Biomedical subjects

J McCluskey

Publications and source records attributed to J McCluskey.

At least 19 recordsLinked to original sources

PubMed1996

Hierarchical self-tolerance to T cell determinants within the ubiquitous nuclear self-antigen La (SS-B) permits induction of systemic autoimmunity in normal mice.

Systemic autoimmune diseases are frequently associated with clustering of high titer autoantibody responses towards nuclear self-antigens. Little is known, however, about the extent of immune tolerance to the target nuclear antigens or the events leading to the complex autoantibody responses that are characteristic of systemic autoimmunity. To address these issues, we have examined the mouse immune response to La autoantigen (mLa) and the homologous human La antigen (hLa), which are components of the La(SS-B)/Ro(SS-A) ribonucleoprotein (RNP) complex targeted in systemic lupus erythematosus and primary Sjögren's syndrome. The findings reveal the presence of hierarchical T cell tolerance involving multiple autodeterminants within the La autoantigen expressed by normal H-2k and H-2a mice. At one end of this spectrum, there was no detectable T or B cell autoimmunity observed in mice that were immunized with the immunodominant mLa287-301 determinant, which differed by a single residue in its core sequence from the homologous but highly immunogenic human La288-302 determinant. Interestingly, the mLa287-301 peptide acted as an altered peptide ligand that specifically antagonized the activation of an hLa288-302-specific T cell hybridoma. In contrast to the tolerogenic mLa287-301 determinant, a range of autoimmune potential was identified among poorly tolerizing, subdominant self-peptides present within mouse La autoantigen. Notably, immunization of normal mice with the autologous subdominant La25-44 and La106-129 determinants resulted in limited or no detectable autoantibody response. In contrast, immunization with the subdominant mouse La13-30 determinant induced a proliferative T cell response associated with the appearance of specific autoantibodies recognizing multiple intrastructural (La) and intermolecular components (Ro) of the murine La/Ro RNP. The findings suggest how diversified autoimmunity might follow initiation of immunity to simple peptide mimics of poorly tolerogenic determinants that are present within ubiquitous self-antigens.

Amino Acid Sequence↗

PubMed1996

The immune response to 52-kDa Ro and 60-kDa Ro is linked in experimental autoimmunity.

Clustering of autoantibody specificities is a consistent finding in patients with systemic autoimmune diseases. Patients with Sjögren's syndrome frequently have autoantibodies to La, 60-kDa Ro(SS-A) protein (Ro60), and 52-kDa Ro(SS-A) protein (Ro52). In the case of anti-Ro60 and anti-La, there is evidence that these specificities occur together because of the physical association of the Ro60 and La proteins that form a ribonucleoprotein particle (RNP). Thus, the autoantibody response may spread from a single epitope to involve new epitopes located within other components of the RNP. The physical association of Ro52 with the Ro/La RNP has remained controversial, implying that Abs to Ro52 are not a consequence of intermolecular spreading and may be triggered independently of the anti-Ro60 response. To examine this relationship of the immune response to Ro52 and Ro60, mice were immunized with recombinant Ro52, Ro60, or La, and examined for autoantibody production. Immunization with Ro52 resulted in rapid, high titer Ab production to Ro52, followed 7 to 14 days later by lower titer autoantibody production to Ro60. Immunization with Ro60 led to anti-Ro60, which was also followed 7 to 14 days later by a lower titer anti-Ro52 response. Cross-reactivity of affinity-purified Abs from immune mouse sera was not observed. These observations suggest that the autoimmune responses to Ro60 and Ro52 are linked intrinsically, despite previous evidence suggesting they are not associated in vivo. The mechanism of linkage remains unclear, but the data are most consistent with some physical association of Ro52 and Ro60 allowing autoimmunization, presumably as a result of normal cell turnover or specific injury in vivo.

Animals↗

PubMed1996

CTL recognition of an altered peptide associated with asparagine bond rearrangement. Implications for immunity and vaccine design.

The extent to which peptides containing chemically and post-translationally modified amino acid side chains are recognized by primed CTL has not been clearly defined. We report on the CTL recognition of a MHC class I-restricted peptide containing a cyclized asparagine (succinimide) residue. This modification of the asparagine side chain is a common intermediate structure during deamidation, isomerization, and bond rearrangements of amide-containing amino acids and also occurs as a side reaction in peptide synthesis. The CTL specifically recognized the succinimide-containing peptide showing only weak cross-reactivity at high concentrations of the parent peptide containing unmodified asparagine. Similarly, CTL raised against the parent peptide did not recognize the succinimide derivative of this peptide. Naturally processed forms of these structures are likely to occur given the importance and frequency of deamidation both in vitro and in vivo. Moreover, since succinimide intermediates of deamidated peptides can occasionally be very stable, these peptides have the potential to act as altered self-Ags with significant implications for autoimmunity. In addition, unwanted and potentially hazardous specificities may be elicited when using synthetic peptides in subunit vaccines in which succinimide residues may form spontaneously during storage or chemical synthesis.

