Purification and Biochemical Characterization of

Nuclear Ribonucleoprotein Antigen using

Purified Antibody from Serum of a

Patient with Mixed Connective Tissue Disease

MAKOTOTAKANO, PAUL F. AGRIS, and GORDONC. SHARP, Division of Itmtmnutnologyand Rheumatology, Department of Medicine and the Division of BiologicalSciences, University of Missouri-Columbia, Columbia, Missouri 65212

A B S T RA C T We have achieved a high degree ofpurification of nuclear ribonucleoprotein antigen fromcalf thymus nuclear extract through antibody affinitychromatography. Antibody to nuclear ribonucleopro-tein was purified from the serum of a patient with mixedconnective tissue disease and Sepharose 4B was co-valently coupled with the purified human antibody.The sodium thiocyanate eluate from the affinity columncontained active ribonucleoprotein antigen and thespecific activity of the antigen in this eluate was 488times higher than the original nuclear extract. Theprotein component of the eluate consisted of six poly-peptides determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Two of them wereshown to be antigenic by a hemagglutination inhibi-tion test. The molecular weights of these two peptideswere -13,000 and those of the other four were 13,000,13,000, 30,000, and 65,000. The ribonucleic acid com-ponent of the eluate was shown by urea-polyacryl-amide gel electrophoresis to contain five polynucleo-tides. Two of the five, estimated to contain 40 and 60nucleosides, had antigenic activity. The other threepolynucleotides which had more than 77 nucleosideshad no antigenic activity. No modified nucleosideswere found in these ribonucleic acid molecules. Evenin this highly purified ribonucleoprotein antigen, Smantigen was detected by immunodiffusion. Thisevidence indicated that there are some molecular asso-ciations between ribonucleoprotein and Smantigens ashas previously been suggested.

Received for publication 6 September 1979 and in revisedform 24 January 1980.

INTRODUCTION

Antibody to nuclear ribonucleoprotein (RNP)' is almostalways detected in very high titers by passive hemag-glutination testing of the serumn of patients with mixedconnective tissue disease (MCTD) (1). This antibody isalso found, although less fre(luently and in lower titers,in patients with systemic lupus erythematosus, pro-gressive systemic sclerosis, or mild, undifferentiatedconnective tissue disease (2-5). Patients with antibodyto RNPwho are diagnosed as having systemic lupuserythematosus, progressive systemic sclerosis, and un-differentiated connective tissue disease tend to havesome of the clinical features of MCTD(6). Thesefindings suggest that immune reactions to RNPmayplay an important role in the development of some ofthe disease manifestations of patients with MCTD.

Although a precise pathological role has yet to bedetermined, the high frequency of detectable serumimmune complexes in MCTD(7) and the deposition ofantibody to RNPin the glomerular lesion in systemiclupus erythematosus (8) suggest that this antibody, inthe form of immune complexes, may contribute to im-mune injury. The recent observation of Alarcon-Segovia (9, 10) that antibody to RNPcan enter livingTy cells through their Fc receptors and bind to theirnuclei suggests that another possible disease mech-anism in MCTDmight be an alteration of the biological

1 Abbreviations used in this paper: CTE, calf thymusnuclear extract; ENA, extractable nuclear antigen; MCTD,mixed connective tissue disease; NaSCN, sodium thiocyanate;PBS, phosphate-buffered saline; RNP, nuclear ribonucleopro-tein; SDS, sodium dodecyl sulfate.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/80106/1449/08 $1.00Volume 65 June 1980 1449-1456

1449

function of RNP, which is bound to its cognate anti-body. Therefore, studies of the biological role of RNPmight result in a better understanding of the patho-genesis of MCTD.

To perform such studies it is first necessary to purifyand characterize the RNP antigen. An RNP antigenhas been isolated and purified from rat liver extract(11). The purpose of this paper is to report our meth-ods, which have achieved a high degree of purifica-tion of nuclear RNPantigen from calf thymus nuclearextract and the biochemical characterization of thispurified antigen.

