Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.     

In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits

We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the alpha subunit of integrins. We found that affinity chromatography of cell extracts with this peptide followed by elution with EDTA resulted in the isolation of a 60-kDa protein (p60). The N-terminal amino acid sequence of this 60-kDa polypeptide was found to be highly homologous to the Ro/SS-A antigen, a 60-kDa protein homologous to calreticulin and Aplysia "memory molecule". The binding of p60 was found to be specific for the KLGFFKR sequence since this polypeptide did not bind to a peptide with a scrambled amino acid sequence (KLRFGFK), and it was also specifically eluted from the KLGFFKR affinity matrix ith soluble KLGFFKR peptide but not with the scrambled peptide. Solid phase in vitro binding assays demonstrated specific interaction of p60 with integrin alpha 3 and alpha 5 subunits but not with the beta 1 subunit. Furthermore, p60 could be copurified with alpha 3 beta 1 following coincubation in vitro. These interactions could be inhibited by KLGFFKR peptide and also by EDTA, indicating sequence-specific and divalent cation dependent binding. Despite the fact that calreticulin is thought to be localized in the endoplasmic reticulum, a pool of Ro/SS A antigen homologous 60-kDa polypeptide was found to be present in the soluble cytoplasm, indicating the feasibility of an interaction of p60 with the integrin alpha subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin alpha subunits via the highly conserved KLGFFKR amino acid sequence.     

Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.     

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.     

Evolutionary study of multigenic families mapping close to the human MHC class I region

Summary During a search for novel coding sequences within the human MHC class I region (chromosome 6p21.3), we found an exon (named B30-2) coding for a 166-amino-acid peptide which is very similar to the C-terminal domain of several coding sequences: human 52-kD Sjögren's syndrome nuclear antigen A/Ro (SS-A/Ro) and ret finger protein (RFP), Xenopus nuclear factor 7 (XNF7), and bovine butyrophilin. The first three of these proteins share similarities over the whole length of the molecule whereas butyrophilin is similar in the C-terminal domain. The N-terminal domain of butyrophilin is similar to rat myelin/oligodendrocyte glycoprotein (MOG) and chicken B blood group system (B-G) protein. These domains are components of a new subfamily of the immunoglobulin superfamily (IgSF). Butyrophilin is thus a mosaic protein composed of the MOG/B-G Ig-like domain and the C-terminal domain of 52-kD SS-A/Ro, RFP, and XNF7 (1330-2-like domain). Moreover, in situ hybridization shows that RFP, butyrophilin, and MOG map to the human chromosome 6p2l.3-6p22 region and are thus close to the MHC class I genes. It is therefore possible that the butyrophilin gene is the product of an exon shuffling event which occurred between ancestors of the RFP and MOG genes. To our knowledge, this is the first example of the colocalization of a chimeric gene and its putative progenitors. Finally, regulatory protein T-lymphocyte 1 (Rpt-1) shares similarities with the N-terminal halves of RFP, 52-kD SS-A/Ro, and XNF7, but not with the B30-2-like domain. We show that the ancestral Rpt-l gene evolved by overprinting. Correspondence to: P. Pontarotti     

Homology of the B50 murine melanoma antigen to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium-binding proteins

B50 is a murine melanoma-associated antigen found in tight association with B700, a melanoma-specific antigen. B700-like molecules are produced by all melanomas tested to date, including those of murine, human, swine and hamster origin. We have used rabbit antibodies to B50 to determine whether B50 expression is also restricted to melanomas. The results demonstrate that B50 is a commonly occurring protein, or is immunologically cross-reactive to a commonly occurring protein; 29 of 29 cell lines tested bound anti-B50 antibodies. N-terminal amino acid sequence analysis indicates that B50 has significant homology to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium binding proteins; hence B50 is likely to be an RNA and/or calcium-binding protein.     

Characterization of the autoantigen calreticulin

Anti-Ro/SS-A antibodies are commonly found in the sera of patients with Sj?gren's syndrome and SLE. These antibodies also occur in the mothers of children with neonatal lupus and congenital heart block. Ro/SS-A is a ribonucleoprotein complex whose cellular function remains unknown. To study its cellular function and to characterize its immunoreactivity, we have used an oligonucleotide designed after the published amino terminal sequence of a putative 60-kDa Ro/SS-A autoantigen to isolate its cDNA. This cDNA encodes a polypeptide that is the human homologue of calreticulin, a calcium binding protein of the endoplasmatic reticulum. The encoded polypeptide also shows a 64.4% identity with RAL-1, an Ag of the river blindness pathogen Onchocerca volvulus. Contrary to the data published by other authors, our results indicate that calreticulin is not a Ro/SS-A autoantigen. Moreover, we show that anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis.     

