterns observed wlth a representative anti-PM-Scl
serum (S (25) are shown In Figure 1. [n human HeLa ce[[s
(Fig. [a and b). monkey Vero cells (Fig. Je and d). and
mitogen-stlmu[ated human periphera[ b[ood [ymphocytes
(Fig. 1 e and J). anti-PM-Scl antibodies strongly (+++)
stained the Interior of nuc)eo[i. Weak nuc)eoplasmic
staining (+) was also observed with most of these sub-
strates. The homogeneous nuc[eolar sta[ning pattern was
uniformly observed in all substrates tested. including
cells such as human Molt 4 cells. rat RV cells. marsuplal
PtK2 cell . and rat liver sections. In unstlmulated perlph-
eral blood lymphocytes. PM-Scl staining was consider-
ably weaker (+) when compared with Con A-stimulated
lymphocytes. which gave a (+++) stainlng (Fig. 1e andJ).
1n addition. Pm-Scl staining was also present In fish FHM
rells. turtle TH-l ceJls. and a duck embryo cell line.
su~e ting that the epitopes recognized by anti-PM-Sc1
anUbodies are widely conserved In evolution. All anti-
PM-Scl sera tesled so far produced comparab1e nuc[eo1ar
• Abbrcvlallons used In lhls paper: PM5F. phenylmethylsulfonylfluor-
Ide: DRB. 5.6 dlchloro-ß-o-rlbofuranosylbenzlmldazole.
Figure I. Immunofluorescence stalning of HeI..Il cells (0). Vel'o cells
(cl. and eon A-sllmulaled human perlpheral blood lymphocytes (e) \Vllh
3utolmrnune hllman antl-PM-Scl anllbodles [5125). All nucleoll dernon-
strale brlght homogeneous nucleolar stalnlng 10 all cells tesled. Wcak
nllcleoplasrnlc slalnlng Is also present. especlally In Vero cells. Note
prenucleolar body slalnlng (arrows) of a telophase HeLa cell In a. Slmllar
results were obtalned wlth all the sera tcsted. For comparlson. lhe cells
are shown In phase contrasl (b. d. and!). Bars 10 "m. X 1450.
stainlng patterns in different cell substrates.
Anti-PM-Scl staining during cell cycle. Figure 2a and
b shows indirect immunofluorescence wlth rat kangaroo
PtK2 cells used as substrate when reacted with a repre-
sentative antl-PM-Scl serum (S 125) from a patient with
scleroderma. In interphase cells. immunofluorescence
staining was predominantly wlthln the nucleolus with
weak (+) staining of the nucleoplasm. In metaphase cells.
diffuse statning was observed in the area of the con-
densed chromosomes. In anaphase cells. anti-PM-Scl
slaining was diffusely dlstrtbuted around the chromo-
somes and in the penchromosomal cell plasm.ln telo-
phase. anti-PM-Scl antih:>dies stalned the prenucleolar
bodies of the reconstituling nucleoll. as shown for HeLa
cells In Figure 1a (arrows).
Subnucleolar localization oJ PM-Scl by electron mi-
croscoptc immunocytochemistry. For subnuc1eolar 10-
callzation of the reactive antigen. electron microscopic
immunocytochemtstry was performed with the tmmu-
nogold label technlque. Ftgure 3a. b. and c shows secUons
through nucleol! of regenerattng rat liver. IgG from a
representatfve antl-PM-Sc1 serum (S 125) was localtzed
primarlly in the granular component of nucleol1. Gold-
antlbody complexes were selecttvely enrlched over lhe
granular component wlth accentuation over the perlph-
ery of the nucleoli. Immunogold was virtually absent In
the dense f1brillar component or ftbrillar centers. It
should be noted that gold-antibody complexes were also
observed In distlnct clusters dtspersed throughout the
nucleoplasm as potnted out by arrows (Flg. 3a and cl.
Flgure 2. Immunolocaltzatlon 01' anU-PM-5cl antlbodles (5125) In rat
kangaroo PtK2 cells durlng different phases 01' the cell cycle. as vlsuallzed
by Immunofluorescence mlcroscopy (a) and phase contrasl (b). In Inter-
phase cells. br1ght nucleolar slalnlng and weak Ilueleoplasmlc slalnlng
by anll-PM-Scl Is present. Note lhat nucleolar stalning rclocatcs as the
chromosomes condense durlng metaphase (arrow 1) giving rise Lo diffuse
sialnlng In the area of the condenscd chromosomes. in laIe anaphase cell
(a r row 2). PM-Sei stalning Is diffusely dislr1buted on the surface of the
chromosomes and In the per1chromosomal cell plasm. Individual chro-
mosomes can be disllngulshed in phase-contrast optics (b) as elongated
IIghter structures surrounded by dark borders. Bar 20 ~m . x I 040.