METHODS

Selection of seraA reference serum monospecific for antibody to nuclear

RNPwas obtained from a patient with MCTD.The titer of anti-body in whole serum to extractable nuclear antigen (ENA)by passive hemagglutination was 1:1,260,000. When sheeperythrocytes coated with ENA were digested with ribo-nuclease A (RNase) (1) before reaction with the positivereference serum, the hemagglutination titer was eliminated.This serum gave a single precipitin line against calf thymusnuclear extract (CTE) in agarose immunodiffusion. This pre-cipitation reaction was abolished by treatment of CTE withRNase. Negative control serum was drawn from a healthysubject. In this serum, no antibody activity to ENA, CTE, orDNAwas detected by hemagglutination or immunodiffusion.Reference sera for antibodies to Sm, PM-1, MA, Scl-1, SS-B,and DNA were used to determine antigen specificity ofpurified antigen.

IgG preparationIgG was prepared by ammonium sulfate precipitation fol-

lowed by DEAE-cellulose column chromatography accordingto the method described by Fahey and Horbett (12).

Purification of antibody to RNPPurified antibody to RNPwas obtained from immune pre-

cipitates made by mixing CTE and serum monospecific forantibody to RNP. The precipitates were washed three timeswith ice cold 0.01 M phosphate-buffered saline (PBS) con-taining 0.15 M NaCl, pH 7.4, and dissolved in 0.01 M HCIsolution. This solution was incubated at 56°C for 30 min to de-stroy RNP antigenic activity. Further purification wasachieved through ammonium sulfate precipitation. The pre-cipitate at 25% saturation was discarded; the next precipitateat 50%saturation collected by centrifugation was dissolved inthe smallest volume of PBSand dialyzed against the same buf-fer for 36 h. The purified antibody was shown to be pureimmunoglobulin (Ig)G by immunoelectrophoresis and wasmonospecific for RNP in that it did not contain activity forSm, PM-1, MA, Scl-1, SS-B, or DNA.

Nuclear antigen preparationENAand CTEwere prepared by described methods (1, 13).

After ultracentrifugation at 106,000 g for 2 h, the CTE super-nate was further fractionated using ammonium sulfate pre-cipitation. The precipitate at 30% saturation was discarded,and the next precipitate at 60% saturation dissolved in PBS

and dialyzed against PBS for 48 h. The final protein concen-tration of this crude CTE was 15-20 mg/ml.

Agarose gel column chromatographyAgarose gel column chromatography (Sepharose 6B,

Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) was usedfor partial purification of RNPantigen. The column was 2.5 cmDiam and 90 cm in length. PBS, 0.01 M, pH 7.4, containing0.5 MNaCl was used as running buffer. Blue dextran (2,000,000mol. wt) and 7S IgG (160,000 mol wt) were used for molecu-lar weight standards.

Affinity chromatographyIn affinity chromatography used to purify RNP antigen,

0.5 g of cyanogen bromide-activated Sepharose 4B was re-acted with 2 ml (5.2 mg) of purified antibody to RNPor thesame amount of IgG from negative control serum in 0.1 MNaHCO3containing 0.5 M NaCl solution at 4°C for 18 h bygentle stirring with a magnetic stirrer. Remaining activegroups were blocked with 1.0 Methanolamine and unboundprotein was washed away from the gel according to the manu-facturer's instruction. Under these conditions, the Sepharose4B gel was coupled with >97% added IgG. The gel wasthen poured into a column, 0.9 cm Diam and 15 cm in length,and equilibrated with 0.01 M PBS containing 0.5 M NaCl,pH 7.4.

