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Deciphering the mysteries of RNA-containing lupus antigens.

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76 I
DECIPHERING THE MYSTERIES OF RNA-CONTAINING
LUPUS ANTIGENS
ETHAN A. LERNER, MICHAEL R. LERNER, JOHN A. HARDIN, CHARLES A. JANEWAY, JR., and JOAN A. STEITZ
A n t i r i b o n u c l e o p o r t e i n (RNP) and anti-Sm a n t i b o d i e s
from p a t i e n t sera and monoclonal anti-Sm a n t i b o d y
d e r i v e d f r a n MRL/1 spleen c e l l s were used t o character i z e t h e s t r u c t u r e and f u n c t i o n o f t h e RNP and Sm
antigens.
THe RNP and Sm detenninants a r e present on
d i s c r e t e small r i b o n u c l e o p r o t e i n complexes (snRNPs).
A
One o f these snRNPs i s i n v o l v e d i n RNA s p l i c i n g .
c o m p e t i t i o n r a d i o i m n o a s s a y employing t h e monclonal
anti-Sm a n t i b o d y t o t i t e r p a t i e n t sera i s presented. A
screen o f a n t i n u c l e a r a n t i b o d y (ANA) p o s i t i v e sera
r e v e a l s a d d i t i o n a l RNP s p e c i f i c i t i e s .
A c h a r a c t e r i s t i c o f connective t i s s u e disease i s
the production o f autoantibodies against c e l l u l a r
canponents.
DNA i s t h e b e s t known a n t i g e n w i t h which
lupus a n t i b o d i e s r e a c t , b u t t h e r e a r e many o t h e r a n t i gen-antibody systems which occur i n p a t i e n t s w i t h
connective t i s s u e disease.
The ones we have s t u d i e d
most i n t e n s i v e l y i n c l u d e t h e Sm, RNP, Ro and La systems
(1-3).
The f i r s t two o f these form an i n t e r e s t i n g
p a i r . Anti-Sm a n t i b o d y i s v i r t u a l l y unique t o p a t i e n t s
Anti-RNP antibody i s
w i t h lupus and MRL mice ( 4 . 5 ) .
seen n o t o n l y i n lupus b u t o t h e r autoimnune diseases
i n c l u d i n g most cases o f mixed c o n n e c t i v e t i s s u e disease
(6).
The Sm and RNP a n t i g e n s were shown t o be n u c l e a r
by i n d i r e c t immunofluorescence and c e l l f r a c t i o n a t i o n ,
and p h y s i c a l l y associated w i t h each o t h e r by immunodif'fusion (7.8). Both Sm and RNP a r e s e n s i t i v e t o t r y p s i n
and a r e c o n v e n t i o n a l l y d i s t i n g u i s h e d by t h e s u s c e p t i b i l i t y o f RNP b u t n o t Sm t o ribonuclease (9-11).
From the Departments o f Cell Biology, Internal Medicine, Molecular Biophysics and Biochemistry, and Pathology,
Yale University School o f k d i c i n e , New Haven, Connecticut
06510
MRL i s currently i n the Department o f Internal Medicine, Washington University School o f Mediclne. S t . Louis,
Mlssouri. JAH i s a senior investigator o f the A r t h r i t i s
Foundation. CAJ i s a Howard Hughes Medical I n s t i t u t e In-
vestigator.
This work was supported by United State Public Health
Service Grants f r a n the N I H . by a grant f r a n the Kroc Foundation, and by a Culpeper Fellowship t o MRL.
Arthritis and Rheumah, Vd. 25, No. 7 (July 1982)
Using a monoclonal anti-Sm antibody, we have
confirmed t h e n u c l e a r l o c a t i o n o f t h e Sm antigen.
Biochemical techniques r e v e a l t h a t t h e a n t i g e n i c s i t e s
recognized by anti-Sm l i e on d i s c r e t e p a r t i c l e s composed o f small n u c l e a r RNAs complexed w i t h p r o t e i n ,
designated snRNPs (pronounced snurps). The a n t i g e n i c
s i t e bound by anti-RNP l i e s on one o f these snRNPs b u t
t h e RNP a n t i g e n i c determinant i s d i s t i n c t from t h a t
recognized by anti-Sm. The monoclonal anti-Sin antibody
has been used i n a simple radioimrmnoassay which may
serve as a general model f o r a new s e t o f assays t o
t i t r a t e a u t o a n t i b o d y a c t i v i t y i n p a t i e n t sera.
