~ chemistry vol. 254, no. 20, issue of october 25, pp ... · identification of a nuclear protein...

THE JOURNAL OF B~m.ocrc/\~ CHEMISTRY Vol. 254, No. 20, Issue of October 25, pp. 10514-10522, 1979 Prrnted in lJ.S A.

Identification of a Nuclear Protein (Scl-70) as a Unique Target of Human Antinuclear Antibodies in Scleroderma*

(Received for publication, May 17, 1979, and in revised form, June 27, 1979)

Angeline S. Douvas,$ Marilyn Achten, and Eng M. Tan From the Division of Rheumatic Diseases and Dewartment of Biochemistry, Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80262

A protein antigenic to antibodies from scleroderma patients was isolated from rat liver nuclei using a com- bination of biochemical and immunologic methods. A 250-fold purification was attained by: (a) sonication and differential centrifugation of the nuclei; (6) KC1 extraction of a chromatin fraction; (c) cation exchange chromatography of extracted proteins. Final identification of the antigen, Scl-70, was made by immunoprecipitation from the 250-fold enriched fraction with IgG from scleroderma sera. Quantitative estimates based on chemical assays and optical scans of polyacrylamide gels indicate that there are approximately 26,000 copies of Scl-70 (Mr = 70,000) per haploid genome in the rat liver nucleus.

The specificities of precipitating antibodies from five genetically unrelated scleroderma patients were compared. Immunoglobulin G (IgG) antibodies from two patients formed complexes with the same protein, Scl- 70, in immunoprecipitation experiments. Sera from the other three patients had the same specificity as the first two when compared by immunodiffusion using crude and purified antigen. The specificity of IgG antibodies from two of the scleroderma sera was also investigated by immunofluorescence using cells from a human epidermal cell culture line, HEp-2, as substrates. The IgG antibodies responsible for nuclear fluorescence could be absorbed completely by the Scl-70 antigen isolated from rat liver nuclei. This result indicated that these antibodies are effectively monospecific for Scl-70.

On the basis of their clinical features, autoimmune diseases have been divided into a number of different syndromes including systemic lupus erythematosus, rheumatoid arthritis, mixed connective tissue disease, and others (1, 2). One of the more well known characteristics of autoimmune diseases is the spontaneous occurrence in the blood of antibodies directed against nuclear macromolecules (3-5). Although there is some overlap, the specificity of certain autoimmune antibodies appears to be characteristic of the syndrome in which they occur. For example, antibodies against nuclear RNA. protein complexes are characteristic of mixed connective tissue disease although they are found occasionally in patients with systemic lupus erythematosus (6). In order to understand the significance of these different specificities we are attempting to investigate some of the antigen/antibody systems in detail and to biochemically isolate the antigenic nuclear molecules.

* This work was supported in part by National Institutes of Health Grant AM 21839. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed.

Once the specificity of the antibodies has been characterized they may prove to be useful tools for investigating the nuclear distribution and functions of their target macromolecules.

In a previous communication, we described some of the properties of nuclear RNA-protein complexes which react with antibodies from patients with mixed connective tissue disease (7). We found that antibodies from four genetically unrelated patients were directed against the same subset of nuclear RNA. protein complexes and specifically against two RNA-associated polypeptides of molecular weights 13,000 and 30,000. The high degree of specificity found in the mixed connective tissue disease antibodies prompted us to investigate a second antigen/antibody system associated with the autoimmune syndrome scleroderma (also known as progres- sive systemic sclerosis).

Approximately 20% of patients with scleroderma have precipitating antibodies (detectable by double diffusion in agar) to an antigen found in mammalian nuclei (8). Little was known about the biochemical characteristics of the antigen; however, its sensitivity to proteases but not to nucleases suggested that it was a protein (or proteins) as confirmed in the current investigation. Scleroderma antigen/antibody reactions can be distinguished unambiguously in double diffusion tests from other autoimmune reactions such as anti-hi&one and anti- DNA (associated primarily with systemic lupus erythematosus) (4,9) and anti-ribonucleoprotein (found in mixed connective tissue disease) (6, 10, 11). Although little has been pub- lished on this precipitating system, sera containing the antibodies are routinely identified during screening of scleroderma patients in the clinical laboratory at this institution. We obtained five such sera which in preliminary tests showed a single precipitin reaction against a crude protein extract from rat liver nuclei. Moreover, the reactions of all five sera were immunologically identical. This preliminary result indicated that it would be feasible to use the human antibodies to isolate and characterize the scleroderma antigen.

One objective of the current investigation was to determine if sera which appear to be immunologically identical when tested against crude nuclear extracts also have the same specificity when tested against a purified antigen. This prob- lem was approached by biochemically isolating the antigenic activity in rat liver nuclei and comparing the immunologic specificities of five sera at each stage of the purification by double diffusion. Final identification of the antigen was made by immunoprecipitation with IgG’ from two of the sera.

The second objective was to determine if antibodies from scleroderma sera could be used to investigate the nuclear distribution of the precipitating scleroderma antigen by in situ immunofluorescence. Previous immunofluorescence data show that the nuclear antigens reacting with scleroderma sera

I The abbreviations used are: IgG, immunoglobulin G; SDS, sodium dodecyl sulfate.

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Antibodies to a Nuclear Protein 10515

are not tissue specific (12-14). Therefore a variety of mammalian, and possibly even nonmammalian cells could be used for such studies. However it was first necessary to determine if a one-to-one correlation could be established between the precipitating scleroderma antigen and the antigen(s) detected by immunofluorescence. For this purpose IgG pools from two sera were absorbed with the precipitating antigen, then as- sayed by immunofluorescence for the presence of residual anti-nuclear activity.

