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Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

A cDNA encoding the C-C chemokine MDC was isolated from a human macrophage cDNA library by differential hybridization using monocyte- and macrophage-specific cDNA probes. During monocyte to macrophage differentiation in vitro, MDC expression is first detected after 1 day of culturing and reaches maximum levels after 6 days when macrophages have fully matured, as judged from the expression of known macrophage marker genes. Exposure of macrophages to lipopolysaccharide (LPS) results in a dose-dependent increase in MDC mRNA levels, with maximum induction occurring after 6-8 h, whereas expression levels of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2, interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) respond much faster to LPS. Furthermore, MDC expression in macrophages is enhanced by the inflammatory mediators TNF-alpha and IL-1beta. Similar to other TNF-alpha/IL-1beta-inducible genes, costimulation of macrophages with both cytokines leads to higher MDC expression levels than stimulation with a single cytokine. By contrast, both resting and activated monocytes do not express MDC mRNA.

Proteins subject to proteolysis or phosphorylation during apoptosis are commonly precipitated by autoantibodies found in the serum of patients with systemic lupus erythematosus (SLE). We screened a panel of murine monoclonal and human monospecific sera reactive with known autoantigens for their ability to selectively precipitate phosphoproteins from apoptotic Jurkat T cell lysates. Sera known to recognize the U1-small nuclear ribonucleoprotein (snRNP) complex (confirmed by their ability to precipitate U1-snRNA) selectively precipitated a phosphoprotein complex (pp54, pp42, pp34, and pp23) from apoptotic lysates. Monoclonal antibodies reactive with U1-snRNP proteins precipitated the same phosphoprotein complex from apoptotic lysates. The phosphorylation and/or recruitment of these proteins to the U1-snRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, gamma irradiation, or UV irradiation), and is blocked by overexpression of bcl-2. The U1-snRNP-associated phosphoprotein complex is immunoprecipitated by monoclonal antibodies reactive with serine/arginine (SR) proteins that comprise a structurally related family of splicing factors. The association of phosphorylated SR proteins with the U1-snRNP complex in cells undergoing apoptosis suggests a mechanism for regulation of alternative splicing of apoptotic effector molecules.

Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.

The Ro and La autoantigens, which were originally identified by sera from autoimmune patients, consist of RNA-protein complexes (RNPs), in which the protein components carry most of the autoantigenic determinants. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY1, hY3, hY4 and hY5 [35], associated with several proteins, Ro60, Ro52 and La [8, 35, 106] and possibly other, yet unidentified polypeptides. Controversial data have been published on the association of the protein calreticulin with Ro RNPs [53, 58, 75, 78], but recently in vitro evidence has been obtained that non-phosphorylated calreticulin is able to interact with hY RNAs directly [17]. The physiological significance of this interaction remains to be established. In addition to its association with hY RNAs, La is (in most cases transiently) associated with all other RNA polymerase III transcripts. The hY RNAs are the only known cellular RNAs stably bound by La. While the Ro RNPs were found to reside mainly in the cytoplasm, the other La RNPs are mainly nuclear. In this review recent progress made on the intracellular localization and transport of Ro RNP components and of assembled Ro RNPs will be discussed.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called "telomeres" to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

The U1 small nuclear ribonucleoprotein (snRNP) particle consists of an RNA backbone and eleven associated proteins. Both the RNA and some of the proteins are immunogenic in patients suffering from systemic lupus erythematosus or mixed connective tissue disease. An overview is given of the U1 snRNP particle with respect to its structure and function as well as its autoantigenicity.

The U1 small nuclear ribonucleoprotein particle (snRNP) has an important function in the early formation of the spliceosome, the multicomponent complex in which pre-mRNA splicing takes place. The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. Here we show by microinjection of Xenopus laevis oocytes that the third U1 snRNP-specific protein, U1C, passively enters the nucleus. Furthermore, we show that in both X. laevis oocytes and cultured HeLa cells mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP.

The phage display approach has proven to be a major step forward in studies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from these libraries resemble original in vivo pairings. Here we describe a novel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantigen-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as soluble factors were shown to be essential. This B cell culture system combined with a selection on antigen (the U1A protein, a frequent autoantigenic target in patients with systemic lupus erythematosus) and single cell sorting resulted in the isolation of U1A-specific human B cells and the subsequent expression of an U1A-specific single chain variable fragment (scFv). Our method circumvents laborious plating and screening and has the advantage that original heavy/light chain pairings can be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied using this system.