In
a d d i t i o n , a survey o f ANA p o s i t i v e sera r e v e a l s RNP
s p e c i f i c i t i e s d i s t i n c t from Sm and RNP.
METHODS FOR STUDYING snRNPs
I n o r d e r t o study t h e s t r u c t u r e o f snRNPs i t i s
s i m p l e s t t o examine t h e RNA and p r o t e i n components
To examine &t
RNA, HeLa c e l l s a r e
separately ( 2 ) .
b i o s y n t h e t i c a l l y labeled w i t h
P-orthophosphate which
i s i n c o r p o r a t e d i n t o t h e phosphate backbone o f RNA.
The c e l l s a r e sonicated, p a t i e n t antibody added, and
t h e antigen-antibody complexes p r e c i p i t a t e d w i t h formal i n f i x e d p r o t e i n A b e a r i n g Staphylococcus (Pansorbin).
The complexes a r e subjected t o phenol e x t r a c t i o n , which
denatures and removes p r o t e i n b u t leaves RNA i n t a c t ,
and then electrophoresed on 10% acrylamide 7M urea g e l s
t o separate t h e RNA species.
r t h e examination of p r o t e i n s , c e l l s a r e l a b e l e d
w i t h "S-methionine
and subjected e i t h e r t o a procedure
s i m i l a r t o t h a t f o r t h e c h a r a c t e r i z a t i o n o f RNA o r t h e
i m n e complexes may be adsorbed t o and e l u t e d from a
p r o t e i n A-Sepharose column (Pharmacia).
The p r o t e i n s
a r e concentrated by p r e c i p i t a t i o n w i t h acetone and
f r a c t i o n a t e d on 15% polyacrylamide g e l s c o n t a i n i n g SDS.
ANTI RNP AND ANTI-Sm ANTIBODIES PRECIPITATE SMALL
NUCLEAR RNAs (snRNA)
Anti-RNP and anti-Sm a n t i b o d i e s were i s o l a t e d f r a n
p a t i e n t sera by p r e c i p i t a t i o n w i t h s a t u r a t e d ammogjum
sulfate.
When t h e a n t i b o d i e s were mixed w i t h
Pl a b e l e d c e l l e x t r a c t s and t h e RNAs examined as des c r i b e d above, g e l s l i k e t h a t shown i n F i g u r e 1 were
obtained.
Anti-RNP sera o n l y p r e c i p i t a t e s one band,
762
LERNER ET AL
l a b e l e d U1, whereas anti-Sm a n t i b o d y p r e c i p i t a t e t h e
f i v e small RNAs c a l l e d U1, U2, U4, U5 and U6. These
small RNAs turned o u t t o be d i s c r e t e molecules c a l l e d
snRNAs t h a t had p r e v i o u s l y been s t u d i e d b i o c h e m i c a l l y
b u t were o f unknown f u n c t i o n (12). They range i n s i z e
from about 100 ( U 6 ) t o 200 (U2) bases; a l l have r e c e n t l y been completely sequenced (13).
Examination o f F i g u r e 1 a l s o suggests t h a t U1-U6
a r e i n v o l v e d i n separate RNA-protein complexes. N e i t h e r
anti-Sm n o r anti-RNP a r e a b l e t o p r e c i p i t a t e snRNAs
a f t e r d e p r o t e i n i z a t i o n o f c e l l e x t r a c t s w i t h phenol,
c o n f i r m i n g previous r e p o r t s t h a t both Sm and RNP r e q u i r e p r o t e i n f o r a n t i g e n i c i t y (9,lO). Anti-RNP prec i p i t a t e s o n l y a subset o f t h e snRNAs o f t h e Sm "fami l y " , s p e c i f i c a l l y , U1; t h e r e f o r e , i t must r e s i d e i n a
r i bonucleoprotein separate from t h e o t h e r snRNAs.
P r e c l e a r i n g t h e c e l l e x t r a c t s w i t h anti-RNP a n t i b o d y
f o l l o w e d by p r e c i p i t a t i o n w i t h anti-Sm antibody r e s u l t s
i n t h e appearance o f snRNAs U2. U4, U5 and U6 b u t n o t
U1.