Amino Acid Sequence↗

PubMed1996

Perfect wound healing in the keratin 8 deficient mouse embryo.

It is generally believed that the strength and structural integrity of both adult and embryonic epithelia comes, at least in part, from their internal cytoskeletal network of keratin filaments and associated cell:cell junctions. Indeed, recent keratin depletion experiments in Xenopus suggest that the capacity of embryonic epithelia to undergo natural morphogenetic movements such as gastrulation, or artificially triggered epithelial movements such as wound closure, are severely compromised in the absence of the predominant embryonic keratin, K8 [Torpey et al., 1992: Nature 357:413-415; Klymkowsky et al., 1992: Proc. Natl. Acad. Sci. USA 89:8736-8740]. These experiments contrast with studies of genetically K8 deficient mouse embryos which undergo gastrulation quite normally and, dependent upon background strain, can survive until beyond birth [Baribault et al., 1993: Genes Dev. 7:1191-1202; Baribault et al., 1994: Genes Dev. 8:2964-2973], but to date no wound healing investigations have been carried out on mK8-mice. In this article, we report our studies of healing in embryonic day 11.5 mouse embryos, wounded by amputation of the hindlimb bud and then cultured in roller bottles. In wild-type embryos, wound closure puts severe strain on the embryonic epidermis since it is under tension and gapes immediately upon wounding; subsequently, epithelial cells tug on one another by means of an actin purse-string in order to close the defect. Even given these extremely challenging conditions, we show here that the mK8- epidermis performs no differently from wild-type epidermis, assembling an actin purse-string in the wound marginal cells and closing the wound with identical timecourse to its wild-type counterpart.

Actins↗

PubMed1996

Nonprecipitating anti-La(SS-B) autoantibodies in primary Sjögren's syndrome.

Anti-La(SS-B) precipitin-negative sera show a restricted epitope recognition and can be easily overlooked in routine laboratory testing. We have therefore determined the prevalence of nonprecipitating anti-La(SS-B) antibodies in patients with primary Sjögren's syndrome and studied their clinical and immunological associations. Clinical details were obtained from 68 patients with primary Sjögren's syndrome, and serum samples were examined by enzyme-linked immunosorbent assay using purified recombinant La, 60-kDa Ro, and 52-kDa Ro proteins and by counterimmunoelectrophoresis. Thirteen patients (19%) were identified with anti-La antibodies which were nonprecipitating. These patients had similar clinical findings to other groups of patients with Sjogren's syndrome, but had significantly lower rheumatoid factor and serum IgG levels than patients with anti-La precipitins. None of the patients with nonprecipitating anti-La antibodies had previously contained anti-La precipitins in their sera. Furthermore, they tended to have lower levels of antibodies directed against denatured 60-kDa Ro (but not 52-kDa Ro) compared with anti-La precipitin-positive patients. Patients with Sjögren's syndrome associated with nonprecipitating anti-La antibodies represent a stable serological and clinical subset in which there appears to be limited diversification of the autoimmune response to the Ro60 and La proteins of the Ro/La ribonucleoprotein.

Antibodies, Antinuclear↗

PubMed1996

Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

Allergens↗

PubMed1996

Structural differences between the human and mouse 52-kD Ro autoantigens associated with poorly conserved autoantibody activity across species.

Anti-nuclear autoantibodies found in human autoimmune diseases frequently cross-react with homologous autoantigens in distant species, supporting the notion that autoantibodies target conserved functional domains. However, the 52-kD Ro(SS-A) protein is an exception, in that human autoantibodies are not known to recognize any equivalent antigen in the cells of rodents and other non-primate species. To understand this lack of cross-reactivity we have isolated cDNAs encoding the mouse 52-kD Ro molecule. The cDNA encoding mouse 52-kD Ro revealed an open reading frame of 470 amino acids, with 70% sequence identity to the human 52-kD Ro antigen. The putative leucine-zipper and zinc-finger motifs present in human Ro52 were conserved in the mouse protein. Recombinant mouse 52-kD Ro protein reacted with human autoantibodies by ELISA and immunoblot, but with approximately 10-fold lower reactivity than recombinant human 52-kD Ro protein under the same conditions. Detection of both human and mouse 52-kD Ro by immunoblot was dependent on antigen concentration which was limiting in the cell equivalents generally used in immunoblot assays. Differential chaotropic disruption of antibody binding suggested a lower avidity of human autoantibody binding to the mouse 52-kD Ro protein compared with the human antigen. Thus the poor reactivity of native mouse 52-kD Ro with human autoantibodies is associated with species divergence diffusely distributed throughout the primary structure of the 52-kD Ro molecule.