Previously, we described a DP-65 encoded heavy chain variable (VH) gene restriction in anti-U1A antibodies. The U1A protein (a component of the U1 ribonucleoprotein particle) is an important autoantigenic target in certain systemic lupus erythematosus (SLE) patients. Here we examined the effect of randomizing amino acids in the heavy chain complementarity determining region 3 (CDR3) of this germline encoded recombinant antibody fragment on binding to the U1A protein. A phage display library was constructed using the DP-65 VH domain with four randomized CDR3 residues and our results showed that a high frequency (10%) of the randomized mutants in the unselected library were able to bind the U1A protein. This corroborates our previous finding that this VH domain provides an appropriate structure for U1A binding, although the nature of the CDR3 residues appears crucial in determining whether or not this VH domain binds U1A. After two rounds of selection U1A binders show a consensus sequence in their randomized CDR3 residues i.e. S(K,R,S)XG, in which X is an uncharged residue. This consensus is partially present in an antibody which was derived from an SLE patient indicating that this consensus, to some extent, is also followed in vivo. Clones which match the consensus sequence obtained up to 25-fold higher affinities compared with the original clones, illustrating the importance of the VH CDR3 residues in determining the affinity of these antibodies.

We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52+ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1+ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti-Ro52+ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long alpha-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1+ and anti-Jo-1- IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.

Objective: To determine whether the E, F, and G Sm proteins present antigenic determinants recognized by systemic lupus erythematosus (SLE) patient sera, and if so, whether the antigenicity depends on the native conformations of the polypeptides and/or is E-F-G complex restricted., Methods: Radioimmunoprecipitation, epitope tagging, expression polymerase chain reaction, in vitro translation, in vitro reconstitution, and immunoblotting., Results: Most of the anti-Sm SLE patient sera tested reacted with one or more of the E, F, and G proteins in immunoprecipitation studies but not on immunoblots. All sera, however, highly efficiently immunoprecipitated the E-F-G complex. This complex recognition was detected exclusively in anti-Sm patient sera but not in patient sera with other serotypes., Conclusion: We demonstrate the presence of a novel and abundant anti-Sm autoantibody class in SLE patient sera which exclusively or predominantly recognizes conformational Sm epitopes present on the E-F-G complex but not on the denatured proteins. This complex recognition is highly specific for sera of the anti-Sm serotype and may be relevant for clinical diagnosis as well as for understanding the etiology of anti-Sm autoantibody production.

The Ro ribonucleoprotein particle (RoRNP) is the target of a variety of specific anti-protein autoantibodies produced by patients with systemic autoimmune diseases. RoRNPs, the function of which remains elusive, appear to be rather heterogeneous in terms of their molecular composition. Among several Ro proteins, a protein of 52 kD (Ro52) has been identified, but its association with RoRNPs remains questionable. In this study, we first mapped the Ro52 regions that are accessible at the surface of the isolated protein, by means of antibodies raised in rabbits, against 39 overlapping synthetic peptides covering the whole Ro52 molecule and using several different methods, including various ELISA formats and Western blotting (based on the use of recombinant Ro52) and immunoprecipitation of in vitro translated Ro52. Then, the whole set of anti-peptide antibodies was used to attempt to immunoprecipitate RoRNPs from a cell extract of K562 cells. RoRNPs, especially Ro(hY3)RNPs, were effectively immunoprecipitated by certain anti-peptide antibodies, indicating that Ro52 protein is associated with at least a subset of RoRNP particles. As expected, a number of epitopes available on the isolated Ro52 molecule are no longer accessible when Ro52 is associated with the RoRNP. Conversely, neotopes, among them at least one corresponding to a previously characterized epitope recognized by patients' antibodies, are only present at the surface of the particle and not detectable on the isolated Ro52 protein.