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.     

Functional similarities of recombinant OLP and cytokinin-binding protein 2

CBP1 and CBP2 are cytokinin-binding proteins isolated from tobacco callus. In particularly, CBP2 is a 26-kDa protein with high affinity (Kd=1.08 x 10(-6) M) for cytokinin [Kobayashi et al. Plant Cell Physiol.41(2): 148-157 (2000)] and the N-terminal amino acid analysis of CBP2 showed high sequence homology (92.9%) to tobacco osmotin-like protein (OLP). To compare the properties of OLP and CBP2, recombinant OLP was purified, and binding to benzyladenine (BA) was examined. The inclusion bodies of recombinant OLP were solubilized in 8 M urea and purified on an SP-Sepharose column. SDS-PAGE analysis of the purified recombinant OLP revealed a single band of 26 kDa. The Kd of solublized recombinant OLP to BA obtained from a Scatchard plot was 1.10 x 10(-6) M, which was similar to the Kd of CBP2 to BA (1.08 x 10(-6) M).     

SS-A/Ro52, an autoantigen involved in CD28-mediated IL-2 production

An autoantibody against SS-A/Ro52 (Ro52) is most frequently found in the sera of patients with Sj?gren's syndrome, systemic lupus erythematosus, and congenital heart block from anti-Ro52 Ab-positive mother. However, the physiological function of the autoantigen SS-A/Ro52 has not yet been elucidated. In this study, we describe the role of Ro52 protein in T cell activation. Overexpression of SS-A/Ro52 in Jurkat T cell resulted in enhanced IL-2 production following CD28 stimulation. Furthermore, transfection of anti-Ro52-specific small RNA duplexes partially blocked the expression of native and overexpressed Ro52 in Jurkat T cell, resulting in decreased IL-2 production via CD28 pathway in these cells. Finally, intracellular localization of Ro52 dramatically changed following CD28 stimulation. Our data reveal a novel function of Ro52 in CD28-mediated pathway, which eventually contributes to cytokine production and expression of the T cell biological programs.     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies

Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.     

Ro60 and Y RNAs: structure,functions, and roles in autoimmunity

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3′ to 5′ exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren’s syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

52 kD Ro/SS-A localizes to punctate structures in the cytoplasm of epithelial cells

Autoantibodies directed against 52 kD and 60 kD Ro/SS-A are frequently found in the sera of patients with lupus erythematosus and Sj?gren's syndrome-related disorders. Their location in the cell is subject to continuous debate in literature. It has been postulated that 52 kD Ro (52 Ro) co-localizes with the 60 kD Ro autoantigen in the nucleus, while others demonstrated that 52 Ro is primarily cytoplasmic. In order to resolve this controversy, 52 Ro protein was tagged with green fluorescence protein, overexpressed in A431 keratynocytes, and its location determined using fluorescence confocal microscopy. The intracellular location of the fusion protein was revealed via GFP autofluorescene and indirect immunofluorescence microscopy, using purified anti-52 Ro antibodies. The cellular locations of native 52 Ro in normal human keratinocytes, and in human A431 keratinocyte and HepG2 hepatocyte cell lines were similarly determined by utilizing 2 human anti-52 Ro antibodies purified from two different non-overlapping fragments of recombinant 52 Ro. In addition, colocalization of 52 Ro with mitochondria, lysosomes and endosomes was evaluated. It was found that both the 52 Ro-GFP fusion protein and the native 52 Ro localize in discrete cytoplasmic punctate structures separately from the mitochondria, lysosomes and endosomes. Furthermore, human autoantibodies that are reactive with denaturation-sensitive epitopes on 52 Ro recognize these cytoplasmic punctate structures, whereas antibodies directed against denaturation-resistant 52 Ro epitopes do not. This explains why the previously used antibody against denaturation-resistant 52 Ro epitopes failed to detect the protein in such punctate structures.     

Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive β-1,4-endoglucanase from Cellulomonas flavigena

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60°C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.     

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas)

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.