The third objective of this investigation was to determine how much of the protein in rat liver nuclei reacts with the precipitating scleroderma antibodies. In the process of isolating the scleroderma antigen, we partitioned the total nuclear protein into a number of fractions. The distribution of nuclear protein and histone in these fractions was determined by chemical assay. The rationale for quantitating histones was

that the antigen co-isolated with histone Hl up to the penul- timate purification step. As all of the antigen was partitioned into a single nuclear fraction, it was possible to calculate the fold-purification of the antigen in this fraction. The ratio of antigen to total protein in this fraction was estimated by quantitative polyacrylamide gel electrophoresis (15). From these quantitations and from a knowledge of the molecular weight of the antigen, we have attempted to calculate the number of copies of this protein per haploid genome.

EXPERIMENTAL PROCEDURES

Nuclear Fractionation-All procedures were carried out at 0 to 4°C. Nuclei were isolated from 200 to 240 g of frozen rat livers (Pel- Freez, Rogers, Ark) as described previously (7). This procedure yields nuclei having a protein:DNA:RNA ratio of 3:1:0.45. Nuclei from approximately 200 g of tissue were suspended in 60 ml of 10 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.5, and sonicated on ice in six to eight 15-s pulses using a Heat Systems sonicator at a setting of 4, then fractionated as outlined in Fig. 1. Following sonication and centrifugation at 12,062 x g for 10 min, the low speed pellets were suspended and homogenized with 50 ml of 1 M KCl, 5 mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, using a motor-driven Potter- Elvehjem homogenizer. The suspension was stirred for 1 h, then centrifuged at 12,062 x g for 10 min. The resulting pellets were suspended in 1 M KCl, 5 mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, to a final volume of 30 ml and homogenized as before. The supernatant was centrifuged at 113,000 X g for 18 h to pellet DNA and undissociated protein. The pellets were resuspended with 30 ml of 1 M KCl, 5 mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0. All fractions not processed further were stored at -20°C.

Quantitative Assays-Proteins were quantitated by the methods of Lowry et al. (16) and Sedmak and Grossberg (17) using bovine serum albumin and histones as standards. Perchloric rather than hydrochloric acid was used as a dye solvent in the Sedmak and Grossberg procedure to avoid gross underestimation of histones. Nucleic acid determinations were performed by a modification of the Schmidt-Tannhauser procedure (18).

Histones-Histones were isolated either by extraction with 0.4 N sulfuric acid as described elsewhere (19) or by chromatography over Bio-Rex 70 as described below. Quantitative and qualitative compar- isons of the two methods are presented under “Results.” Identification of acid-extractable proteins as histones was made on the basis of their electrophoretic mobilities in acid/urea gels (21).

Selection and Testing of Human Sera, IgG Isolation, and Immu- noprecipitation-Sera from five scleroderma patients (GC, MC, JH, AT, LJ) were obtained from the Division of Rheumatic Diseases, University of Colorado Medical Center. Identification of sera contain. ing the scleroderma-specific antibodies was made by comparison with reference sera. The absence of antibodies to cytoplasmic components was confirmed by indirect immunofluorescence on mouse kidney sections (1). Normal sera from laboratory personnel were used as controls. Serum from a mixed connective tissue disease patient, MP, having antibodies directed against nuclear ribonucleoprotein (7), was used as an autoimmune control.

For detection of the precipitating antigen/antibody system, double diffusion assays were conducted in plates of 0.4% agarose, 0.1% sodium azide in 10 mM phosphate buffer, 0.15 M NaCl, pH 7.5 (7). IgG was

isolated from 5 ml of serum from each of the scleroderma patients, GC and MC, as described previously (7). The yield from 5 ml of GC serum was 24 mg at a concentration of 5.2 mg/ml, and 20.6 mg from MC serum at a concentration of 3.2 mg/ml in 15 mM phosphate buffer, pH 7.4. Previously isolated anti-ribonucleoprotein IgG from the patient MP (7) was used at a concentration of 5.9 mg/ml. For isolating antigen.antibody complexes, IgG was dialyzed into 0.65 M NaCl, 10 mM Tris-HCI, 5 mM 2-mercaptoethanol, pH 7.0, and mixed with the antigen in the same buffer. Antigen protein concentration ranged from 0.17 to 0.26 mg/ml. Antigen and either GC or MP IgG were mixed in 1:l (v/v) ratios, and in 1:1X ratios using MC IgG. The mixtures were incubated for 10 days at 0°C then centrifuged at 12,062 g for 30 min to sediment the immunoprecipitates. Precipitates were suspended in sample buffer for polyacrylamide gel electrophoresis (see below).

Bio-Rex 70 Chromatography-All Tris-HCl buffers contained 5 mM 2-mercaptoethanol. The cation exchange resin Bio-Rex 70 (Bio- Rad Laboratories, Richmond, Calif.) was dissolved and washed exten- sively in 4.0 M NaCl, 10 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 8.0. The resin was allowed to settle to 30 cc and the supernatant fluid was removed. Resin combined with 50 to 60 mg of crude antigen in 50 ml of 1 M KCI, 10 mM Tris-HCl, pH 8.0 was placed in a dialysis bag in a stoppered glass cyclinder and dialyzed against several changes of 0.4 M NaCl, 10 mru Tris-HCI, pH 7.0, over a period of 17 h, while affixed to a rotating device which prevented settling of the resin, The dialyzed slurry was then packed into a column and the effluent protein collected at a flow rate controlled by a Gilson Minipuls pump set at 175 (approximately 30 ml/h). After extensive washing with the same buffer, bound protein was eluted with 2 M NaCl, 10 mM Tris-HCI, pH 7.0. The 2 M NaCl eluate was chromatographed again with 10 cc of fresh resin, as outlined in Fig. 4. Recovery of protein from Bio-Rex 70 columns ranged from 92 to 100%.