In this study we have used a number of monoclonal antibodies with various anti-Sm specificities originating from MRL/lpr mice to map B cell epitopes of the Sm-B/B' and Sm-D1 proteins. Selection of Sm-B subfragments reactive with the Sm-B/B'-specific monoclonal antibody ANA125 from a DNaseI fragment expression library revealed that the epitope recognized by this monoclonal antibody is located between amino acids 146 and 158: GRGTVAAAAAAAT. The epitopes recognized by two distinct Sm-D1-specific monoclonal antibodies, 7.13 and ANA127, appeared to be located in the carboxy-terminal region of the protein as revealed by immunoprecipitation of in vitro translated deletion mutants of Sm-D1. These epitopes are probably identical and not simply composed of a GR repeat, which is a characteristic feature of this part of the protein. Immunoprecipitation of in vitro translated deletion mutants of both Sm-B and Sm-D1 was also employed to determine the sequence requirements for recognition by two monoclonal antibodies that are cross-reactive with several Sm proteins, Y12 and ANA128. The epitope recognized by these two monoclonal antibodies is probably also identical and composed by the juxtaposition of several regions in the folded protein. The low, but significant, level of immunoprecipitation of truncated versions of both Sm-B and Sm-D1, suggests that the Sm domain, which is shared by all Sm proteins, in particular the amino-terminal part of the Sm1 motif of Sm-B and Sm-D1, plays an important role in formation of the cross-reactive epitope and might contribute to cross-reactivity with other Sm proteins. The results of immunoprecipitation experiments with cellular extracts show that the epitopes recognized by all anti-Sm monoclonal antibodies used in this study are accessible in the assembled snRNPs.

Objective: To define the fine specificity of the 10 reference sera used for determination of antinuclear antibodies (ANA) and ANA subsets which are available from the Arthritis Foundation (AF) and from the Centers for Disease Control and Prevention (CDC)., Methods: AF/CDC sera were assessed by experienced laboratory staff, using indirect immunofluorescence and Western blotting., Results: The original assignment of fluorescence patterns to 4 reference sera was confirmed, and the fluorescence intensities were determined using reference fluorescent beads. On Western blots, sera AF/CDC2 (anti-SS-B/La) and AF/CDC7 (anti-SS-A/Ro) did not detect Ro antigens, sera AF/CDC9 and AF/CDC10 appeared to be monospecific anti-Scl-70 and anti-Jo-1 sera, respectively, serum AF/CDC4 (anti-U1 small nuclear RNP) recognized the 70-kd band, and serum AF/CDC5 recognized the Sm antigen with its multiple bands. Semiquantitative analyses revealed that AF/CDC5, AF/CDC2, and AF/CDC10 were strongly reactive sera, whereas AF/CDC4 and AF/CDC9 were much weaker and should be used at lower dilutions on Western blots., Conclusion: The AF/CDC ANA reference sera, originally described as reference reagents for indirect immunofluorescence and double immunodiffusion techniques, are also useful for Western blotting. The data presented herein further support the use of these sera for reference purposes.

The structure of the conserved region of the U1A pre-mRNA (AgRNA) and its complex with U1A protein was investigated. The previously proposed secondary structure of Ag RNA, derived from enzymatic probing and analysis of the structure and function of mutant mRNAs, is now confirmed by chemical probing data and further refined in the regions where the enzymatic data were not conclusive. The two unpaired nucleotides in the internal loops opposite of the Box sequences as well as the tetraloop could not be cleaved by ribonucleases, but are accessible to chemical probes. Concerning the RNA-protein complex, the protection experiments showed that the Box regions are largely protected when the U1A protein is present. All stem regions in the 5' part of the structure seem protected against ribonucleases. Unexpectedly, the nucleotides of the tetraloop become accessible to ribonucleases in the RNA-protein complex. This result indicates that the tetraloop undergoes a conformational change upon U1A protein binding. The 3' part of the Ag RNA sequence, containing the polyadenylation signal in a hairpin structure, showed hardly any protection, a finding that agrees with the fact that U1A does not interfere with the binding of the cleavage polyadenylation specificity factor (CPSF) to the polyadenylation signal.

During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.

Genes for the snRNP proteins U1-70K, U1-A, Sm-B'/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3' poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.