These electron microscopic findlngs glve support to the
observation that in indirect immunofluorescence there is
weak nucleoplasmic [n addition to nucleolar staining. In
[he control experiment. antibodies against RNA polym-
erase I (12) were used that only stained the fibrillar
centers but not the granular component of nucleolL
E;[fect oJ actinomycin D and DRB on the distributi.on
oJ anti-PM-Sc! stainlng. To rurther study the nature of
the PM-Sei antigen. Vero cells were exposed to the nu-
cleolus-segregatlng drugs actinomycin D and DRB. After
drug treatment and fixation. cells were incubated with
severaJ anti-PM-Scl sera and stained with a f1uorescein
anti-human IgG conJugate. AcUnomycin D-inactivated
nucleoli are compact and show a typical segregation of
the fibrillar component from the granular component.
usually resulting in the formation of distinct nucleolar
hemispheres (15- 17). F[gure 4 shows the results ob-
tained with prototype serum S125. Actlnomycin D at 0.1
IlgJml considerably reduced nucleolar staining by antl-
PM-Scl antibodles. Residual staining was predominantly
Flgure 3. Immunolocalizatlon of antl-PM-Scl anllbodtcs (5 125)ln sub-
nuclcolar compartments tn the hepatocytes or regenerattng rat Itver, as
shown by the electron mlcroscoptc tmmul1ogold label technlque. For
orlentatton. frame a of thls composite shows seetIons of two nucleoll and
surroundtng hctcrochromaltn at low magniftcatlon. F'rames band c dem-
onstrate at high magntfteallon two sectlons of nucleoll. The 5-mm gold
partlcles (small black dors) Indlcatlng gold-anttbody-antlgen complexes
are selectlvely enriched over the granular component IGC) and are dls-
trlbutcd at hlgher denslty In the perlphcry 01' these nucleoll. The dense
fibrlliar componenl (DFC) and the ftbr1JJar centers (F'C) are free of gold
partlcles. Note that gold-antibody-anUgen cornplexes are also present In
clusters throughout the heterochromaUn structurcs as Indlcated by ur-
rows In a and c. Bars I /Lm, x20.000 (a). 0.2 jJm. x 70.000 (b). 0.2 ~ 1 l 1
x I 25.000 (c).
localized in the granular component (Flg. 4b). whlch ap-
peared dark in phase optlcs (Fig. 4c). When DRB was
added to Vero cells growing in culture. the normally
compact nucleoll unraveled Into extended beaded
strands. again segregatlng the nucleolus Into granular
and flbrfllar regions as has been descrtbed In prevtous
studies (18- 20). Treatment of Vero cells wtlh DRB at 50
IlgJml for 4 to 6 hr substantially removed PM-Sc I statning
of segregated nucleolar structures (Fig. 4e andJ). How-
ever. diffuse nucleoplasmtc slainlng of low intenslty (+).
which was not reproduced weil in the micrograph. was
still present. In contrast. a mouse monoclonal IgG2a an-
tibody agalnst a M, 34.000 nucleolar protein associated
with the U3 rlbonucleoprotein particle (Reimer ct aL.
manuscript In preparatlon) still strongly reacted wlth the
flbrllJar component of act!nomycin D- and DRB-segre-
ga ted nucleoli (Flg. 4a and d). These observations show
lhat actinomyctn D and DRB significantly affect expres-
sion of PM-Scl antigen. leavlng another nucleolar protein
Nuclease digestion studtes. To evaluate the effect of
nuclease digestion on PM-Scl antigen. HeLa cells were
Incubated with DNase and RNase be fore tncubatlon with
antl-PM-Scl serum. Nelther of these nucleases substan-
tially altered intenslty of nucleolar lmmunofluorescence
stalntng when compared wtth controls In which buffers
wlthout enzyme were used. In other control experiments.
nuclear staining by a monoclonal anti-native DNA anti-
body (11) was abolished by DNase pretreatment of cells.