Sodium dodecyl sulfate (SDS)-polyacrylamideslab gel electrophoresisElectrophoresis of crude and purified RNPantigen in SDS-

polyacrylamide slab gels was performed according to themethods of Laemmli (14) and Weber and Osborn (15). The gelswere formed as 1.5 x 120 x 106 mmslabs, which consisted ofseparating gels containing 8-13% gradient acrylamide and0.1% SDS in Tris-HCI pH 8.8 with 15 mmof stacking gelscontaining 5%acrylamide and 0.1% SDSin Tris HCG, pH 6.8.To retain their antigenic activity, samples were not boiled.Gels were run at 150 V, constant voltage at 4°C. The proteinswere stained with 0.04% Coomassie Brilliant Blue in 25%isopropyl alcohol, 10% acetic acid solution overnight. RNase(13,700 mol wt), chymotrypsinogen (25,000 mol wt), oval-bumin (43,000 mol wt), and bovine serum albumin (67,000mol wt) were used as molecular weight standards.

Urea-polyacrylamide gel electrophoresisRibonucleic acid content of purified RNPantigen was de-

termined by polyacrylamide gel electrophoresis. Slab gels(1.5 mmthick; 16.5 x 16.5 cm) consisting of 10% acrylamideand 7 Murea running gels were prepared and run as described(16). Escherichia coli tRNAmet (Sigma Chemical Co., St.Louis, Mo.), 90% pure, was used as a molecular weight stand-ard. Bromphenol blue and xylene cyanol FF were added to allsamples; BPB marks the electrophoretic front and xylenecyanol FF migrates at a rate comparable to 5S RNAunderthe conditions used (16). Gels were run at 15 mAconstantcurrent. RNAwas detected by staining the gels with ethidiumbromide (0.1 mg in 200 ml H20) for 40 min followed by rinsingwith H20. Gels were photographed under a near ultra-violet lamp.

Immunological methodsImmunodiffusion. The Ouchterlony double immuno-

diffusion method was uised to demonstrate and identify the

1450 M. Takano, P. F. Agris, and G. C. Sharp

precipitation reaction between the antibody to nuclear pro-tein antigens in the reference serum and crude or purifiedantigen. 10 ml of 0.4% agarose (Marine Colloids Div., FMCCorp., Rockland, Maine) in 0.01 NI PBS, pH 7.4, containiing0.15 MNaCl and 0.1% NaN3, were pipetted onto glass plates(8 x 10 cm). Wells of 7 mmDiam were placed 4 mmapart.The precipitin reaction was observed after 24, 48, and 72 h.

Hemagglutination test. A passive hemagglutination testusing ENA-coated sheep erythrocytes was performed by pub-lished methods (17).

Hemagglutination inhibition test. To detect antigenic ac-tivity of purified fractions, a hemagglutination inhibition testwas performed in microtiter plates (U plate; Dynatech Labora-tories, Inc., Alexandria, Va.). 50 ,l of 1:1,500 diluted serumimonospecific for antibody to RNP, which had been inac-tivated at 56°C for 30 min and absorbed with sheep erythro-cytes, were mixed with 50 p.l of various concentrations ofantigens. After 30 min of incubation at room temperature,100 ul of ENA-coated sheep erythrocytes were added. Theresults were determined after 18 h incubation at 4°C.

Protein estimationProtein concentration was estimated with the method of

Lowry et al. (18).

Analysis of RNPnucleoside compositionQualitative and quantitative analysis of the major and minor

nucleosides of the RNAof RNP antigen was accomplishedaccording to our previously published methods (19).

Amino acid comnposition of RNPA Beckman 121 Mamino acid analyzer, with a 45 x 2.8-cm

column packed with Beckman AA-20 resin (Beckman Instru-ments, Inc., Fullerton, Calif.), was used for the amino acidanalysis. A single column, isothermal (51°C), 3 buffer (Na')system and 3-h chromatography were used. The data handlingsystem was a model 3352 B laboratory system from Hewlett-Packard Co., Palo Alto, Calif. Analvsis was performe(d asdescribed (20).

RESULTS

Purification of nuclear RNP. Partial purification ofRNP from crude CTE was performed by Sepharose6B column chromatography. As shown in Fig. 1, RNPantigenic activity was detected in the early fractionsof the second peak of material (OD curve at 280 nm.)eluting from the column. Since this antigenic activityand 7S IgG were eluted in the same fractions, themolecular weight of this antigen is considered to be-160,000.