Nuclease and Protease Digestions-RNase A (Worthington, RAF grade) was preboiled to inactivate deoxyribonuclease activity. DNase I (DPFF grade, RNase-free) and trypsin (267 units/mg), from Worth- ington, were dissolved in 10 mM Tris-HCl, pH 8.0, to 1 mg/ml. DNase and RNase were added to the antigen (1.2 mg/ml in 0.65 M NaCl, 10 mM Tris-HCI, 5 mM 2-mercaptoethanol, pH 8.0) to final concentrations of 50 pg/ml. Trypsin was added to 10 pg/ml. To the DNase I reaction mixture, 1 IIIM MgClr and 0.1 mM CaCh were supplied as counterions. Reaction mixtures, including a control aliquot without enzymes, were incubated at 22°C for 15 min. Reactions were termi- nated by transferring the samples to 0°C.

Polyacrylamide Gel Electrophoresis and Quantitation of the An- tigen-Polyacrylamide disc gel electrophoresis (10% acrylamide w/v) in sodium dodecyl sulfate was performed according to King and Laemmli (20) as described in detail elsewhere (7, 19). Gels were fixed and stained with Coomassie brilliant blue (19) and destained exhaus- tively against 10% acetic acid, 5% methanol. The gels were scanned at a wavelength of 600 nm using a Varian 634 spectrophotometer equipped with scanning attachment and chart recorder.

Acid extracts or Bio-Rex 70 fractions were dialyzed against 2.5 M urea (Schwarz/Mann, ultrapure), 0.9 N acetic acid, (v/v) 2-mercaptoethanol and electrophoresed on gels (0.8 x 13 cm) by the method of Panyim and Chalkley (21). The gels were stained with amido black and scanned as described above.

Quantitations from optical scans of gels were performed as follows: Samples were applied to either SDS or acid/urea gels; at least two protein loads were run and quantitated for each sample. Loads on acid/urea gels ranged from 5 to 48 pg, as determined by chemical assay. Within this range, the amplitude of the peaks in the scans were linearly related to the protein load. The lower end of this range (~20 pg) was used for samples which did not contain the full spectrum of histones. Loads on SDS-gels ranged from 40 to 100 pg. Each gel was scanned in duplicate, and the scans were reproduced in triplicate by photocopying. The area of each scan rising above the baseline was cut out and weighed on an analytical balance to establish a quantity for the total amount of protein. The area of the peak was then cut out and weighed and compared to the total.

Zmmunofluorescence-Immunofluorescence was performed using cells from a human epidermal cell line, HEp-2, as antibody substrates (22, 23). HEp-2 cells were obtained affixed to slides from Antibodies Incorporated (Davis, Calif.) and were stored at -20°C before use. The slides were brought to room temperature using a blow drier; IgG in a volume of 15 ~1 was applied to each well; the slides were then incubated in a humidity chamber for 30 min, and transferred to a staining dish for 10 min of washing with two 125-ml changes of 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.5. Goat anti-human y-globulin


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10516 Antibodies to a Nuclear Protein

conjugated with fluorescence isothiocyanate was applied to each well (15 ~1 of 0.82 mg/ml). The slides were incubated in the humidity chamber, washed again, then counterstained for 30 s with 0.05% Evans blue in 0.01 M phosphate buffer, 0.15 M NaCI, pH 7.5, and washed again with the same buffer. Coverslips were applied over 50% glycerol. The slides were viewed and photographed using a Leitz Ortholux II fluorescence microscope equipped with an Orthomat automatic cam- era.

RESULTS

Nuclear Fractionation-Fiat liver nuclei were partitioned initially into two fractions by sonication followed by low speed centrifugation, as outlined in Fig. 1. The low speed supernatant contained most of the nuclear RNA (60%), and the pellet most of the DNA (95%). The low speed pellet also contained essentially all of the histones and had a protein to DNA ratio of 1.7:1. Thus it is analogous to chromatin isolated by other methods (24). In order to dissociate some of the protein, this pellet was extracted with 1 M KC1 then centrifuged twice, at low followed by high speed, to clear the extract of DNA and undissociated protein. The distributions of protein, DNA, and histones in all of the resulting nuclear fractions are shown in Fig. 1. Dissociated protein was contained in the high speed KC1 extract, composed of a 2.5:1 mass ratio of nonhistone to histone protein and more than a 3O:l mass ratio of protein to DNA. The histone content of the nuclear fractions was in most cases determined by quantitating the acid soluble proteins by chemical assay (16, 17). These proteins were also resolved by electrophoresis on acid-urea gels. Shown in Fig. 2 are gels of acid extracts of the four fractions derived from KC1 extraction of the chromatin pellet (low speed pellet). Two fractions, the low speed KC1 pellet and high speed KC1 extract, contained significant quantities of acid-soluble nonhistone protein. By quantitating optical scans of the gels in Panels B

FIG. 1. Nuclear fractionation. Nu- clei were isolated and sonicated as described under “Experimental Proce- dures” at a protein concentration of approximately 30 mg/ml in 0.01 M phosphate buffer, pH 7.5,0.15 M NaCl (NaCI/ Pi, pH 7.5). Following sonication and centrifugation, the low speed pellet was resuspended to a protein concentration of 12 mg/ml and extracted in 1 M KCL, 5 mu 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0. Protein concentrations of the low speed KC1 extract and the low speed KC1 pellet averaged 8.6 and 7.7 mg/ml, respectively. The high speed KC1 extract and high speed KC1 pellet had protein concentrations averaging 3.0 and 7.0 mg/ ml, respectively. Protein contents were determined by the methods of Lowry et al. (16) and Sedmak and Grossberg (17). Data obtained by the Lowry et al. (16) method are included here and compared to the other method under “Results.” Nuclear fractions were dialyzed against 1 N NaOH before protein quantitation. Acid extracts were dialyzed against NaC1/Pi, pH 7.5, before histone quanti- t&ion. Numbers for nuclear protein (np) represent the average and range of three experiments, and for histones the average of two. Where two fractions are derived from a single cruder fraction, the percentages reflect the average distribution of protein in the resulting fractions assuming 100% recovery. Recovery was actually ~~90% for all procedures. DNA was quantitated by the modified Schmidt-Tannhauser procedure of Ts’O and Sato (18).