The eukaryotic endonucleases RNase P and RNase MRP require both RNA and protein subunits for function. Even though the human RNase P and MRP RNAs were previously characterized, the protein composition of the particles remains unknown. We have identified a human a Caenorhabditis elegans sequence showing homology to yPop1, a protein subunit of the yeast RNase P and MRP particles. A cDNA containing the complete coding sequence for the human protein, hPop1, was cloned. Sequence analysis identifies three novel sequence motifs, conserved between the human, C. elegans and yeast proteins. Affinity-purified anti-hPop1 antibodies recognize a single 115 kDa protein in HeLa cell nuclear extracts. Immunoprecipitations with different anti-hPop1 antibodies demonstrate an association of hPop1 with the vast majority of the RNase P and MRP RNAs in HeLa cell nuclear extracts. Additionally, anti-hPop1 immunoprecipitates possess RNase P enzymatic activity. These results establish hPop1 as the first identified RNase P and MRP protein subunit from humans. Anti-hPop1 antibodies generate a strong nucleolar and a weaker homogeneous nuclear staining in HeLa cells. A certain class of autoimmune patient serum precipitates in vitro-translated hPop1. hPop1 is therefore an autoantigen in patients suffering from connective tissue diseases.

Objective: Salivary glands of patients with Sjögren's syndrome (SS) have been shown to be a site of anti-SS-B/La antibody production. The present study investigated differences in the localization of the SS-B/La antigen in labial salivary gland (LSG) tissue between SS and non-SS patients, which may explain the local antigen-driven anti-SS-B/La response., Methods: Distribution of SS-B/La was studied immunohistologically in the LSG biopsy samples of 9 SS patients, 10 non-SS patients, and in normal tissues obtained at autopsy within 2 hours after death, using a mouse monoclonal antibody directed to SS-B/La. In 3 SS and 3 non-SS patients, LSGs were also studied with affinity-purified biotinylated human antibodies directed against SS-B/La., Results: In the non-SS patients, SS-B/La was primarily observed in the nucleoli of acinic cells of the LSGs. Patients with either primary SS or secondary SS showed an accumulation of SS-B/La in the nucleoplasm of acinic cells. In the SS patients, SS-B/La was also detected in the cytoplasm as a diffuse or perinuclear staining. Sometimes, SS-B/La was found along the membrane of acinic cells as well. This aberrant nuclear and cytoplasmic distribution of SS-B/La in SS patients correlated well with abnormalities in the composition of the plasma cell population in the LSGs, but not with a lymphocytic focus score > 1., Conclusion: The accumulation and redistribution of SS-B/La in the LSGs may play an important role in the local antigen-driven anti-SS-B/La response in SS, and can also be used to improve the diagnostic possibilities of the LSG biopsy.

After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients. So far, data on the antigen-specificity and therefore the function of these H-Ig are not available. Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT. In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens. The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens. However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen. An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

The La (SS-B) autoantigen is a 47-kDa protein which binds to the 3' termini of nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) mRNAs. In this study we have addressed the intracellular trafficking of the La protein and characterized cis-acting elements involved in nuclear import and retention in Xenopus laevis oocytes by microinjection of in vitro translated La protein. The steady-state distribution of recombinant human La protein was, like the endogenous Xenopus La protein, mainly nuclear. Nuclear import of La appeared to be energy-dependent and is governed by a nuclear localization signal (NLS) located in the extreme C-terminal part of the protein, resembling the consensus bipartite NLS. Another sequence element in La, which completely corresponds to the bipartite NLS consensus, appeared to be nonfunctional in nuclear import of the La protein. Nuclear accumulation of La was found to be mediated by retention in the nuclear compartment. The N-terminal RNA binding domain of La is not involved in this retention, but sequence elements in the central region of the polypeptide (amino acids 165 to 337) appear to be required. Amino acids 266-269 as well as 313-337 were found to be of major importance for retention in the nucleus.

This is the first study describing recombinant human antibody fragments directed to the U1 RNA-associated A protein (U1A). Three anti-U1A antibody fragments (Fab) were isolated from a semi-synthetic human Fab library and one anti-U1A single-chain variable fragment (scFv) was isolated from a library which was derived from the IgG-positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE-overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ-line gene DP-65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C-terminal RNP motif of the U1A protein. The DP-65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti-U1A antibody fragments use this gene. This points to a restricted VH gene usage in the case of U1A, suggesting that the DP-65 heavy chain has a natural shape complementarity to the U1A protein.

Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal.