These antigen-positive fractions were pooled andammonium sulfate added to make a final concentrationof 60% saturation. The resulting precipitate was dis-solved in the smallest possible vol of 0.01 M PBS,pH 7.4, containing 0.15 M NaCl and dialyzed againistthe same buffer for 36 h followed by dialysis against0.01 M PBS, pH 7.4, containing 0.5 M NaCl for 24 h.The protein concentration of this partially purifiedCTE antigen was 13.2 mg/ml.

5. 0

4.0

>- 3.0

z

a 2.0

I 1.00-X10

I BlueDextran 7S

50 RNP

50 100 150 200

FRACTION NUMBER

FIGURE 1 Partial purification of CTE with Seplharose 6Bcolumiin chromatography. CTE (6 ml, 20 mg protein/ill) thathad been obtainedIby ammiloniumlll stulfate precipitatioin (NMeth-ods) was subjected to agarose gel chromnatography. RNPantigenic activity determinie(d l)y imimultnodifftusion analvsiswas detected in earls fractions of the secondl peak of 'an opticaldensity curxe. This activity was 1present in the samiie frac-tions as 7S IgG.

To fturther purify RNP antigen, 2 ml of partiallypurified antigen were applied to the antibody affinitychromatography columnlli. PBS, 0.01 MI pH 7.4, containi-ing 0.5 Ml NaCl was use(d as a runnliing buffer. Theflow rate was acljusted to be 15 mil/hr. After the proteinconcentration of' the columlln fractions wenit to zero, thecolumn was eluted with 3 MI sodium thiocvanate(NaSCN) solution. The NaSCNeluate was (lialyzed for24 h against 0.01 NI PBS, pH 7.4, conitaining 0.15 MlNaCl immniediately after eltution fromi the coltumniii. TableI shows the percentage of recovered protein alnd aniti-genic activity of the dialyzed NaSCN eluiates. TheNaSCNeltuate from the antibodv affinitv columni con-tained 1.41% of the total applied protein and had anti-genic activity, as determined by double immintiiodiffii-sion and hemiiagglutinationi inhibition tests. On theother hand, the eluate from the coltumni that wascouipled wvith IgG fromii negative control serumiii con-

TABLE IPercent Proteitn Recoverj antd Atntigenic Acticities* il 3 .X1NaSCNEluate fromn the Affiniitij Chromatographyj Columnio7z

\NtSCN eltiate

Sepharose 4B Applieid Recovere(d Percenit Antigenic*couiple(d with protein lprotcin recovere(l activitv

il1g itRg

Pturified antibodyto RNP 26.4 0.372 1.41 Positive

IgG from negativecontrol serumiii 26.4 0.024 (.(9 Negative

* Determinecl by immunodiffuisioni test andl hemnagglutinlatiooninhibition test.

Purification and Characterization of Nuiclear Ribon ucleo protein Antigen 11451

FIGURE 2 Completely identical precipitin lines aimongcrude CTE, partially purified RNP, and purified RNPantigenagainst reference serum for antibody to RNP. Well 1 con-tained crude CTE; well 2, the NaSCN eluate; and well 3,Sepharose 6B-fractionated CTE. Wells 4-6 contained thesame samples as in wells 1-3, respectively, but thesesamples had been pretreated with RNase. The center well con-tained reference serum for antibody to RNP. Precipitin linesdid not appear when the antigen was pretreated with RNase.

tained <0.1% of the total applied protein and had noantigenic activity. Fig. 2 shows completely identicalprecipitin lines among crude, partially purified andpurified antigens against reference serum for antibodyto RNP. These lines were abolished by RNase treatmentof antigens. Quantitative antigenic activities for thesethree antigens are compared in Table II. The NaSCNeluate was found to have very strong antigenic ac-tivity, 0.375 ng of protein from this purified RNPinhibiting the hemagglutination test, compared to 183ng of protein from crude CTE. The degree of inhibitionshowed that the NaSCN eluate was 488 times moreactive for RNPantigen than crude CTE.