and nucleolar and cytoplasmic stainlng produced by hu-
man and monoc\onal antl-RNA antibodles was abolished
by RNase pretreatment. These ftndtngs suggested that
the antigenictty of PM-SeIls not dependent on DNA or
VIgil re 4
IgG2a anubody 3/i!alnst flbrilluin. a basic nllcleolar protein. and human
anU-PM-Scl anllbodles (S 125) was reuelcd simllltMleously wtth acUno-
mycln D- 10 and b) and DHß- (d and e) lreated Vero cells. Thc IgG2a mouse
monO<'lonal antlbody (0 and d) was deteeted wtth a SPN'lfic f!uoresceln
conJlIg"tc; thc human antlbodle . wllh an antl-rgG rhodamlne conJugate
(b and e). Stalning Inlcnslty of f1brlllar components of nucleolf by mono-
cl(>l'la I antlbocly to flbrillarin was nOI slgnlflcantly affccted byactlnomycln
o (al or DRB (cl). In eonlrast. anll·PM-Sel anllbodles thai reaeled wlth t11C
granular eomponent (arrow) showed conslderably reduced stalnlng Inten·
slty after actlnomycln D trealment (b) when eompared wlth unlrealed
nuclcolf (for eomparlson see rlg. l e). DRB treatment almost completcly
abollshecl anll-PM-Scl lalnlng of dlspersed nucleolar structures (e). ror
rC"fcrf'nce. cells are also hown In phase·contrast opucs Ic andJ). Bar 10
pm. x 1450.
Double ,mmunofluorescence stalnlng. 11 mouse monoclonal
Ftgure 5. Autoradiogram of Immunopreclpltated f"Slmethlonlne-la-
beled HeLa eell proteins electrophorellcally rcsolvecl In a 15% SOS-poly-
acrylamide gel. Lane 1. normal human serum; lane 2 to 7. antl-PM-Scl
sera as Identlfted prevlously In Immunodiffusion; lane 8. molecular
welght markers. A group of dlstlncl polypeptides were unlformly preclp-
Itated by all anll-PM-Scl sera but not by normal control serum.
MolecuLar characterization OJ the PM-Scl antigen. To
identlfy the molecular composition of the PM-Sei antigen.
we performed protein A-faeilltated immunopreeipitatlon
of radiolabeled antigen followed by gel analysis. Immu-
nopreeipitation with slx anti-PM-SeI sera. using [35Slme-
lhionine-Iabeled HeLa cell extract. followed by SDS-
PAGE analysis are shown in Figure 5. lanes 2 to 7. All
sera wlth PM-ScI specificity by double Immunodiffusion
uniformly precipltated at least II polypeptides (PI-PlI)
with Mr 110.000. 90.000. 80.000. 39.000. 37.000.
33.000. 30.000. 27.000. 26.000. 22.000. and 20.000.
Figure 6. lane 2. shows In detail the polypeptides preclp-
itated by representative anti-PM-ScI serum S125. On
eloser examination of Figure 5 it ean be nottced that there
are additional polypeptides present in lanes 3. 4, and 5.
but the common polypeptides preclpilated by all sera
examined comprlsed the PI-PlI complex as shown in
HeLa cells were labeled with e2Plorthophosphate. and
the cell Iysate was used In immunopreclpitatlon to deter-
mine wh ether the PM-Scl antigen might be phosphory-
lated or associated with tightly bound RNA. The Mr
80.000 (P3) and 20.000 (P I I) polypeptides that were
precipltated by anti-PM-Sel antibodies were phosphopro-
teins (Fig. 7. lane 4). RNA was not part of the PM-Sei
antigen as shown by absence of RNA in urea-polyacryl-
amide gels. We also incubated deprotelnized RNA from
HeLa cells wlth anti-PM-Scl antibodles but falled to dem-
onstrate any binding with RNA. suggesting that RNA
itself is not a target (data not shown). The latter data are
In agreement with the in situ nuelease digestion results
obtalned in tissue culture HeLa cells. where no effect of
RNase pretreatment of eell substrates on immunostain-
Ing w.lth antl-PM-Scl was observed.
Several attempts were made to identify the antlgenlc
epitopes of the PM-Sei complex by immunoblottlng ex-
periments. Extractable whole HeLa cell proleins as well
as lsolated nucleolar proteins were separated by SDS-
PAGE and were transferred lo nitrocellulose for immu-
noblotting. At the present time. no conclusive results
have been obtained. although a protein of approxlmately
Mr 80.000 has been reacttve wlth some sera. Vartous
explanatlons are possible. such as denaturation of the
antigens and the requlrement of tertiary conformation
for antigentcity. Definitive identtty of the antigenie com-
ponent of the PM-Sei compIex awaits further study.