The La (SS-B) autoantigen is an evolutionarily conserved 47-kDa protein which binds to nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) messenger RNAs. Here, we show that La in the cytoplasm is associated with a subset of small ribosomal subunits, possibly by direct association with 18S ribosomal RNA. This association is likely to be related to the putative role of this protein in translation regulation.

The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif which is required for the binding of the U1C protein to the U1 snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study we demonstrate that the human U1C protein is able to form homodimers as well. U1C homodimers are found when (i) the human U1C protein is expressed in Escherichia coli, (ii) immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when (iii) the yeast two hybrid system is used. Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization.

Many intracellular proteins and nucleic acids, that are involved in important biosynthetic pathways, are targeted by autoantibodies occurring spontaneously in the sera of patients with systemic autoimmune diseases. Frequently, the autoantigens are assembled into multicomponent complexes containing both nucleic acid(s) and proteins. Recently, progress has been made in the study of autoantigenic ribonucleoprotein complexes, the most important of which are spliceosomal ribonucleoproteins, nucleolar ribonucleoproteins, Ro/La ribonucleoproteins and complexes of aminoacyl-tRNA synthetase and tRNA. In addition to new structural and functional information, important results have been obtained on epitope spreading, as well as on a potential role for apoptosis during the development of an autoimmune response against these complexes.

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti-La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre-tRNA, pre-5S rRNA, hY RNAs, pre-U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7-2 RNA. Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross-competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3. Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA-binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts.

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All four MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb 1D8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs are highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.

Objective: To determine any clinical or genetic markers of differentiation and outcome in a previously described cohort of 46 patients with mixed connective tissue disease (MCTD)., Methods: Patients were clinically evaluated, chart notes reviewed, and HLA subtyping and immunology profiles performed where possible. Eleven had died and 7 were lost to followup., Results: MCTD had differentiated into systemic lupus erythematosus in 12 patients and into systemic sclerosis in 13. The latter was associated with HLA-DR5 (P = 0.038), and nondifferentiation was associated with HLA-DR2 or DR4 (P = 0.007). Three HLA-DR4 positive patients had MCTD that evolved into rheumatoid arthritis. Erosive and/or deforming arthritis was associated with HLA-DR1 or DR4 (P = 0.015). HLA-DR3 was associated with interstitial lung fibrosis (P = 0.044) and keratoconjunctivitis sicca (0.001 < P < 0.01). Severe Raynaud's phenomenon predicted higher mortality (0.01 < P < 0.05)., Conclusion: We suggest that MCTD is, for most patients, an intermediate stage in a genetically determined progression to a recognized connective tissue disease. Those whose disease remains undifferentiated might be considered a distinct subset.

Objective: To investigate the occurrence and features of anti-56K/annexin XI autoantibodies in sera from patients with various systemic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, polymyositis and Raynaud's phenomenon., Methods: Anti-56K/annexin XI autoantibodies were detected by an enzyme linked immunosorbent assay using the bacterially expressed recombinant protein as antigen., Results: Anti-56K/annexin XI autoantibodies were found in a substantial number of patient sera (4.1-10.1%), but are rarely detected in sera from healthy controls and from patients with infectious diseases. Disease specificity was not observed. The majority of these autoantibodies belong to the IgG class and their titers in positive sera are at least as high as those of other well defined autoantibodies., Conclusion: Anti-56K/annexin XI autoantibodies frequently occur in systemic autoimmune diseases in contrast to infectious diseases and healthy individuals and are primarily of the IgG isotype, consistent with an antigen driven mechanism of autoantibody production.

We investigated the role of humoral factors in the pathogenesis of Coxsackie B1 virus-induced murine myositis (CB1-myositis). At 2, 4 and 8 weeks after inoculation, serum was studied for circulating immune complexes (CIC) (Raji-cell assay), haemolytic complement activity (CH50 titre) and anticytoplasmic autoantibodies (Western blotting, immunoprecipitation) in relation to degree of mononuclear cell infiltration and muscle fibre necrosis. At 2, 4 and 8 weeks, cell infiltration correlated positively to muscle fibre necrosis. From 2 weeks on, moderate quantities of CIC were found in nearly all CB1-inoculated mice, but without correlation to histological changes. Except for a positive correlation of CH50 titre to muscle necrosis at 4 weeks (r = 0.60; P = 0.02), CH50 titres did not correlate to muscle lesions. Anticytoplasmic and other known autoantibody specificities were absent. In conclusion, first, in CB1-myositis CIC occurred from 2 weeks on, but no correlative evidence was found for their involvement in pathogenesis, neither for that of complement nor for anticytoplasmic autoantibodies. Secondly, cell infiltration correlated positively to muscle necrosis, underscoring the importance of cellular mechanisms. Thus, our data do not support, or conclusively exclude, a role for humoral processes in CB1-myositis.