Protein analysis of NaSCNeluate. As shown in Fig.3, SDS-polyacrylamide slab gel electrophoresis ofthe NaSCNeluate gave six polypeptide bands. Four ofthe six polypeptides comprised of two darkly stain-

ing major bands and two lightly staining minor bandshad molecular weights estimated as 12,000-13,000.The molecular weights of the larger polypeptides were-65,000 and 30,000.

Elution of the individual polypeptides from the gelwas performed as possible to determine which poly-peptides contained antigenic activity. The whole gelwas cut into 1-mm wide slices from the bottom to thetop. Each gel slice was homogenized in 100 ,ul of0.01 M PBS, pH 7.4, containing 0.15 MNaCl and thencentrifuged at 1,086 g for 10 min. The supernatantfluid was examined for antigenic activity by hemagglu-tination inhibition. Antigenic activity was found inthree areas in the gel. Two of these corresponded tothe polypeptide bands that were located around the13,000-mol wt area and the other was seen to be in thelower molecular weight portion of the gel, an area notstained with Coomassie Brilliant Blue.

Amino acid analysis was performed on the NaSCNeluate containing the peptides shown in Fig. 3. Thetotal amino acid composition is depicted in Table III.No extraordinary amount of basic amino acids, that maybe expected in a nucleic acid-protein complex, werefound.

RNAanalysis of NaSCNeluate. Purified RNPwasenzyme-hydrolyzed to nucleosides and the nucleo-sides subjected to high performance liquid chromatog-raphy for determination of composition (19). Theantigen's RNAwas found to contain only the four majornucleosides. Relative amounts of the nucleosides were:adenosine, 3.11; uridine, 5.73; guanosine, 6.07; andcytosine, 6.65. The (A + U)/(G + C) ratio was 0.695.

Urea-polyacrylamiide gel electrophoresis of theNaSCNeluate showed the purified RNPantigen to becomprised of five polynucleotides (Fig. 4). One poly-nucleotide chain, designated band 1, and two similarlysized chains, designated band 2 in Fig. 4, were largerthan E. coli tRNAmet, which has 77 nucleosides. Twosmaller polynucleotides, bands 3 and 4, could be es-timated to be of 60 and 40 nucleosides in length,

TABLE IIComparison of Quantitative Antigenic Activities among Crude,

Partially Purified, and Purified Antigens

Hemaggltutination inhibition test

Minimum inihibiting Minimunm inhibiting Pturificationiprotein concentrationi amount of proteini index*

pglrlll rfig

Crude CTE 3.66 183 1Sepharose 6B-

fractionated CTE 0.32 16 11.4NaSCNeluate 0.0075 0.375 488

* Minimum inhibiting amount of protein of crude CTE/minimum inhibitingamouint of protein of purified fraction.

1452 M. Takano, P. F. Agris, and G. C. Sharp

Sepharose 6BCrude fractionatedCTE CTE

#i _

U:

I

MolecularNaSCN weighteluate standard

XCFF

tRNAmet NaSCNf pil IMP

BLANK 1

BAND 1

BAND 2

BAND 3

BLANK 2

BAND 4

-67, 000

-43, 000

-25,000 BRP

-13, 700

FIGURE 3 SDS-polyacrylamide slab gel electrophoresis ofcrude, partially purified, and purified antigens. The NaSCNeluate gave six polypeptide bands stained with 0.04% Coomas-sie Brilliant Blue. Four of the six bands had molecular weightsestimated at 12,000-13,000. The molecular weights of thelarger polypeptides were -65,000 and 30,000.

respectively. Hemagglutination inhibition tests of thefour bands and in addition two blanks of gel remotefrom any ethidium bromide-staining material, showedthat only bands 3 and 4 were antigenic.

