Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.     

Multiple phosphorylation of human SS-B/LA autoantigen and its effect on poly(U) and autoantibody binding

The metabolic turnover rates and the effect of in vitro phosphorylation on poly(U) and autoantibody binding of human SS-B/La ribonucleoprotein, an autoantigen expressed in various autoimmune disorders, were studied. The determination of the metabolic turnover rates of SS-B/La protein, SS-B/La protein phosphorylation and RNA binding yielded values of 12.1 h, 3.6 h and 3.7 h, respectively, indicating a possible functional correlation of RNA-binding and phosphorylation. This assumption was confirmed by studies of in vitro phosphorylation using purified SS-B/La protein and purified casein kinase type II as a model system. A high degree of phosphorylation of the SS-B/La protein (molecular mass 49 kDa) substantially diminished its binding capacity for poly[3H]U. However, binding of human autoantibodies against SS-B/La antigen increases 2-fold with increased SS-B/La phosphorylation. Complete phosphorylation in vitro led to partial molecular transformation, yielding an antigenically cross-reacting component with an apparent molecular mass of 51 kDa which could not be detected during in vivo phosphorylation.     

Analysis of the molecular composition of Ro ribonucleoprotein complexes. Identification of novel Y RNA-binding proteins.

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional proteins including the Ro52 protein and calreticulin has been suggested, but clear-cut evidence is still lacking. Partial purification of Ro RNPs from HeLa S100 extracts allowed characterization of several subpopulations of Ro RNPs with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro60 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA-associated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several proteins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the latter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to these proteins were found in sera from patients with systemic lupus erythematosus. The proteins bound preferentially to Y1 and Y3 RNA but, with the exception of the 53-kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53-kDa proteins by antibodies to Ro60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the binding sites for these proteins on Y1 RNA were clearly distinct from the Ro60-binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53-kDa proteins, as well as the stem 3-loop 3 and stem 2-loop 1 regions. Interestingly, truncation of the La-binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient association. Taken together, these results suggest the existence of further subpopulations of Ro RNPs or Y RNPs, consistent with the heterogeneous characteristics observed for these particles in the biochemical fractionation experiments.     

Antigenic relatedness of small ribonucleoprotein particles

We have examined the relationships among small ribonucleoprotein particles found in eucaryotic cells by an antigen depletion technique using autoimmune antibodies. We have confirmed that the (U1) ribonucleoprotein particle antigen is found on the same complex as the Sm antigen. We have also shown that the Ro antigen is found on the same complexes as the La antigen. However, both Sm and La antigens are also found on complexes that are never associated with (U1) ribonucleoprotein particle and Ro, respectively. Further, U1 containing complexes can exist that contain the Sm antigen but not the (U1) ribonucleoprotein particle antigen. In a similar manner, we find several La-Ro RNA containing complexes that carry the La antigen but do not always carry the Ro antigen. Sm and La antigen are quantitatively associated with their specific ribonucleoprotein complexes.     

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.     

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

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.     

Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25-40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE-Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE-keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.     

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.     

Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting

Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.     

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316-335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128-285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316-335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316-335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.     

Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.     

Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein.

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.