Antinucleolar antibodies are part of the spectrum of
autoantibodies present in scIeroderma and were found to
Figure 6. Autorudlograrn of f"Sjrnethlonlne-labeled HeLa cell proteins
Immunopreclpllated by antl-PM-S I antilXldles (S 125) and resolved In a
17.5% SDS-polyacrylamlde gel. Lunc I shows proteins preclp!tated by
normal control serum. Lane 2 demonstratcs In detail the racllolabclccl
pl'ote!ns from HeLa cells that are seieclively brought down byantl-PM-
Sei antlbodles and are named P I -P 11 .
Figure 7. Autoradiogram of Immunopreclpllaled. I"Slmelhlonlne-
[Iane land 3J and 131Plorthophosphate· [Iane 2 and 4J labeled HeLa cell
proteins. Immunopreclpltates as obtalned wlth normal human serum are
shown In lane l and 2 and wHh anll-PM-Scl serum In lane 3 and 4. The
polypeptides of M, 80.000 [P3) and 20.000 IPI I J. whlch are labeled wlth
e·Slmcthlonlne. are phosphorylated. Thc dark smear on top of lane 2 and
4 as weil as the signal jusl above marker or M, 46.000 were sensitive to
RNase treatment and most probably rcprcsents coprrClpllating RNA In
normal human and anll-PM-Scl serum.
be hlghly assoclated wlth this systemlc autoimmune dls-
ease (21-24). Nucleolar 4-6 S RNA was shown to be one
of the targets (24). More recently. a novel nucleolar ribo-
nucleoprotein complexed wlth 7-2 RNA was detected with
antlnucleolar scleroderma antlbodles (25. 26). U3 ribo-
nucleoprotein was also shown to be part of a complex
precipitated by certaln antinucleolar antlbodles from
scleroderma sera (25). These antlbodles were shown to
recognize a M, 34.000 basic nucleolar protein rlch in NG•
NG-dimethylarglnine. and glycine (27). Because of its pre-
dominant locaflzation in the flbrillar component of nu-
cleoli. Ochs el al. (28) named this nucleolar protein fl-
brillarin. We recently showed that a subgroup of antf-
nucleolar anttbodles that produce speckled or punctate
nucleolar immunofluorescence stainlng In tlssue culture
cells Immunopreclpitated the RNA polymerase I complex
(29). Naturally occurring autoimmune antibodles against
nucleolar proteins and RNA protein complexes thus far
have proved to be valuable tools to dissect nucleolus
structure and function.
In the present study. we have characterized the antigen
reactlve wlth anti-PM-Scl antlbodies that were previously
shown to be associated wlth polymyositIs. scleroderma.
or polymyosltis/scleroderma overlap syndromes. The pre-
dominant nucleolar presence of the PM-Scl antigen was
demonstrated by indirect immunofluorescence per-
formed by uslng tissue culture cells [rom a variety of
species. The wide distribution of the epltopes recognlzed
by antf-PM-Scl autoantibodies suggests that the reactlve
protein is a conserved and important cellular component.
The nucleolar localization of the PM-Scl antigen as ob-
served In this study Is in keeplng wlth a recent report by
Targoff and Reichlin (9). who also found this antigen to
be nucleolar. However. from our Immunolocallzation
studies It appears that the PM-Scl antigen Is also present
In the nucleoplasm.
Sy electron microscoplc Immunocytochemlstry. anti-
PM-Scl anUbodles predomlnantly stalned the granular
component of nucleoli and clustcred partlcles dispersed
throughout the heterochromatin. In actinomycin D-seg-
regated nucleol!. anti-PM-Scl stalning was substantially
reduced but malnly localized in the granular regions.