Antigenic specificity of the NaSCN eluate. TheNaSCNeluate gave no immunodiffusion precipitin line

TABLE IIIAmino Acid Comnposition of NaSCNEluate

Amino acid \Micrograms* Nanomoles Moles

ASP 13.2 99.2 10.4Thr 5.7 47.9 5.0Ser 5.8 55.3 5.8Glu 17.4 118.3 12.4Pro 5.4 47.1 4.9Gly 6.2 82.3 8.6Ala 5.7 64.0 6.7Cys 1.3 5.5 0.6Val 7.8 64.6 6.8Met 2.5 16.8 1.8Ile 6.9 52.7 5.5Leu 11.5 87.7 9.2Tyr 4.5 25.0 2.6Phe 5.8 35.1 3.7His 3.5 22.4 2.3Lys 11.4 78.0 8.1Arg 9.7 55.9 5.8

* Micrograms per 0.5 g of NaSCNeluiate, approximate values.

FIGURE 4 Urea-polyacrylamide gel separation of RNAcon-tained in the NaSCNeluate. The NaSCNeluate (5 ,tg RNA)and E. coli tRNAPet (10 ,ug) were subjected to urea-poly-acrylamide gel electrophoresis. The gel was stained withethidium bromide revealing the presence of four bands ofpolynucleotides in the purified RNPantigen and the locationof tRNAfmet. Areas designated by bands 1-4 and blanks 1 and2 were assayed for antigenicity by the hemagglutinationinhibition test. Only bands 3 and 4 RNA molecules withmolecular weights of -18,000 and 12,000, respectively, gavepositive results.

against reference sera for antibody to PM-1, MA, Scl-i,and SS-B but did contain Sm antigenic activity asshown in Fig. 5. However, investigation of materialeluted from SDS-polyacrylamide gel showed no Smac-tivity could be detected by hemagglutination inhibi-

FIGURE 5 Demonstration of Smantigen in the NaSCNeluateby immunodiffusion. The center well contained referenceserum for antibody to RNPand Sm. Well 1 contained crudeCTE digested with RNase A; well 2, crude CTE; well 3,reference serum monospecific for antibody to RNP; well 4, thesame serum as in well 3; well 5, the NaSCNeluate; and well6, the NaSCNeluate digested with RNase. Sm-anti-Sm pre-cipitin lines between the center well and wells 1, 2, 5, and 6are completely identical.

Purification and Characterization of Nuclear Ribonucleoprotein Antigen 1453

tion testing of the various peptides in contrast toresults for RNPantigen.

DISCUSSION

This paper describes a methodology for achieving ahigh dlegree of purification of CTE nuclear RNPanti-gen. Biochemical and immunological analyses ofthis antigen are also described. A dialyzed NaSCNeluate from the anti-RNP affinity column contained im-munologically active RNPantigen that was 488 timeshigher in specific activity than crude CTE. In controlexperiments, when partially purified RNPanitigen waspassed through a column of Sepharose 4B coupledwith IgG from negative control serum and eluted with3 MNaSCN, <0.1% of the total applied protein couldbe eluted and no detectable antigenic activity wasfound. This finding indicates that affinity chromatog-raphy with purified antibody was the method of choiceto obtain purified RNPantigen. However, when theNaSCNeluates had not been dialyzed against PBS, noprecipitin line was observed in immunodiffusion tests.Therefore, it is important to dialyze this eluate againstPBS as soon after elution as possible.