low speed supernatant

43+3% of np; 5% of DNA; ~5% of histone

10~ speed Kii extract

36.6+2.6% of np; 69% ;;f DNA; 87.7% of histone

high speed KC1 extract

9.6+1.5% of np; 0.9% of DNA; 9.5% of histone

and D, as described in “Experimental Procedures”, we estimated that acid-soluble non-hi&ones account for 40 and lo%, respectively, of the protein applied to these gels. These quantities have been subtracted from the histone values shown in Fig. 1. The mass ratio of histone to DNA in the low speed (chromatin) pellet, excluding acid-soluble non-hi&one protein, was 1:l.

The nuclear fractions described in Fig. 1 were tested for immunologic activity against antisera from five scleroderma patients, as shown in Fig. 3, Panels A and B. None of the scleroderma sera reacted with the chromatin-free low speed

H1\ H3--\

H2B-

H2A-

H4J

A B C D FIG. 2. Acid/urea polyacrylamide gels of nuclear fractions.

Nuclear fractions were acid-extracted, dialyzed into 0.9 N acetic acid, 2.5 M urea, 1% (v/v) 2-mercaptoethanol, then electrophoresed as described under “Experimental Procedures.” The gels in Panels A and B contain 18 pg of low speed KC1 extract and 48 M of low speed KC1 pellet, respectively. The gels in Panels C and D contain 25 M of high speed KC1 pellet and 10 M of high speed KC1 extract, respectively.

Nuclei I

sonicate ; centrifuge l2,062g, 10 min

low speed pellet I

57+3% of np; 95% of DNA; >9% of histone -

extract in 1 M KCl, pH 8.0; centrifuge l2,062g, 10 min

low speed KC1 pellet (resuspend in 1M KCl, pH 8.0)

23+4% of np; 252% of DNA; 7.3% of histone

centrifuge 113,OOOg, 18 hrs

high speed KC1 pellet (resuspend in 1 M KCl, pH 8.0)

20.7+2.9% of np; 68.1% of DNA; 78.2% of histone


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supernatant. This fraction reacts with anti-ribonucleoprotein antibodies found in patients with mixed connective tissue disease (7). The low speed KC1 extract was antigenically active against all five scleroderma sera as shown in Panel A. A single precipitin line was observed with each serum, and the conflu- ence of all five precipitin reactions indicated that they were immunologically identical. The low speed KC1 pellet was inactive (Panel B). The high speed KC1 extract, which as shown in Fig. 1 is composed essentially of dissociated protein, contained the antigenic activity. The high speed pellet was inactive (Panel 13). Both high and low speed pellets have been re-extracted with 1 M and 3 M KC1 with no further recovery of immunologic activity (data not shown). The high speed KC1 extract represents a lo-fold enrichment in the antigen over total nuclear protein. As shown in Panel C of Fig. 3, normal sera from laboratory personnel and from autoimmune patients with anti-ribonucleoprotein antibodies (see Ref. 7) are unreactive against the high speed KC1 extract.

Biochemical Isolation of Antigenic Actkity-To further purify the antigenic activity, the high speed KC1 extract was fractionated by cation exchange chromatography on a column of Bio-Rex 70 (see “Experimental Procedures”). In 0.4 M

sodium chloride buffered to pH 7.0 this resin binds all of the nuclear histones while allowing the majority of the nonhistones to flow through (25). As outlined in Fig. 4, the bulk of

A B

the applied protein was recovered in the first 0.4 M NaCl effluent, and was composed of nonhistones, as illustrated below (Fig. 5). The bound histone protein plus contaminating nonhistones were eluted with 2.0 M NaCl, 10 mM Tris-HCl, pH 7.0. In order to separate nonhistone contaminants from the histone, the 2 M NaCl eluate was chromatographed over Bio-Rex 70 a second time in 0.4 M NaCl. Following collection of the effluent, step elutions of 0.65 M NaCl followed by 2.0 M NaCl were performed. The second 0.4 M NaCl effluent contained no protein. The 0.65 M NaCl eluate, comprising 0.4%> of the nuclear protein, was composed of nonhistones. The absence of histones from this fraction was determined by acid-urea-gel electrophoresis (not shown) and was confirmed with SDS-gels, presented below (Fig. 7). The post 0.65-M NaCl fraction, eluted with 2.0 M NaCl, consisted entirely of histone protein (gel not shown). As shown in Fig. 2, Hl was the only histone in the high speed KC1 extract, and the purified histone is labeled accordingly in Fig. 4. This fraction comprises 8.4% of the nuclear histone, a number which compares reasonably well with the 9.5% estimated from acid extraction of the high speed KC1 extract (Fig. 1).