P r e c l e a r i n g w i t h anti-Sm a n t i b o d y f o l l o w e d by
anti-RNP y i e l d s no p r e c i p i t a b l e snRNAs.
From t h e i r
s i z e , we e x t r a p o l a t e t h a t U2, U4, U5 and U6 l i k e w i s e
r e s i d e i n separate snRNPs ( 1 4 ) .
ANTI-RNP AND ANTI-Sm ANTIBODIES ALSO PRECIPITATE
NUCLEAR PROTEINS
When a i-RNP and anti-Sm a n t i b o d i e s were used t o
precipiate " S
methionine l a b e l e d snRNPs, seven prot e i n s l a b e l e d A-G w i t h m o l e c u l a r weights o f 12,000 t o
35,000 were found on 15% polyacrylamide gels ( 2 ) .
C l e a r i n g elgyeriments w i t h anti-RNP and anti-Sm a n t i S-JQbeled c e l l e x t r a c t s , analagous t o t h e
bodies on
ones f o r t h e
p l a b e l e d e x t r a c t s revealed t h a t a l l
seven p r o t e i n s were associated w i t h U 1 snRNA w h i l e o n l y
p r o t e i n s B, D, E, F and G were found w i t h t h e U2, U4,
U5 and U6 snRNPs.
I n summary, an i n d i v i d u a l snRNP can be envisaged
t o c o n t a i n one RNA molecule.
Thus t h e r e a r e f i v e
d i s t i n c t U RNA c o n t a i n i n g snRNPs. U 1 associates w i t h a
s e t of seven p r o t e i n s , w h i l e U2, U4, U5 and U6 each
a s s o c i a t e w i t h a l l o f a subset o f f i v e p r o t e i n s .
Anti-RNP a n t i b o d y r e a c t s o n l y w i t h t h e U 1 c o n t a i n i n g
snRNP and anti-Sm r e a c t s w i t h a l l f i v e snRNPs. Thus,
i f t h e U RNA c o n t a i n i n g snRNPs a r e considered a f a m i l y ,
anti-Sm recognizes a l l members o f t h e f a m i l y w h i l e
anti-RNP recognizes o n l y one member o f t h i s f a m i l y .
The U 1 snRNP i s a p a r t i c l e w i t h two a n t i g e n i c s i t e s ,
one f o r anti-RNP, t h e o t h e r f o r anti-Sm a n t i b o d y (Figu r e 2).
It w i l l be shown below t h a t t l i e a n t i g e n i c
s i t e s on t h e U 1 snRNP recognized by a n t i - h and a n t i RNP a r e indeed d i s t i n c t , as would be expected from t h e
above f i n d i n g s .
snRNPS ARE ABUNDANT AND HIGHLY CONSERVED
F i g u r e 1. Polyacrylamide g e l e l e c t r o p h o r e s i s o f 32P-labeled
n u c l e i c a c i d s f r a n HeLa c e l l s . T o t a l c e l l s o n i c a t e ( l a n e
T ) . Bands i n o t h e r l a n e s were o b t a i n e d by immunopreclpitat i o n o f c e l l e x t r a c t s w i t h p a t i e n t anti-rRNA ( l a n e R ) ,
normal human serum ( l a n e N), p a t i e n t a n t i - L a ( l a n e La ,
p a t i e n t a n t i - h ( l a n e sm). p a t i e n t a n t i - w p ( l a n e RNpI.
p a t i e n t anti-Ro ( l a n e Ro). and p a t i e n t anti-tRNA ( l a n e
tRNA). The bands l a b e l e d 5.85 and 55 a r e c h a r a c t e r i s t i c o f
rRNA. See t e x t f o r t h e d e s c r i p t i o n o f o t h e r bands.
The snRNAs a r e v e r y abundant w i t h U1 RNA being t h e
mobt commn snRNA i n mammalian c e l l s , present i n over
10 copies per nucleus. ( T h i s i s about 1/10 t h e number
o f ribosomes i n a mammalian c e l l . )
U2 i s almost a5
abundant as U1.
U4, U5 and U6 each number some 10
copies per nucleus.