Recently, Douvas et al. (11) described the char-acterization of nuclear RNPantigen isolated with af-finity chromatography. There are some significant dif-ferences in the methods employed by Douvas et al. andourselves. They used rat liver nuclear extract as acrude nuclear antigen, whereas we have used CTE.But more importantly, (a) they coupled Sepharose 4Bgel with IgG from the serum of patients with MCTD,whereas we used purified antibody to RNP instead ofthe IgG fraction; and (b) they eluted RNP antigenfrom the coluimn with 0.01 M HCl solution containing0.15 Ml NaCl, whereas we used a 3 MNaSCNsolution.By coupling Sepharose 4B gel with purified antibodyto RNP, we could reduce nonspecific adsorption of pro-tein to the column. When our affinity column waseluted with 0.01 M HCI, we also found antigenic ac-tivity for RNP in this eluate. However, after HCI elu-tion, we could obtain an additional amount of moretightly bound antigen by eluting with 3 MNaSCN. Theratio of A280/A260 for the HCI eluate tended to be muchhigher than that of the subsequent NaSCN eluate.Furthermore, the specific activity for RNPantigen ofthe HCI eluate was much lower than that of the NaSCNeltuate. These findings suggested that HCI dissociatesnonspecifically bound proteins and some specificallybound RNPantigen from antibody but the greater por-tion of the nucleic acid-rich RNPmolecules were noteluted by the HCI solution. The HCI eluate in thestudy of Douvas et al. produced a precipitin line par-tially identical with that of crude antigen. Whereas inthis study, precipitin lines of the dialyzed NaSCNeluate and CTEantigen against the serum of a patient

with MCTDwere completely identical. Thus, theNaSCNeluate probably contained less degraded, morehighly purified RNP antigen molecules than theHCI eluate.

SDS-polyacrylamide gel electrophoresis of theNaSCNdisclosed that the protein comnponent of thisantigen consisted of six polypeptides. The molecularweights of four of the six polypeptides were estimatedto be - 13,000 each and that of the remaining two were65,000 and 30,000. Some of these peptides seemed tobe very similar in molecular weight to peptides con-tained in the HCI eluate described by Douvas et al.They reported two main polypeptide components ofmolecular weights 13,000 and 30,000 were found in theHCI-eluted RNPantigen. By indirect means they wereable to attribute the immunoprecipitating ability of theRNPantigen to the 13,000-mol wt comnponent in thatthe affinity of this component for column-bound anti-RNP antibodies was RNase sensitive. We have con-firmed and further elucidated this finding. Antigenicactivities of gel-separated polypeptides were examineddirectly by the hemagglutination inhibition test. In re-peated experiments we have consistently found thatonly two of the four peptides located in the 13,000-mol wt area had antigenic activity. Wecould also findla third region of antigenic activity in the gel at a locationrepresentative of a small molecular weight materialbut no protein band was evident. This may be at-tributable to the higher sensitivity of the hemagglutina-tion inhibition test compared to the sensitivity ofthe Coomassie Blue staining for protein. This thirdantigenically active portion seemed to be of very sm-allmolecular weight. It might be somiie degraded part ofthe whole RNPmolecule.

A nucleic acid-protein complex such as the RNPantigen might be expected to have protein containing alarger than average number of basic amino acids.Amino acid composition analysis of the RNPantigenshowed that the amounts of lysine and arginine werenot high. However, the antigen of the NaSCNeluate iscomposed of some six polypeptides. Analysis of thecomposite may have hidden the fact that one or more ofthe peptides have higher than average amounts of basicamino acid residues and are responsible for the protein-nucleic acid interaction.

The RNA component of the NaSCN eluate wasseparated into five polynucleotides upon the urea-polyacrylamide gel electrophoresis. Three poly-nucleotide chains of lengths larger than 77 nucleosidesdid not exhibit antigenic activity. Whereas two smallerpolynucleotides, estimated to be 40 and 60 nucleosidesin length, were antigenic. The larger RNAmoleculesmay not contribute to the structure of the antigenicsite for RNP. Alternatively, they may have lost theirantigenic activity by being selectively stripped of pro-tein during urea-gel electrophoresis. The smallest

1454 M. Takano, P. F. Agris, and G. C. Sharp

RNAcomiponent, estimiated at 40 nucleosides in length,could have been generated from the next largest mole-cule by nonspecific ribonuclease action during puri-fication. Sequence analysis of the two antigenic mole-cules, nlow in progress, will determinie if they arerelated.