Polyacrylamide gels in SDS of the high speed KC1 extract, the nonhistone effluent, and the 2 M NaCl eluate from the first Bio-Rex 70 column are shown in Panels A, B, and C of Fig. 5. Histone Hl was the quantitatively major component of

FIG. 3. Immunologic reactions of nuclear fractions against scleroderma and control sera. Nuclear fractions: nuclear fractions are numbered I to 5. Fraction 1, Panel A, is the low speed superna- tam, 3 mg/ml of protein, in NaCI/P,, pH 7.5. All other fractions are in 1 M KCl, 5 mM 2-mercaptoethanol, 10 mM Tris-HCI, pH 8.0; Fraction 2, Panel A, the low speed KC1 extract, 3.4 mg/ml of protein; Fraction 3, Panel El, the high speed KCI extract, 4.1 mg/ml of protein; Fraction 4, Panel B, the low speed KCI pellet, 4.7 mg/ml of protein; Fraction 5, Panel B, the high speed KCI pellet, 5.4 mg/ml of protein. In addition, dilutions of 1:1, 1:2 and 1:4 of the above fractions were tested against the sera at the concentrations given below (data not shown). Scleroderma and control sera: .sera from sclcroderma patients, diluted in NaCI/P,, pH 7.5, were as follows: GC, 1:2, MC, 1:4; JH, 1:l; AT, l:2; LJ, 1:4. These dilutions were found to be optimal for the

antigen concentrations shown. LJndlluted sera, and dilutions up to 1: 20, were also tested agamst the same antigen concentrations as well as antigen dilutions listed above. Although the sharpness and strength of reacttons varied at different dilutions of antigen and antibody, multiple reactions were not observed. The undiluted Set-a of laboratory personnel CP, PR, MM, and MW were tested against the high speed KCI extract (4.1 mg/ml) in Panel C. Also in Panel C are the anti-ribonucleoprotem sera from two autoimmune patients Ml’ and MK, diluted 1:1, in NaCI/P,, pH 7.5. Reaction conditions: agarose plates (0.4% agarose, 0.1% sodium axide in NaCl/P,) were used at room temperature. The wells contained 100 ~1 of solution. Plates were allowed to develop 18 to 24 h, then washed with 54 sodium citrate followed by drstilled H,O, 0.1% sodium azide.


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10518

FIG. 4. Bio-Rex 70 chromatography. In a typical experiment, 58 to 60 mg of high speed KC1 extract protein in 40 ml of 1 M KCl, 5 mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, were mixed with 40 cc of packed Bio-Rex 70, then dialyzed and poured into a column as described in “Experimental Proce- dures”. The 2.0 M NaCl eluate fractions were pooled and stored at -20°C for up to 3 weeks before the second chromato- graphic step. In the second procedure, approximately 22 mg of 2 M NaCl eluate protein in 17 ml of 2.0 M NaCl, 5 IIIM 2. mercaptoethanol, 10 mM Tris-HCl, pH 7.0. were combined with 10 ml of packed Bio-Rex 70 pre-equilibrated in thk same solution. Protein quantitations were performed by the method of Lowry et al (16). Numbers shown represent the average and range of three experiments, and are expressed as the per cent of nuclear protein (np).

Antibodies to a Nuclear Protein

High speed KC1 extract + Bio-Rex 70

dialyze to 0.4 M NaCl, 10 mM Tris-HCl, pH 7.0

0.4 M NaCl effluent 1 = nonhistones 6.4 + 0.3% of np -

2MN

0.4 M NaCl effluent 2

elute with 2 M NaCl, 10

mM Tris-HCl, pH 7.0

the 2 M NaCl eluate from this column (Panel C). The gel in Panel C was heavily loaded to reveal the minor nonhistone contaminants, having a broad molecular weight distribution. Panel D shows an acid-urea gel of the same 2 M NaCl eluate, the histone moiety of which is composed entirely of Hl. An acid-urea gel of the histone moiety of the low speed pellet, containing all five major histones, is shown for comparison in Panel E.

All of the fractions resulting from the Bio-Rex 70 protocol (Fig. 4) were tested for antigenic activity against scleroderma sera by double diffusion, as reported in Fig. 6. The first 0.4 M NaCl effluent was inactive and the 2 M NaCl eluate, which is 88% by mass histone Hl, was active (Panel A). However histone Hl was itself inactive, and the antigen was contained in the 0.65 M NaCl eluate. This eluate, comprising 0.4% of the nuclear protein, represents a 250-fold purification of the antigen. The serum used in Panel A was from the patient GC. The same results were obtained with the other four sera (data not shown). All five scleroderma sera have identical specificity against the 0.65 M NaCl eluate, as demonstrated in Panel B. In Panel C, a comparison of the 0.65 M NaCl eluate to the crude fractions from which it was derived demonstrates that within the limits of sensitivity of the double diffusion assay, the original extract and final product contain the same anti- genie determinants. Preliminary data indicated that the precipitating scleroderma antigen is a protein (or proteins) due to its sensitivity to proteases but not to deoxyribonucleases or

h I

is1 - 2.8 + 0. -.

elute with 2 M NaCl, 10 mM Tris-HCl, pH 7.0

1 eluate

combine with Bio-Rex- 70; dialyze to 0.4 M NaCl

elute with 0.65 M NaCl, 10 mM Tris-HCl, pti 7.0

-- 0.65 M NaCl eluate

0.4 + 0.1% of np -

Lone 0 ./o of r!p

ribonucleases (8). We confirmed this observation by treating the 0.65 M NaCl eluate with DNase I, pancreatic RNase A and trypsin (Panel D).