The degree o f e v o l u t i o n a r y cons e r v a t i o n o f these a n t i g e n i c s i t e s can be examined by
!gins anti-RNP and anti-Sm a n t i b o d i e s t o p r e c i p i t a t e
P-labeled m a t e r i a l from c e l l s o f v a r i o u s species.
RNA-CONTAINING LUPUS ANTIGENS
I
with antbodies to
other smailRNAs
in porticles of
some class
2
Srn= 0 UIRNP;IS
763
3
4
Ro= I
La= 0
f o r m a t i o n making t h e DNA f o r t h e gene l o n g e r than t h e
corresponding mRNA which i s t r a n s l a t e d i n t o p r o t e i n .
A n a l y s i s o f a number o f genes has r e v e a l e d t h a t t h e
r e g i o n s o f e x t r a i n f o m a t i o n , c a l l e d i n t r o n s (which can
be thousands o f bases l o n g ) , do n o t have a conserved
sequence.
However, t h e sequences a t e i t h e r end o f
these nonsense segments, termed s p l i c e j u n c t i o n s , a r e
v i r t u a l l y i d e n t i c a l from gene t o gene. Since i n t r o n s
a r e present i n t h e n u c l e a r hnRNA b u t absent from t h e
mature mRNA found on ribosomes i n t h e cytoplasm, i t was
necessary t o propose t h a t t h e I n t r o n sequences were
" s p l i c e d " o u t by c u t t i n g and r e j o i n i n g a t t h e " s p l i c e
j u n c t i o n s " d u r i n g t h e m a t u r a t i o n o f mRNA.
What c e l l u l a r components m i g h t be i n v o l v e d i n t h e process o f
RNA s p l i c i n g were i n i t i a l l y a mystery.
We (14) and o t h e r s ( 1 5 ) noted t h a t t h e 5 ' end o f
t h e U 1 snRNA had a sequence t h a t was e x a c t l y complement a r y t o c a n m n s p l i c e j u n c t i o n sequences o f hnRNA and
t h e r e f o r e proposed t h a t t h e U 1 snRNP was i n v o l v e d i n
RNA s p l i c i n g .
T h i s idea has r e c e i v e d experimental
support by u s i n g anti-RNP and anti-Sm a n t i b o d i e s i n an
-i n v i t r o s p l i c i n g system; t h e two antibodies, both o f
which recognize and p r e c i p i t a t e t h e U 1 snRNP, indeed
were observed t o b l o c k RNA s p l i c i n g (16). The d e t a i l e d
cellukr RNAa
u4
u5
U6
Y2
4.5,s and 4.5S
y3
virolRNAa
EeER2,VALVAXX
Dlagrammatic r e p r e s e n t a t i o n o f a n t i g e n i c d e t e r F i g u r e 2.
minants on r l b o n u c l e o p r o t e i n (RNP) p a r t i c l e s recognized by
Sm and RNP a r e described i n t h e t e x t .
autoantibodies.
Anti-Ro a n t i b o d i e s r e a c t w i t h small cytoplasmic r i b o n u c l e o p r o t e i n p a r t i c l e s (scRNPs) whlch c o n t a i n small RNAs i n c l u d i n g Y1, Y2 and Y3 (3). Anti-La a n t i b o d i e s r e a c t w i t h r i b o n u c l e o p r o t e i n p a r t i c l e s which c o n t a i n numerous small c e l l u I n addition, anti-La
l a r RNAs i n c l u d i n g 4.55 species.
antibodies
immunoprecipitate RNP
p a r t i c l e s containing
Epstein-Barr v i r u s encoded RNAs EBER 1 and EBER 2 from
Epstein-Barr v i r u s i n f e c t e d c e l l s and p a r t i c l e s which cont a i n t h e adenovirus encoded RNAs V A I and V A I I f r a n adenov i r u s i n f e c t e d c e l l s (3).
We have r e c e n t l y demonstrated
t h a t Ro p a r t i c l e s bear both t h e Ro and La determinants;
hence Ro i s a subset o f La. T h i s o b s e r v a t i o n i s depicted by
p l a c i n g t h e La ( o v a l ) determinant on t h e Ro p a r t i c l e s (data
n o t shown).
