The characteristics of eukaryotic ribosomal proteins PO, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). PO, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the-presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel fitration on a Sephadex G-200 column. PO may, therefore, be the eukaryotic equivalent ofEscherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and PO and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia saUna (eL12). Sixteen SLE sera containing antibodies to PO, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 anmno acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).Systemic lupus erythematosus (SLE) is an autoimmune disease generally characterized by serum antibodies directed against nuclear proteins and nucleic acids (1). That some patients' sera contain antibodies against ribosomal constituents (2, 3) has long been known, but the identity of these ribosomal antigens has only recently been determined (4). SLE anti-ribosome antibodies show almost exclusive reactivity against three 60S ribosomal subunit phosphoproteins called PO, P1, and P2 (4, 5). These same three proteins are also recognized by a mouse monoclonal antibody raised against chicken ribosomes (6). These "P" proteins have molecular sizes of -38, 19, and 17 kDa, respectively. P1 and P2 are believed to be the eukaryotic equivalent of the Escherichia coli ribosomal protein L12 and have been shown to contain sequences that are highly conserved among eukaryotes (6). Thus, P2 (7) from rat liver shows a high degree of amino acid sequence homology with Artemia salina ribosomal protein eL12 (8) and yeast ribosomal protein YPA1 (9). P1 and P2 also appear to be the functional counterparts of Artemia ribosomal proteins eL12' and eL12 and yeast ribosomal proteins YPA1/YPA2 (10-12). In order to further evaluate the remarkable specificity of the SLE anti-PO, -P1, and -P2 (anti-P) antibodies, we have mapped the antigenic determinant on the P proteins. This determinant is present on all three proteins and is contained within a common sequence of 22 amino acids at the carboxyl terminus of the Artemia r...

Although antibodies against nuclear antigens are considered the hallmark of systemic lupus erythematosus (SLE) 1, antibodies against cytoplasmic constituents have also been identified (1, 2). Despite their recognition more than 25 years ago, the prevalence, antigenic specificities, and disease associations of antiribosomal antibodies (ARA) are all controversial (3-10). Since these problems may relate to detection of different ribosomal or ribosome-associated antigens, we attempted to define more precisely the identity of the ribosomal protein antigens. The results of this study indicate that ARA reactive with ribosomal proteins occur in ~5-10% of SLE patients, but are highly specific, binding to epitopes on 3 out of a total of-80 proteins. Materials and MethodsAntibody Screening Procedures. Cellular localization of antigens was determined by indirect immunofluorescence using Hep-2 cells as substrate. The effect of the enzymes RNase (50 #g/ml), trypsin (5 ~zg/ml), and proteinase K (0.5 ~g/ml) on the intensity of immunofluorescence was quantitatively analyzed using a FIAX fluorometer, kindly loaned by Dr. L. J. Kagen (The Hospital for Special Surgery). Reactivity with saline-soluble extracts of human spleen and rabbit thymus was determined by counterimmunoelectrophoresis (CIE) (11) using standard reference serum to Ro, La, Sm, and ribonucleoprotein (RNP) (12). An anti-Jo-1 reference serum was kindly provided by C. C. Bunn and G. R. V. Hughes, Hammersmith Hospital, London, Great Britain.Isolation oJ Ribosomes. Ribosomes were isolated from dog, rat, and chicken livers essentially by the method of Fairhurst et al. (13). Briefly, livers were homogenized in 0.25 M sucrose in TK2~M buffer (20 mM Tris HCI, 25 mM KCI, 5 mM MgCI2, pH 7.5). The postmitochondrial supernatant was adjusted to 1.0% deoxycholate and centrifuged through a discontinuous sucrose gradient using an SW40 rotor. In some cases, the KC1 concentration in the sucrose was increased to 500 mM. The ribosomal pellet was washed and resuspended in distilled water or an appropriate buffer.Analysis of Ribosomal Constituents. The isolated ribosomes had an optical density 260/280 ratio of ~ 1.6. The RNA composition was analyzed by sucrose density ultracentrifugation on a 5-20% linear gradient using a SW60 rotor, as well as by a 2% composite gel (14) in a vertical gel apparatus (15). The ribosome was separated into large and small subunits either by incubation in 20 mM EDTA for 10 rain at room temperature, or by This work was supported by grants AM 32845 and AM 14627 from the National Institutes of Health, Bethesda, MD, and by grants from the Lupus Foundation of America. Address correspondence to Keith B. EIkon, The Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021.i Abbreviations used in this paper: ARA, antiribosomal antibodies; CIE, counterimmunoelectrophoresis; CM-cellulose, carboxymethylcellulose; NEPHGE, nonequilibrium pH gel electrophoresis; RNP, ribonucleoprotein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLE, sys...

SummaryFas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/Ipr mice results in massive lymphoproliferation (Ipr) and accelerated autoimmunity.To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wildtype Fas message was produced at ~10-fold lower levels in the Ipr compared with the + + substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts Materials and Methods Mice. MRL/Ipr (MRL/MpJ-lpr/lpr), MILL/+ + (MRL/MP-+/+), NZB/W F~, and C3H (C3H/HeSnJ) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred at the Hospital for Special Surgery.Northern and Southern Blotting. Single-ceU suspensions were prepared from the thymus, spleen, lymph nodes, and bone marrow of MRL and C3H mice. Total RNA was isolated by the acid guanidinium method (4) and poly(A)+-enriched KNA was obtained by the method of Badley et al. (5) using oligo(dT) cellulose (Collaborative Research, Bedford, MA). RNA was blotted to nylon membranes (Gene Screen; New England Nuclear, Boston, MA) and hybridizations were performed according to the manufacturer's instructions. PCR products were also blotted to nylon membranes and hybridized with DNA or oligonucleotide probes. DNA probes were labeled with [32p]dCTP by the random primer method (6) and oligonucleotides were biotinylated at the 3' termini using terminal transferase (Tdt) (7). After hybridization with oligonucleotides, blots were developed with the chemiluminescent substrate, AMPPD (Tropix, Bedford, MA), and fluorography. All blots were washed under high stringency conditions. PCR and DNA Sequencing. cDNA was synthesized from total KNA with Moloney murine leukemia virus (MoMULV) KT and oligo(dT) as described (8). cDNA was amplified by PCK on a thermal cycler (480; Perkin Elmer Corp., Norwalk, CT) with Taq polymerase (Bethesda Research Laboratories) unless indicated otherwise. The primers used for PCK analysis are shown in Table 1. The conditions used for amplification were: denaturation at 94~ for 1 rain, annealing at 5~ below the melting temperature (Tm) (calculated as described [91) for 1 min, and extension at 72~ for

Autoantibodies directed against a ribosomal small subunit protein of 20,000 molecular weight were found in sera from 5 of 44 patients with systemic lupus erythematosus (11%) and 5 of 48 MRLllpr mice (10%). This ribosomal protein was identified as S10 on the basis of two-dimensional gel electrophoresis and immunoblotting, as well as immunoblots of the purified S10 protein. The S10 protein antigen was readily extracted from ribosomes at low salt (300 mM KCI) and low magnesium (0.5 mM) concentrations, consistent with the highly exposed location proposed for this protein on the 40s subunit. Anti-SlO antibodies were observed significantly more frequently in lupus sera containing both anti-Sm and antiribosomal P protein antibodies and in MRLlZpr sera with anti-Sm activity, suggesting a linked pattern of autoantibody response. Together with anti-Sm and antiribosomal P protein antibodies, anti410 represents a third autoantibody highly specific for lupus in humans and MLRllpr mice.