The subcellular distribution of Ro ribonucleoprotein particles (Ro RNPs) and their constituents was determined using mammalian tissue culture cells and Xenopus laevis oocytes. Cell fractionation revealed that Ro RNPs are restricted to the cytoplasm, while the Ro RNP-specific proteins were also present in the nucleus. Transport studies indicated that the protein components of Ro RNPs are actively imported into the nucleus while the RNA component accumulates in the cytoplasm. These results suggest a model for the assembly of Ro RNPs.

Ro ribonucleoprotein particles (Ro RNPs) are evolutionarily conserved cytoplasmic complexes of unknown function. They are composed of several proteins and a small, RNA polymerase III-transcribed Ro or Y RNA. Abs directed against the protein moiety of Ro RNPs are often found in sera of patients suffering from certain autoimmune disorders. The association of one of the Ro proteins, a protein of 52 kDa (Ro52), with Ro RNPs is still questionable. In this study, we have used anti-Ro52 Abs isolated from autoimmune sera to locate the antigenic determinants of Ro52 and to analyze the correlation between regions of Ro52 recognized by these Abs and their ability to immunoprecipitate Ro RNPs. The results indicate that the autoimmune response against Ro52 is heterogeneous and that the exclusive recognition of certain epitopes does not result in immunoprecipitation of Ro RNPs.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

The secondary structures of human hY1 and hY5 RNAs were determined using both chemical modification techniques and enzymatic structure probing. The results indicate that both for hY1 and for hY5 RNA the secondary structure largely corresponds to the structure predicted by sequence alignment and computerized energy-minimization. However, some important deviations were observed. In the case of hY1 RNA, two regions forming a predicted helix appeared to be single-stranded. Furthermore, the pyrimidine-rich region of hY1 RNA appeared to be very resistant to reagents under native conditions, although it was accessible to chemical reagents under semi-denaturing conditions. This may point to yet unidentified tertiary interactions for this region of hY1 RNA. In the case of hY5 RNA, two neighbouring internal loops in the predicted structure appeared to form one large internal loop.

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.

Antibodies to the RNA polymerase III transcription termination factor La are frequently found in the serum of patients with various autoimmune diseases. The mechanisms by which autoimmune responses are evoked remain largely obscure, but the presentation of autoantigens on the cell surface during stress conditions has been reported as a possible factor. In this study we analysed the effects of adenovirus infection on the binding of anti-La antibodies to the surface of several human cell lines and on the levels of the membrane-expressed glycoproteins HLA class I, CD44 and the CD3 complex. In addition, we studied the relative amount and the intracellular distribution of the La protein as well as its association with the major species of non-coding virus-associated (VAI) RNA. While immunofluorescence patterns revealed a redistribution and possibly cell surface expression of the La protein during infection, this could not be confirmed by other techniques. In contrast, surface levels of HLA class I proteins and CD3 complex were severely affected. The data suggest that the subcellular distribution of the La protein is not detectably influenced by adenovirus infection.

In this study we report the detection of autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoprotein (RNP) particles in sera from patients with connective tissue diseases. The method described employs radioactively labelled antisense U3- and Th RNA which are hybridized to immunoprecipitated U3- or Th RNA from a HeLa cell extract. Of the 66 sera that were screened with this method seven sera (11%) precipitated only Th RNP, 16 sera (24%) precipitated only U3 RNP and 4 sera (6%) precipitated both U3- and Th RNP. Both anti-U3 RNP and anti-Th RNP activity appeared to be mostly associated with scleroderma or scleroderma-associated diseases. Using this method we also showed that some of the Th RNP particles in a cell extract are associated with the La autoantigen. We conclude that for the identification of immunoprecipitated RNAs this method is very sensitive and provides unambiguous data.