Identification of the Scleroderma Antigen by Immunopre- cipitation and Immunofluorescence-The antigenically active 0.65 M NaCl eluate is composed of a number of nonhistone polypeptides, as shown in Fig. 7, Panel A. To determine which of these polypeptides react with the scleroderma antibodies, the 0.65 M NaCl eluate was immunoprecipitated with IgG from two patients, GC and MC, in separate experiments. The other immunoglobulin classes contained no precipitating antinuclear antibodies. As demonstrated in Panels B or C of Fig. 7, immunoprecipitates from both experiments contained a polypeptide, M, = 70,000, corresponding to a quantitatively major polypeptide in the 0.65 M NaCl eluate. This was the only polypeptide in the immunoprecipitate not also present in the control IgG in Panel D, as confirmed by superimposing gel scans of immunoprecipitates and control IgG, shown in Fig. 8. Ratios of IgG to 0.65 M NaCl eluate were calculated to be at equivalence so no free antigen or antibody would result (26). To determine if equivalence was attained, immunoprecipitate supernatants were subjected to gel electrophoresis and tested against fresh antigen and antibody by double diffusion. It is evident in the heavily loaded gel in Panel E, Fig. 7, that the supernatant shown lacks the 70,000- M, polypeptide, but does have some of the circa M, = 23,000 components of the 0.65 M NaCl eluate. An autoimmune con-


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Antibodies to a Nuclear Protein

68000-

45000-

31000-

25000-

---Hz2

\HZA

LH4

ABC DE FIG. 5. Polyacrylamide gel electrophoresis of the high speed

KC1 extract and derived fractions. The high speed KCI extract was fractionated by Bin-Rex 70 column chromatography mto 0.4 M

NaCl effluent and 2 M NaCl eluate as described in Fig. 4. The extract and derived column fractions were electrophoresed m SDS bv the method of Kmg and Laemmh (20) through loci polyacrylanride (Panels A to Cl. Panel A, 2101(g of high speed KCI extract, Panel H, 175 ng of 0.4 M NaCl effluent; Panel C, 190 pg of 2 M NaCl eluate The molecular weights indicated to the left of Panel A were calculated from the relative mobilities of the followmg molecular weight markers: bovine serum albumin, 68,090; pyruvate kinase, 57, Ooo; human heavy chain IgG, 50,ooO. ovalhumm, 43,ooO; DNase I, 31,ooO. trypsm, 23,009. Arrows to the rlghl of Panel C indicate the positions of histone H 1. An acid/urea gel of the 2 M NaCl eluate, 19 ng of protein is shown in Panel D. An aliquot of this &ate was dialyzed directly into 0.9 N

acetic acid, 2.5 M urea, 2-mercaptoethanol. Panel E shows an acid extract of the low speed pellet, 28 pg of protem, containing the full spectrum of rat liver histones, as labeled to the r&t of Panel B.

trol of anti-RNP IgG incubated with 0.65 M NaCl eluate, shows that the M, = 70,000 polypeptide is not affected non- specifically by experimental conditions (Panel 8’). Double diffusion tests of the supernatants were negative; also when both supernatants were tested against the other three scleroderma sera (JH, AT, IJ) they were unreactive although control-incubated antigen was active (not shown). The absence of free antibodies in the supernatants was confirmed by immunofluorescence (below). We conclude from these experiments, repeated a total of six times, that the M, = 70,000 protein, which we have labeled S&70, is the precipitating scleroderma antigen.

Immunofluorescence experiments, using cultured human epidermal cells (HEp-2) as substrates, were performed to determine if all of the antinuclear activity contained in the IgG of scleroderma patients was bound by the precipitating antigen found in the 0.65 M NaCl eluate. Panels A, B and C of Fig. 9 show cells reacted with control-incubated IgG from two scleroderma sera (GC and MC) and from the anti-ribonucleoprotein serum (MP). In Panels D and E, scleroderma IgGs absorbed with 0.65 M NaCl eluate show essentially no remaining antinuclear activity. Panel F shows that the anti- ribonucleoprotein IgG is not affected by “absorption” with the scleroderma antigen. It was not possible to do the immu- noabsorption experiments with the remaining three scleroderma sera due to insufficient quantities, However, we did react all five with HEp-2 cells and observed the same general pattern of nuclear staining as with the two IgGs. Notable features of the staining patterns were the absence of nucleolar staining and a fine-speckled appearance. The fluorescent stain

FIG. 6. Immunologic reactions of Bio-Rex 70 column fractions against scleroderma sera. Reaction conditions were the Same as in Fig. 3, except that 200 ~1 of sample were apphed to each well. Panel A, the center well contains CC serum, diluted 1:2 with NaCI/P,, pH 7.5. Wells labeled 0.4, 2.0, 0.65 and H. respectively, contam the following Bio-Hex 70 column fractions: 0.4 M NaCl effluent, 0.75 mg/ml of protein in 0.4 M NaCI, 10 mM Tris-HCI, pH 7.0, 2.0 M NaCl eluate, 1.28 mg/ml in 2 M NaCI, 10 mM Tris-HCI, pH 7.0; 0.65 M NaCl eluate, 0.49 mg/ml in 0.75 M NaCI, 10 mM Tris-HCI, pH 7.0; histone, 1.7 mg/ml in 2.0 M NaCI. 10 mM Tris-HCl, pH 7.0. Panel B. the center ulell contams 0.65 M NaCl eluate, 0.49 mg/ml. in 0.65 M NaCI, 10 mM Tris-HCI, pH 7.0. Well C contains as a control the anti-ribonucleoprotein serum MI’, diluted 1:l in NaCI/P,, pH 7.5. The remaming wells contain sera from scleroderma patients diluted in NaCI/P,, pH 7.5, to the concentrations indicated m Fig. 3. Dilutions of antigen to 0.12 mg/ml and of antisera up to I:20 were also tested with qualitatively the same results (not shown). Panel C, the center tuell contains CC serum diluted 1:2 in NaCI/P,, pH 7.5. Wells 2 and 3 correspond to nuclear fractions 2 and 3 (low speed KC1 extract and high speed KC1 extract) described in the legend to Fig. 3. Wells labeled 2.0 and 0.65 are the same Hio-Hex column fractions as m Panel A. Panel D, center well, GC serum, same dilution as Panel c’. The surrounding wells contain 0.65 M NaCl eluate, 1.2 mg/ml, either control incubated (0.65 Cl or incubated with DNase (D), HNase (H) to final enzyme concentrations of 50 g/ml, or trypsin (T), 10 &ml. Incubations were performed in volumes of 200 ~1 in 0.65 M NaCI, 10 mM Tris-HCl, pH 8.0, for 15 min at 22°C.

is associated with both interphase nuclei and chromosomes in mitosis. A cell in mitosis, with brightly staining chromosomes, can be seen in the top center of Panel A.