I
- : :?+
The r e s u l t s from such an experiment reveal t h a t these
two human a n t i b o d i e s p r e c i p i t a t e d snRNPs from men.
mice, chickens, frogs, sea u r c h i n s and even i n s e c t s
(14).
Moreover, t h e snRNAs i n t h e snRNPs a r e almost
i d e n t i c a l i n sequence across t h i s wide range o f species. The abundance and c o n s e r v a t i o n o f snRNPs, t h e r e fore, suggests t h a t these p a r t i c l e s a r e i n v o l v e d i n
important c e l l u l a r processes.
DNA
gene
.1
-----
hnRNA
,I
FUNCTION OF snRNPS
snRNPs a r e complexes o f snRNAs and p r o t e i n .
As
such, t h e RNA component i s d i f f e r e n t from t h e t h r e e
types o f RNA w i t h which we a r e most f a m i l i a r : t r a n s f e r
RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA
(mRNA).
rRNA i s a component o f ribosomes and l i k e
snRNAs i s t i g h t l y associated w i t h p r o t e i n i n c e l l s .
tRNAs, which a r e l e s s than 100 bases long, a s s o c i a t e a t
various times w i t h d i f f e r e n t p r o t e i n s as t h e y perform
t h e i r adaptor f u n c t i o n f o r t r a n s l a t i n g mRNA i n t o prot e i n on t h e ribosome.
I t should be emphasized t h a t
tRNA a c t u a l l y forms base p a i r s w i t h mRNA d u r i n g t h e
process o f decoding t h e message and t h a t t h i s i n t e r a c t i o n takes p l a c e on a RNP p a r t i c l e ( t h e ribosome)
which i m p l i e s v a r i o u s RNA-RNA-protein i n t e r a c t i o n s .
I n m a m l i a n c e l l s , mRNA i s d e r i v e d from a n u c l e a r
precursor c a l l e d h e t e r o n u c l e a r RNA (hnRNA) which i s t h e
f o m o f RNA i n i t i a l l y t r a n s c r i b e d from DNA. This l a s t
statement may appear banal, b u t when DNA sequencing
r e c e n t l y became a v a i l a b l e , i t was noted t h a t w i t h i n t h e
DNA corresponding t o almost every gene, t h e r e e x i s t s
one o r more chunks o f nonsense, t h a t i s , e x t r a i n -
,-.
\I
U, snRNP
aligns splice
junctions
-
4
Cut and ligate
mRNA to cytoplasm
F i g u r e 3 . P o s s i b l e mechanism o f RNA s p l i c l n g . Genanic DNA.
whlch c o n t a i n s i n t r o n s , i s t r a n s c r i b e d i n t o hnRNA i n t h e
nucleus.
The U 1 snRNP a l i g n s w l t h t h e s p l i c e j u n c t i o n s a t
t h e ends o f t h e i n t r o n s and s p l i c e s t h e i n t r o n from hnRNA t o
form mRNA, which i s then t r a n s p o r t e d t o t h e cytoplasm f o r
t r a n s l a t l o n i n t o protein.
LERNER ET AL
764
mechanism f o r s p l i c i n g i s n o t y e t c l e a r , b u t i t i s
reasonable t o propose t h a t U 1 RNA a l i g n s t h e two s p l i c e
j u n c t i o n s a t e i t h e r end o f an i n t r o n by making base
p a i r s w i t h these conserved sequences ( F i g u r e 3 ) . The
i n t e r a c t i o n t h u s resembles t h a t between t R N A and mRNA.
The snRNP p r o t e i n s c o u l d f u r t h e r perform t h e enzymatic
f u n c t i o n o f c u t t i n g t h e i n t r o n s from hnRNA t o produce
mRNA.
I t i s a l r e a d y known t h a t snRNPs a s s o c i a t e w i t h
hnRNA, i n agreement w i t h t h e p r e d i c t i o n (14).
The f u n c t i o n s o f t h e o t h e r snRNPs a r e n o t known.
The f a c t t h a t t h e y possess an a n t i g e n i c determinant i n
common w i t h t h e U 1 snRNP (Sm) suggests t h a t they t o o
may be i n v o l v e d i n t h e n u c l e a r e d i t i n g o f RNA t r a n scripts.