Amino Acid Sequence↗

PubMed1996

Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants.

Anti-Ro (SS-A) antibodies are important diagnostic markers for primary Sjögren's syndrome and systemic lupus erythematosus, but their detection by indirect immunofluorescence (IF) in the diagnostic laboratory is hindered by the low cellular abundance of 60kDa Ro protein (Ro60). The approach we used to overcome this problem was to transfect and over-express the Ro60 gene into HEp-2 cells. In this study we have used a mixture of Ro60 transfectants and untransfected HEp-2 cells (HEp-Ro60) as a substrate for IF-antinuclear antibody (ANA) testing in a hospital laboratory. Screening of 240 routine serum specimens identified 14 Ro transfectant-positive sera which were confirmed by counterimmunoelectrophoresis (CIE); 3 of these sera were ANA-negative on untransfected cells and regular HEp-2. A comparison of HEp-Ro60 and regular HEp-2 showed strong concordance of the different ANA patterns between the 2 substrates. No increase in background staining was observed on the Ro transfectants when reacted with normal human sera. A comparison between HEp-Ro60 and CIE for 53 sera from patients with primary Sjögren's syndrome showed that HEp-Ro60 were a sensitive and specific substrate for detection of anti-Ro antibodies. Masking of positive Ro transfectants was observed rarely in sera containing multiple ANA specificities, but the Ro60 staining on these transfectants were unmasked at higher serum dilutions. We conclude that HEp-Ro60 are a suitable substrate for IF-ANA in the routine laboratory and that they have the additional advantage over regular HEp-2 slides of being able to detect anti-Ro in ANA-negative sera. HEp-RO60 are also a valuable confirmatory test for sera giving equivocal precipitin reactions or ELISA results.

Adult↗

PubMed1996

Stable inheritance of an HLA-"blank" phenotype associated with a structural mutation in the HLA-A*0301 gene.

A serological family study identified an HLA-A "blank" segregating through three generations of apparently healthy individuals. The HLA-A*0301 allele was assigned by DNA genotyping in each of the three individuals. Complete absence of cellular expression of the HLA-A3 antigen was associated with a 6 nucleotide deletion in exon 3 of the A*0301 gene. The in-frame deletion of nucleotides 373-378 results in the absence of residues C101 and D102 from the mature HLA-A heavy chain. Cysteine 101 is involved in the formation of the highly conserved disulfide bridge in the alpha 2 domain of the class I molecule, and deletion of this residue is believed to be highly disruptive to proper folding and function of the class I molecule.

Base Sequence↗

PubMed1995

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

3T3 Cells↗

PubMed1995

Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La (SS-B) and Ro (SS-A).

We have tested the extent of immune self-tolerance to the ubiquitously expressed nuclear/cytoplasmic autoantigens La (SS-B) and Ro (SS-A) in healthy, nonautoimmune mice. Immunization of mice with recombinant mouse La resulted in a specific, isotype-switched autoantibody response, which was initially directed toward the La C subfragment (aa 111-242) but rapidly spread to involve the La A (aa 1-107) and La F (aa 243-345) regions of the La antigen. Intramolecular spreading of the anti-La antibody response was further demonstrated by the appearance of autoantibodies to multiple, nonoverlapping antigenic regions of La, after immunization of mice with the 107-aa La A subfragment. Moreover, immunization of mice with recombinant mouse or human La also elicited specific anti-60-kDa Ro IgG antibodies in all strains tested. Mice immunized with 60-kDa Ro produced a high titer anti-Ro antibody response, which was also associated with intermolecular spreading, resulting in the specific appearance of anti-La autoantibodies. These findings show that the development of autoantibodies to multiple components of the La/Ro ribonucleoprotein complex may follow initiation of immunity to a single component. In addition, the data reveal the incomplete nature of immune tolerance to La and Ro despite their endogenous expression in all nucleated cells. These observations are likely to account for the coexistence of anti-La/Ro antibodies in autoimmune disease and suggest a general explanation for the appearance of mixed autoantibody patterns in systemic autoimmune disorders.

Animals↗

PubMed1995

Analysis of the tissue movements of embryonic wound healing--DiI studies in the limb bud stage mouse embryo.