Estlmatuzg the Number of Copies of Scl-70 per Haploid Rat Liver Genome-In order to determine how many copies of Scl-70 there are per haploid genome, we fust estimated how much of the mass of the 0.65 M NaCl eluate it comprises. A gel scan of this eluate is shown in Fig. 10, Panel A. The weight of the shaded area representing the antigen was compared to that of the total scan as described under “Experimental Pro- cedures.” From this comparison, based on two gels with different protein loads, we calculated that the antigen comprises 8.2% of the 0.65 M NaCl eluate, or 3.3 X lo-“ of the total nuclear protein. The haploid genome of the rat consists of 3 pg of DNA (calculated from its molecular weight of 1.8 X lo”, Ref. 27). The haploid complement of protein in the rat liver nucleus is 9 pg, given the 3:l ratio of protein to DNA. From these numbers and a molecular weight of 70,000 we calculate that there are 25,200 copies of Scl-70 per haploid genome.

The number of copies can be calculated another way taking


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advantage of the co-isolation of histone Hl and the antigen up to the second Bio-Rex 70 column. All of the Hl present in the original chromosomal pellet is partitioned into the iow speed KC1 pellet and the high speed KC1 extract (Fig. 2). The

23.300-

ABCDEF FIG. 7. SDS-polyecrylamide gel electrophoresis of antigen/

antibody inununoprecipitntes and controls. SDS-polyacrylamide gels (10%) were run as described in the legend to Fig. 5. Panel A, 80 pg of 0.65 M NaCl eluate; Panel B, immunoprecipitate using GC IgG, 95 gg; Panel C, immunoprecipitate using MC IgG, 60 pg; Panel D, CC IgG, 130 l.(g; Panel E immunoprecipitate supernatant using GC IgG, 500 pg; Panel F, MP IgG plus 0.65 M NaCl eluate, 106 clg. Immuno- precipitation conditions were described under “Experimental Proce- dures.” Molecular weights to the left of Panel A were calculated from markers described in Fig. 5.

FIG. 9. Immunofluorescence tests of autoimmune IgGs before and after absorption with Scl-70. Cells of the human epidermal cell line HEp-2 were incubated with either control IgG from autoimmune patients, or IgG which had been incubated with antigen, and the immunoprecipitate removed by centrifugation. Absorbed IgG from the patients CC and MC is identical to immunoprecipitate supernatant, an example of which is shown in Fig. 7, Panel E. Im- munoprecipitation conditions were described under “Experimental Proce- dures.” The protein concentrations given here are those of the IgG. The 0.65 M eluate accounted for 13.3% of the input protein in immunoprecipitation reaction mixtures. Following incubation with IgG or absorbed IgG the cells were treated with fluorescence isothiocyanate-conjugated anti-human y-globulin as described in detail under “Experimental Procedures.” Panel A, GC IgG, 0.52 mg/ ml, Panel B, MC IgG, 0.49 mg/ml, Panel C, MP IgG, 0.10 mg/ml, Panel D, absorbed GC IgG, 0.56 mg/ml; Panel E, absorbed MC IgG, 0.66 mg/ml, Panel F, absorbed MP IgG, 0.15 mg/ml.

quantitatively negligible histone found in the low speed supernatant represented a full spectrum of the histones and is ignored in the calculations. Quantitations of gel scans of the low speed KC1 pellet reveal that it contains 7.3% of the nuclear histone (Fig. 1) of which 32.5% is Hl. Therefore, the Hl contained in the low speed KC1 pellet comprises 2.4% of the

0’

i-) anhge” IgG W (+I

~70.000~ heavy cham lqh, cha,”

I50.000) I25 0001

FIG. 8. Optical scans of immunoprecipitate and control IgG polyacrylamide gels. The gels shown in Panels B and D of Fig. 7 were scanned as described under “Experimental Procedures.” - - -, control CC IgG; -, immunoprecipitate using GC IgG. by guest on July 16, 2018

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0.65M NaCl Eluate

FIG. 10. Optical scan of an 0.65 M NaCl eluate polyacrylamide gel. The 0.65 M N&l &ate was applied to SDS-polyacrylamide gels at loads of 40 and 80 pg, and electrophoresed as described under “Experimental Procedures.” An optical scan of the 80 fig protein load is shown here. Arrows designate the beginning and end of the scan for purposes of quantitation, and the horizontal line indicates the baseline of the scan. The cross-hatched area indicates the antigen portion of the scan which was compared to the total.

nuclear histone. The Hl found in the KC1 extract represents between 8.4 and 9.5% of the nuclear histone, depending on whether the value from Fig. 4 (8.4%) or Fig. 1 (9.5%) is used. Using the value in Fig. 1 based on acid extraction, the high speed KC1 extract contains 79% of the histone Hl. Garrard et al. (15) have estimated that there are 9.4 x 10” copies of Hl per haploid genome. Therefore, the 79% of the Hl from the second Bio-Rex 70 column represents 7.4 X 10” molecules. The 0.65 M eluate, of which 8.2% is the antigen, contains one- seventh as much protein as the Hl fraction. From these numbers and taking into account the molecular weight differ- ences between Hl and the antigen (21,500 versus 70,000,

respectively), the number of copies of the antigen is 26,400. This calculation assumes that our quantitations are similar enough to those of Garrard et al. (15) to justify me of their estimation of the number of copies of Hl. By totaling the amount of nuclear histone which is Hl in the low speed KC1 pellet (2.4%) and the high speed KC1 extract (8.4 or 9.5%), Hl represents between 10.8 and 11.9% of the histone mass. In the study of Garrard et al., Hl represents 10.5% of the histone mass (calculated from Table IB of Ref. 15).