AcUnomycin D In the concentratlons used In thls study
suppresses the synthesls of rRNA and the actlvlty of RNA
polymerase II but not the synthesls of rlbosomal proteins
(30- 33). In the absence of rRNA synthesls and desplte
normal protein synthesis. rlbosomal proteins are rapldly
degraded (32). Another drug. the halogenated adenosine
analogue DRS. also had a markedly suppressive effect on
the expression of the PM-Sei antigen. Thls drug almost
completely abollshed antl-PM-Scl stalnlng In tissue cu 1-
ture cells when added to the medium. DRB Is known to
effectively suppress the synthesls of mRNA (34- 36) but
not. or only moderately. transcriptlon of the rRNA genes
(37. 38). DRS causes extensive degradation of the RNA
precursor particles of the large rlbosomal subunlt. prob-
ably due to the deficiency of essential prerlbosomäl pro-
teins. The productlon of the small ribosomal subunlt
appears not to be Impalred (18. 19). Therefore. local!za-
tlon of the PM-Scl antigen predominantly In the granular
region of nucleoll. the Si te of ribosome assembly and
packaglng. and more Importantly sensltivity of the PM-
Scl antigen to actinomycln D and DRB. drugs that cause
degradation of preribosomes. may suggest a relatIonship
of the PM-Scl antigen to prerlbosomes. The distribution
of the PM-Scl antigen durlng cell cycle is compatlble with
a posslble prerlbosomaJ origln or assoclation.
On a molecular level. anti-PM-Scl antlbodies Immuno-
precipltated 1I polypeptides with M, from 110.000 to
20.000 when incubated wlth [35Slmethlonlne-labeled
HeLa cell proteins from the extractable fractlon. The M,
80.000 and 20.000 polypeptides were phosphorylated.
RNA was not preclpitated wlth the PM-Scl antigen. and
therefore. no association of the PM-Scl complex wlth
ribosomal precursor RNA or processed rRNA could be
establlshed that could have facilltated Identiflcatlon of
the putative nature of the PM-Scl antigen. We also at-
lempted to deflne the anUgenlc components of the PM-
Scl antigen complex recognlzed by autoimmune anti-
bodles. Wlth the use of extractable HeLa cell proteins as
weil as Isolated nucleoli as antigen sour e in Immuno-
blotting. the antIgenie polypeptides of the PM-Scl com-
plex could not be concluslvely revealed.
The PM-Scl antigen. however. Is clearly different from
the 7-2 rlbonucleoprotein parUcle recognlzed by certaln
antlnucleolar scleroderma sera (25. 26) slnce none of our
anti-PM-Scl sera coprecipltated 7-2 S RNA. U3 RNA was
also not present in the immunopreclpltates of anti-PM-
Sei anUbodles. suggesting that the U3 rlbonucleoproteln
is not part of the PM-Scl complex. We also incubated
deproteinized RNA wlth anti-PM-Scl antibodies and
showed that RNA was not a target. suggesting that these
antibodles are different from those reported to react wlth
4-6 S nucleolus-specific RNA (24) or any other RNA spe-
Polyclonal and monoclonal antibodles have recently
been used by several Investigators to characterize nu-
cleolar proteins In greater detail. For example, a mouse
monoclonal antibody was shown by HugIe et al. (39) to
Identlfy the largest and most acldlc protein of the small
ribosomal subunlt (SI) wlth Mr43,OOO In the rat. Another
monoclonal antibody characterlzed a nuclear Mr 40,000
protein named ribocharin, whlch appears to be speclflc
to precursor particles of the large ribosomal subunit In
Xenopus laevis and other amphlbla (40). Recently. a Mr
100.000 nucleolar protein was Identlfled as belng asso-
clated wlth preribosomes (41). Other nucleolar proteins
that have been partlally characterized Include protein
C23 and B23. In severa] reports. lt has been suggested
that protein C23 may be a component of the preribosomal
rlbonucleoproteln partlc1es (42, 43) and protein B23 may
be associated wtth ribosome assembly and packaglng (44.
45). It is unclear at the moment whether some of the PM-
SeI polypeptides are identical with known nucleolar pro-
teins, but 1t is conceivable. Further characterlzation of
the PM-SeI complex is needed to answer 11s exact com-
position and functlon in the nucleolus.
Deflnlng the structure and functlon of the PM-Scl au-
toantlgen may be a first step towards understandlng why
patients wlth features of polymyositIs and scJeroderma
eUcH an autoimmune response dlrected against thls nu-
Acknowledgments. We thank Thomas H. Hoger. Insti-
tute of Cell and Tumor Biology. German Cancer Research
Institute. Heidelberg, for performing some of the electron
mlcroscopy presented in thls study. We also thank Dr.
Carol A. Pennlng and Carol L. Peebles. W. M. Keck Foun-
datlon Autoimmune Disease Center. Serlpps ClInlc and
Research Foundatlon. La Jolla. CA. for assistlng wlth
same of the experiments In thls study.
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