Nucleoside composition analysis of RNA frompurified RNP revealed the total absence of modifiednucleosides. The technique we employed for detectingmo(lified nucleosides is capable of detecting less thanone modified nutcleoside per tlhousiand nucleosides(19). NMost RNA, including mRNA, contain modifiednucleosides (21). Since notable exceptions are 5S RNAand heteronuclear RNA, it is possible that RNAfromRNP antigen is derived from these RNAmolecules.The RNAdid conttain almost equal molar amounts ofguianiosine and cytosiine. This could be indicative ofsomiie secondary structure in the RNAof RNP.

Purified RNP, as the NaSCNelutate from the antibodyaffiinity columiin, had Smantigeinic activity as well. Im-munto(liffusion tests showed no antigenic activities forPMi-1, MA, Sdl-i, and SS-B. Sml could have beenretainied and then eluted from the columini witlh RNPin3 NI NaSCNfor a number of reasons: (a) Specific ad-sorption to anti-Smii aintibody that conitamiiinatedl theaniti-RNP antibody use(d in preparation of the coluimIn;(b) nonispecific a(dsorption to any IgG or to the columnmiatrix, Sepharose; or (c) miolecular associationi of Smiiwith RNP. However, purified antibody to RNP, used inaffinity clhromatography, did not conitain antibody toSml (Methods), nor was ainy Smn aintigenlic activity fouindin the NaSCNeluiate fromii the negative control IgG affinity coltumniii. Therefore, RNPacnd Smn miay exist in amlllectular complex as suggested by NIattioli and Reich-lin (22). The separate RNP andl Smii precipitin linesshowni in Fig. 5 mniglht be cauised by dissociation of amiiolecular complex or to the relatively' different coni-centrations of antibodies to RNPaned Smi used. How-ever, Northway ani(d Tani (13) descril)edl these twovaniti-gens as being differenit macromiiolectules. Douvas et al.(11), wlho fouinid i10 precipitini reaction l)etween theirpturifiedl RNP antigen andlc anti-Smii serumiii, suggestedIthat ther-e are n1o physical associationls between thetwo aintigenis.

Eltitioni of biads fromii SDS-polyacrvllamide gel andsubsequent testinlg by hemilaggluitniiatioin inhibition wasnot stuccessfuil ini (lisclosinig wliichl peptides of theNaSCN eluate contributed to aintigeniic activity forSmil. Recently, Lietu aind Tain (23) siuggested that anti-genic activity of Smii was detecte( in a protein of-70,00() miol wt. Therefore, the peptide of 65,000niol wvt in the NaSCN' eluiate may have Smil anitigeniicactiv,ity.

Thutis, RNP aintigeni is miniially comiiposed of twopolyl)eptides of some 13,000 micol wt eaclh, whiclh arecoipiilexedv witlh RNAof' some 60 and(I 40 nuticleosi(les.

Four peptides of 13,000, 13,000, 30,000, and 65,000mol wt, and three RNApolynucleotide chains that havemore than 77 nucleosides may also be part of the com-plex. An Smantigenic site may exist in some of thesemolectules.

The purification and biochemical characterization ofthe RNPantigen achieved by Douvas et al. (11) and inour laboratory should facilitate more incisive studies ofthe possible biological role of the RNPsystem and itsrelationiship to Sm. Such studies may result in a betterunderstanding of the pathogenesis of MCTDandrelated rheumiiiatic diseases.

ACKNOWLEDGNMENTS

WVethanik Susan Golden for her excellent technical assistanceand Dianine Porter and Sharon Mitchell for preparation ofthe manuscript.

This research was supported by a grant from the U. S.Public Health Service (Am 20305), a grant from the ArthritisFouncdationi, a grant from the J. B. Reynolds Founldationi, theUniversity of Missouri Center for Aginig Studies, anid the Divi-sion of Biological Sciences.

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1456 M. Takano, P. F. Agris, and G. C. Sharp