The extinction coefficient of total rat liver histones is E = 3.5 liters/cm g (24). Estimates of histone quantities using the extinction coefficient were within 18% of the average of the two chemical assays used (16, 17), which were in agreement to within 20%.

DISCUSSION

A precipitating antigen/antibody system associated with the autoimmune disease scleroderma has been investigated with the dual purpose of characterizing the immunologic specificity of the human antibodies, and of biochemically isolating the antigen. A nuclear protein, Scl-70, was found to be antigenic to sera from five genetically unrelated scleroderma patients. These sera were monospecific in the sense that after testing several dilutions of each nuclear fraction and antiserum, the only demonstrable precipitating antibodies were those directed against Scl-70 (Figs. 3, 6, 7 and legends). Moreover IgG pools absorbed with the precipitating antigen showed no residual anti-nuclear activity when tested by immunofluorescence (Fig. 9). These experiments show that

scleroderma sera, like the previously characterized mixed connective tissue disease sera (7), may manifest a high degree of specificity for certain nuclear macromolecules. This specificity can be exploited to further investigate the functions of the target macromolecules and may also provide clues as to the etiology of autoimmune diseases.

From quantitative and qualitative data presented in this study, certain inferences can be made about the properties and possible function of the scleroderma antigen. It is associated with the chromatin fraction of sonicated nuclei (Figs. 1 and 3) and is localized on the chromosomes of cells in mitosis (Fig. 9). It is quantitatively extracted from chromatin by 1 M

KC1 and binds to a cation exchange resin at neutral pH, as does histone Hl. These data suggest that Scl-70 is a basic chromosomal nonhistone protein bound to the interphase chromatin via ionic interactions. By calculating the approxi- mate number of copies of Scl-70 per haploid genome it is possible to compare this protein quantitatively to other chro- mosome elements of known distribution or function. The various histones range from 9.4 X 10” to 36.8 X 10” copies per haploid genome (15). A few of the major non-histones are present in excess of 10” copies (15). Quantities of two nuclear enzymes, RNA polymerase (28) and a chromatin-bound protease (29) have been estimated at 5 to 10 x lo” and 4.3 x lo4 copies per haploid genome, respectively. With 2.6 x lo4 copies, Scl-70 falls between the values of the two nuclear enzymes. It is unlikely, therefore, that this antigen is a major structural protein. We have observed that the light chain of IgG in immunoprecipitates with Scl-70 exhibits a reproducible in- crease in molecular weight when compared on SDS-gels to control and immunoprecipitate supernatant IgG (Fig. 7 and unpublished data). The possibility that the IgG in the immunoprecipitate has sustained enzymic modification, perhaps by Scl-70, is under investigation.

The calculations used to estimate the number of copies of Scl-70 are, of course, subject to a number of sources of error, the cumulative effect of which cannot be estimated directly. The two chemical assays for protein (16, 17) differed by as much as 20%. Chemical assays on a number of nuclear fractions were involved in the final calculations. We used both bovine serum albumin and total histones as standards for chemical assays, as well as the extinction coefficient of total histones, to insure that quantitations of histone-enriched fractions were not subject to greater error than crude nuclear fractions. Inactivation of Scl-70 through proteolysis may lead to an underestimation of the quantity of antigen. The antigen was fairly unstable until separated from the bulk of the nonhistones by Bio-Rex 70 chromatography, an observation consistent with that of Chong et al. that Bio-Rex 70 chromatography separates the histones (plus contaminating nonhistones) from a salt extractable chromosomal protease which appears in the nonhistone effluent in 0.4 M NaCl, pH 7.0 (29). Gel scans were used to estimate the amount of Scl-70 contained in the 0.65 M NaCl eluate. Multiple scans of two gels, each with a different protein load, indicated a range of not greater than 10%.

The co-isolation of histone Hl with Scl-70 has enabled us to use the histone as an internal standard for indirectly estimating the cumulative effect of the errors in quantitations discussed above (excluding rates of proteolysis, which may be different for Hl and Scl-70). The histone to DNA ratio in rat liver chromatin, as reported elsewhere, is 1:l (+O.l), and Hl comprises about 11% of the total histone mass (15, 19, 25). The nuclei used in this study also have a 1:l ratio of histone to DNA, as reported under “Results.” Histone Hl purified from the high speed KC1 extract by Bio-Rex 70 chromatography comprises 2.8% of the nuclear protein, and 78% of the


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nuclear Hl recovered in this study (see “Results” and Fig. 4). Given the 3:l ratio of nuclear protein to DNA, of which one- third is histone (see “Experimental Procedures”), it follows that 10.8% of the nuclear histone is Hl. Alternatively, as shown in Figs. 1 and 2, Hl obtained by acid extraction of the high speed KC1 extract comprises 9.5% of the nuclear histone. Another 2.4% is recovered as Hl in the low speed KC1 pellet, for a total yield of 11.9%. The close agreement between our numbers and those in the literature indicate that the succes- sive quantitations of a number of nuclear fractions performed here compare favorably to those based on a one-step quantitative extraction of Hl from chromatin with acid.

Acknowledgments-We wish to thank Dr. David Sadava for criti tally reviewing this manuscript.

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A S Douvas, M Achten and E M Tanantinuclear antibodies in scleroderma.

Identification of a nuclear protein (Scl-70) as a unique target of human

1979, 254:10514-10522.J. Biol. Chem.

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