Anti-56K autoantibodies are present in sera from patients with various autoimmune diseases, predominantly in sera from patients with rheumatoid arthritis, systemic lupus erythematosus, or Sjögren's syndrome. To clarify the molecular structure of this autoantigen, we isolated a 2.0-kilobase pair cDNA clone considered to encode the full-length 56K autoantigen. The longest open reading frame encodes a 505-amino acid polypeptide, with a predicted molecular mass of 54.4 kDa. The in vitro translated protein is recognized by all anti-56K positive patient sera tested. Antibodies affinity-purified using the bacterially expressed recombinant protein recognized the 56K autoantigen in a HeLa cell extract. cDNA sequencing revealed that the 56K cDNA shares a high degree of homology in both nucleotide (87%) and amino acid sequence (92.5%) with bovine annexin XI, indicating that the 56K cDNA encodes the human homologue of annexin XI, a member of the Ca(2+)-dependent phospholipid binding protein family. Anti-56K autoantibody exhibits both a cytoplasmic and a nuclear staining in immunofluorescence experiments. Patients' sera recognize preferentially the N-terminal region of the protein, which is specific for 56K/annexin XI and not shared by other annexins, indicating that the autoimmune response to 56K/annexin XI in these patients is specific for this annexin family member.

The human U1A protein-U1A pre-mRNA complex and the relationship between its structure and function in inhibition of polyadenylation in vitro were investigated. Two molecules of U1A protein were shown to bind to a conserved region in the 3' untranslated region of U1A pre-mRNA. The secondary structure of this region was determined by a combination of theoretical prediction, phylogenetic sequence alignment, enzymatic structure probing and molecular genetics. The U1A binding sites form (part of) a complex secondary structure which is significantly different from the binding site of U1A protein on U1 snRNA. Studies with mutant pre-mRNAs showed that the integrity of much of this structure is required for both high affinity binding to U1A protein and specific inhibition of polyadenylation in vitro. In particular, binding of a single molecule of U1A protein to U1A pre-mRNA is not sufficient to produce efficient inhibition of polyadenylation.

Y RNAs are small cytoplasmic RNAs which are components of the Ro ribonucleoprotein complexes in higher eukaryotes. These complexes are frequently recognized by antibodies present in autoimmune sera. In this study we analysed the occurrence of Y RNAs in various mammalian and human cell lines and erythrocytes by means of hybridization with human Y RNA probes. Y RNAs homologous to their human counterparts, both in length and in sequence, were detected in all mammalian cells analysed. While hY1 and hY3 analogues were found in all cells, Y4 and Y5 RNA could not be detected in rodent cells. In addition, Y5 RNA was absent from bovine cells. Attempts to determine the sequence of rat Y RNAs by genomic cloning resulted in the isolation of a presumptive Y1 RNA pseudogene. Analysis of the hY RNA content of various human cell lines showed that all four human Y RNAs were present in all cell lines examined. However, the relative levels to which these RNAs were expressed showed marked differences.

Antibodies against naked U1RNA can be found in sera from patients with overlap syndromes of systemic lupus erythematosus (SLE) in addition to antibodies directed to the proteins of U1 ribonucleoproteins (U1RNP). We investigated the reactivity of these U1RNA specific autoantibodies with the native U1RNP particle both in vitro and inside the cell. For this purpose a method was developed to purify human autoantibodies directed to specific regions of U1RNA. The antibodies are specifically directed to either stemloop II or stemloop IV of U1RNA and do not crossreact with protein components of U1RNP. Both types of antibody are able to precipitate from cell extracts native U1snRNPs containing most, if not all, protein components. Immunofluorescence patterns indicate that the antigenic sites on the RNA, i.e. the stem of stemloop II and the loop of stemloop IV, are also available after fixation of the cells. Immunoelectron microscopy employing anti-stemloop IV antibodies and purified, complete U1snRNP particles showed that stemloop IV is located within the body of the U1RNP complex, which also comprises the Sm site and the common Sm proteins. The anti-U1RNA autoantibodies described in this paper recognize native U1RNP particles within the cell and can therefore be used as tools to study mechanisms involved in splicing of pre-mRNA.