MONOCLONAL ANTIBODIES TO NUCLEIC A C I D CONTAINING
CELLULAR CONSTITUENTS
The a v a i l a b i l i t y o f m n o c l o n a l a n t i b o d i e s w i t h
s p e c i f i c i t i e s i d e n t i c a l t o those o f p a t i e n t a u t o - a n t i bodies would e l i m i n a t e many u n c e r t a i n t i e s i n e x p e r i mental i n t e r p r e t a t i o n r a i s e d by t h e use o f p a t i e n t
sera. (NZB x NZW)F , BXSB and mice w i t h t h e l p r / l p r
gene such as t h e dRL/MP-lpr/lpr s t r a i n developed by
Edwin Murphy and John Roths a t t h e Jackson Laboratory,
make a u t o a n t i b o d i e s i n a s s o c i a t i o n w i t h autoimmune
s t a t e s resembling lupus (17). I n a screen, we found
t h a t t h e serum o f a s e l e c t e d MRL mouse contained a n t i bodies s p e c i f i c f o r DNA, r R N A and Sm as shown i n F i g u r e
4.
We fused spleen c e l l s from t h i s mouse w i t h t h e
myeloma SP 2/0 and obtained hybridoma d e r i v e d monoc l o n a l a n t i b o d i e s s p e c i f i c f o r DNA, rRNA and Sm (1).
Monoclonal a n t i b o d i e s t o DNA and ribosomal RNA had been
r e p o r t e d p r e v i o u s l y (18,19). Using t h e monoclonal
anti-Sm a n t i b o d y we have confirmed a l l o u r previous
s t u d i e s done w i t h p a t i e n t antibody.
The hybridoma
a n t i b o d y t o Sm p r e c i p i t a t e s t h e same s e t o f f i v e snRNAs
as p a t i e n t Sm a n t i b o d y demonstrating t h a t each o f t h e
f i v e snRNPs c a r r i e s a common a n t i g e n i c p r o t e i n and
e l i m i n a t i n g t h e p o s s i b i l i t y t h a t anti-Sm sera c o n t a i n
&---"
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Sb w
F i g u r e 4.
nn.rlair
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C r u UAI.
U
I I W
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t-..r m ~ a.-,.I,L C ~ I ~ Laiie
.
I,
1
L-A-1
--11
uiiiui~e.
Bands i n l a n e s 2-5 were obtained by imrmnoprecipitaton o f
c e l l e x t r a c t s w i t h MRL/1 mouse serum ( l a n e 2 ) . monoconal
anti-DNA ( t h e smear l a b e l e d PNA n e a r t h e top o f , t h e l a n e i s
L>.
SUSL~PLIOI
r.u~ aeuxvrioonuciease DUC nor t o riDonuciease.
l a n e 3 ) , monoclonal a n t i - r R N A ( l a n e 4 ) and monoclonal anti:
Sm ( l a n e 5 ) .
____
L I L . _
_.L
1
.
lo'
---:--A-
L U L ~ IL ~ I s
I
p
..
I
Polyacrylamide gel e l e c t r o p h o r e s i s o f 32P-labeled
.rill.-
a n u k n ~ t~~ C
,,/'
//
lod
10-2
Log Dilvllon of Serum
Figure 5.
Competition radioimmunoassay t o t i t e r anti-Sm
a c t i v i t y . M-Sm,monoclonal
anti-Sm; Sb, p a t i e n t anti-Sm; Am,
_ ..
Datlent
- r--
antl-<m.
- - - , RNP.
.. . Pc
. -,
natient
r"".-..'
t l n t i - C- m
888,
"I.".
nNA
US.",
Qn.
,\",
N
I.,
normal human serum; Ag, p a t i e n t anti-RNP; M-rRNA, monoclonal
rRNA; M-DNA, monoclonal anti-DNA;
Le, p a t i e n t a n t i - L a .
R NA-CO NTAIN I NG LUPUS ANT IG ENS
f i v e separate antibodies.
I n d i r e c t imnunofluorescence
on Vero c e l l s w i t h t h e monoclonal a n t i b o d i e s r e v e a l s
conventional patterns:
monoclonal anti-Sm g i v e s a
s p e c k l e d n u c l e a r p a t t e r n o f f l u o r e s c e n c e , anti-DNA
s t a i n s t h e i n n e r r i n g s o f t h e n u c l e u s and anti-rRNA
b r i g h t l y s t a i n s n u c l e o l i ( t h e l o c a t i o n o f rRNA b i o s y n t h e s i s ) , w i t h s p e c k l e d c y t o p l a s m i c f l u o r e s c e n c e (1).
A RADIOIMMUNOASSAY SPECIFICALLY DETECTS ANTI-Sm
ACTIVITY
I f monoclonal a n t i b o d i e s r e c o g n i z e t h e same d e t e r m i n a n t s as p a t i e n t a u t o a n t i b o d i e s , t h e f o r m e r c o u l d be
used as a c o n t i n u o u s s o u r c e o f r e f e r e n c e a n t i b o d y f o r
c l i n i c a l assays.
The monoclonal a n t i - S m a n t i b o d y has
been used as such a r e f e r e n c e a n t i b o d y i n a c o m p e t i t i o n
assay t o d e t e r m i n e t h e l e v e l o f anti-Sin a c t i v i t y i n
unknown sera.
Such an assay i s shown i n F i g u r e 5.
Unlabeled
e!oclonal
anti-Sin e f f e c t i v e l y i n h i b i t s t h e b i n d i n g o f
I - l a b e l e d monoclonal a n t i - % t o r a b b i t thymus exA
t r a c t adsorbed o n t o p l a s t i c m i c r o t i t e r p l a t e s .
comparable c u r v e i s o b t a i n e d u s i n g a n t i b o d y fran a
p a t i e n t w i t h anti-Sm.
A n t i b o d i e s fran p a t i e n t s w i t h
l o w e r t i t e r s o f anti-Sm i n h i b i t l e s s w e l l .
Note t h a t
t h e p a t i e n t serum c o n t a i n i n g o n l y anti-RNP, an a n t i b o d y
r e c o g n i z i n g an a n t i g e n i c d e t e r m i n a n t on a p a r t i c l e @ 3 t
a l s o b e a r s t h e Sm a n t i g e n , does n o t i n h i b i t t h e
I
anti-Sm b i n d i n g .
T h i s r e s u l t demonstrates t h e s p e c i f i c i t y o f t h e assay and s u p p o r t s t h e h y p o t h e s i s t h a t
p h y s i c a l l y n o n o v e r l a p p i n g s i t e s on t h e U 1 snRNP a r e
bound by anti-RNP and b y a n t i - h a n t i b o d i e s .
ADDITIONAL RIBONUCLEOPROTEIN COMPLEXES ARE PRECIPITATED BY OTHER PATIENT SERA
U s i n g a n t i b o d i e s o b t a i n e d fran t h e s e r a 3 f f 2 5 0 ANA
p o s i t i v e p a t i e n t s , immunoprecipi t a t i on o f
P- 1a b e l ed
HeLa c e l l e x t r a c t s r e v e a l e d e l e c t r o p h o r e t i c p a t t e r n s
c h a r a c t e r i s t i c n o t o n l y o f t h e snRNPs p r e c p i t a t e d b y Sm
and RNP b u t o f a d d i t i o n a l RNP-protein complexes ( F i g u r e
1) ( 2 0 ) .
A n t i - L a a n t i b o d i e s r e c o g n i z e d d i s c r e t e low
m o l e c u l a r w e i g h t n u c l e a r RNAs a s s o c i a t e d w i t h p r o t e i n
which r e p r e s e n t a c l a s s o f snRNPs d i s t i n c t f r o m t h e Sm
a s s o c i a t e d snRNPs.
Anti-Ro a n t i b o d i e s p r e c i p i t a t e a
t h i r d c l a s s o f RNP p a r t i c l e s . The f i v e members o f t h i s
c l a s s a r e c y t o p l a s m i c and a r e c a l l e d s m a l l c y t o p l a s m i c
RNPs o r scRNPs t o d i s t i n g u i s h them f r o m t h e La and Sm
snRNPs. O t h e r c e l l u l a r components i m m u n o p r e c i p i t a t e d by
a n t i b o d i e s f r o m p a t i e n t s i n t h i s s u r v e y i n c l u d e d DNA,
r R N A (analagous t o t h e monoclonal mouse anti-DNA and
anti-rRNA a n t i b o d i e s d e s c r i b e d above), and tRNA. Some
o f the antibodies resulted i n imnunoprecipitation
p a t t e r n s w h i c h a r e c u r r e n t l y u n c h a r a c t e r i z e d b u t may
c o r r e s p o n d t o o t h e r a n t i g e n a n t i b o d y systems a l r e a d y
d e s c r i b e d i n t h e r h e u m a t i c d i s e a s e l i t e r a t u r e on t h e
b a s i s o f i n u n u n o d i f f u s i o n s t u d i e s ( d a t a n o t shown).
765
o f one o f t h e f i v e s m a l l n u c l e a r RNAs U1, U2, U4, U5 o r
U6 complexed w i t h p r o t e i n .
Anti-RNP a n t i b o d y r e c o g n i z e s a d e t e r m i n a n t p r e s e n t o n l y on t h e U 1 snRNP. As
anti-RNP does n o t c a n p e t e w i t h a n t i - S n i n t h e c a n p e t i t i v e radioimrmnoassay, t h e RNP and Sm a n t i g e n i c s i t e s
a r e n o n o v e r l a p p i n g . The U 1 snRNP has been shown t o be
i n v o l v e d i n t h e s p l i c i n g o f n u c l e a r RNA t o c r e a t e
m a t u r e mRNA.
The f u n c t i o n s o f t h e o t h e r snRNPs a r e
under investigation.
I t i s i n t e r e s t i n g t o s p e c u l a t e t h a t t h e observat i o n t h a t anti-RNP and a n t i - S m a n t i b o d y r e a c t w i t h
abundant, h i g h l y conserved c e l l u l a r components, may be
a g e n e r a l f e a t u r e o f a n t i g e n a n t i b o d y systems i n a u t o imrmne d i s e a s e . A s u r v e y o f a n t i b o d i e s f r o m 250 ANA
p o s i t i v e p a t i e n t s r e v e a l e d , i n a d d i t i o n t o Sm and RNP,
immunoprecipitation patterns c h a r a c t e r i s t i c of other
abundant n u c l e i c a c i d c o n t a i n i n g c e l l u l a r canponents.
Among t h e s e were DNA, La, Ro, rRNA and tRNA.
The a v a i l a b i l i t y o f a l i b r a r y o f monoclonal a n t i bodies w i t h i d e n t i c a l s p e c i f i c i t i e s t o p a t i e n t a n t i b o d i e s would be u s e f u l f o r b o t h m o l e c u l a r b i o l o g y and
c l i n i c a l diagnostics.
The m o l e c u l a r b i o l o g i s t s c o u l d
use t h e monoclonal a n t i b o d i e s as s p e c i f i c probes t o
s t u d y t h e s t r u c t u r e and f u n c t i o n o f h i g h l y conserved
c e l l u l a r c o n s t i t u e n t s , as has been done w i t h t h e monoc l o n a l anti-Sm a n t i b o d y .
C l i n i c i a n s c o u l d use t h e
monoclonal a n t i b o d i e s as s e n s i t i v e r e a g e n t s i n r a d i o imrmnoassays. as demonstrated i n t h i s paper, o r enzyme
l i n k e d immunoadsorbent assays (ELISAs) as a means o f
d e t e c t i n g and t i t e r i n g a u t o a n t i b o d i e s i n p a t i e n t sera.
I n p a r t i c u l a r , as t h e presence o f anti-Sm a n t i b o d y i s
v i r t u a l y d i a g n o s t i c o f lupus, t h e c o m p e t i t i o n r a d i o imrmnoassay u s i n g t h e monoclonal anti-Sin a n t i b o d y c o u l d
be used t o make a l a b o r a t o r y d i a g n o s i s o f l u p u s i n many
cases.
O t h e r uses o f t h e monoclonal a n t i b o d i e s c o u l d
be i n animal models, where a n t i b o d y s p e c i f i c i t y , i s o t y p e and i d i o t y p e s t u d i e s would enhance o u r unders t a n d i n g o f autoimmune disease.
ACKNOWLEDGEMENTS
The authors a r e g r a t e f u l t o Drs.
Edwin Murphy f o r supplying MR!J1 mice.
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1.
2.
3.
4.
DISCUSSION
A monoclonal a n t i - S m a n t i b o d y , p a t i e n t a n t i - S m
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antigens.
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