The tissue movements of epithelial spreading and mesenchymal contraction play key roles in many aspects of embryonic morphogenesis. One way of studying these movements in a controlled manner is to make an excisional skin wound to an embryo and watch the wound heal. In this paper we report our studies of healing of a simple excisional lesion made to the limb bud stage mouse embryo. The wounded, living embryo is cultured in a roller bottle; under such conditions the wound heals with a highly reproducible time course and is completely closed by 24 hr. During the healing period the environment bathing the wound can be simply manipulated by adding drugs or factors to the culture medium. We have used DiI to label mesenchymal cells exposed at the margin of the initial wound and, by following their fate and measuring the area of mesenchyme remaining exposed at various time points during the healing process, we have quantified both the extent of mesenchymal contraction and the extent of reepithelialisation by movement of epidermis over mesenchyme. We show that the two types of tissue movement contribute almost equally (50:50) to the total wound closure rate. We have gone on to investigate the cell machinery underlying these processes. In adult wounds the epidermis migrates by means of lamellipodial crawling, but we show that reepithelialisation in the embryo is achieved instead by purse-string contraction of a cable of filamentous actin which assembles in the basal layer of cells at the free edge of the epidermis. Addition of cytochalasin D to the culture medium blocks formation of this actin cable and leads to failure of reepithelialisation. Contraction of adult wound connective tissue appears to be driven by conversion of dermal fibroblasts into a specialist smooth muscle-like fibroblast, the myofibroblast. However, using an antibody recognising the alpha-isoform of smooth muscle actin and specific for smooth muscle cells and myofibroblasts, we show that a similar conversion into myofibroblasts does not occur at any stage during the embryonic wound healing process. These observations indicate that both of the tissue movements of embryonic wound healing utilise cell machinery fundamentally different from that driving the analogous tissue movements of adult healing.

Actins↗

PubMed1995

Cytoplasmic accumulation of the 52 kDa Ro/SS-A nuclear autoantigen in transfected cell lines.

The 60 kDa Ro/SS-A (Ro60) autoantigen is thought to reside predominantly in the nucleus; however, the intracellular localization of 52 kDa Ro/SS-A (Ro52) in normal cells is controversial, probably due to its low abundance. Therefore, we studied the intracellular expression and localization of the human Ro52 following transfection of a human Ro52 cDNA into cultured cell lines. Immunofluorescence staining of human (HEp-2) and mouse (LTA-5) cell transfectants with affinity-purified anti-Ro52 antibodies revealed that Ro52 antigen was most abundant in the cytoplasm and present to a lesser extent in the nucleus. This relative localization was supported by a preponderance of the Ro52 antigen in the cytoplasmic rather than nuclear fraction of enucleated cell lines detected by immunoblotting. In contrast to the Ro52 autoantigen, Ro60 and La autoantigens were mainly expressed in the nucleus of transfected cells under similar circumstances, indicating distinct localization of the intracellular pools of these autoantigens. The findings indicate that the Ro52 autoantigen lacks intrinsic signals required for nuclear localization and suggest that a significant pool of this autoantigen resides in the cytoplasm. Ro52 may therefore rely upon an association with other molecules for any specific nuclear transport.

Animals↗

PubMed1995

Class II cytoplasmic and transmembrane domains are not required for class II-mediated B cell spreading.

B cells cultured on immobilized anti-class II monoclonal antibody (mAb) change from round to flattened cells, with lamellipodia and filopodia. This change in cell morphology, termed 'spiders', occurs within 30 min upon culture and is mediated through either I-A or I-E molecules. Class II molecules that are defective in mediating protein kinase C (PKC) due to the deletions of both alpha and beta chain's cytoplasmic (Cy) domain sequences can induce spider formation. B-cell transfectants that express chimeric MHC class II/class I molecules, where the ectodomains are class II sequences and the transmembrane and Cy domains are class I sequences also form spiders when cultured on anti-class II mAb. The spider morphology is not induced by either anti-immunoglobulin (Ig) or anti-MHC class I mAb. Treatment of B cells to increase intracellular cAMP, a component of the class II signaling pathway also results in spider formation with the same kinetics and percent change in the responding population as that induced by anti-class II mAb. Cytochalasin A treatment which disrupts cytoskeletal actin filaments and the tyrosine kinase inhibitor, genistein, both inhibit spider formation. Actin redistributes from a concentric ring in round cells to the ends of the filopodia in the spiders. The mechanism of spider induction whether resultant from second messengers following class II signaling or from non-signaling-induced physical interactions of class II with intracellular cytoskeletal components only requires the extracellular domains of class II. The biologic relevance of B-cell spiders is currently not known but has been reported to be associated with class II signal transduction and efficient Ag presentation.

Actins↗

PubMed1995

Evolution in HLA-DRB1 and major histocompatibility complex class II haplotypes of Australian aborigines. Definition of a new DRB1 allele and distribution of DRB1 gene frequencies.

The distribution of HLA-DRB1 alleles was studied in Australian aborigines from different parts of Australia. There were significant differences in the frequencies of DRB1*0412, 1409, and 1410 between the Central Desert and Yuendumu populations and the previously reported Cape York and Kimberley aboriginal populations. A new DRB1 allele, DRB1*1414, present at low frequency in the Central Desert population, was identified. DRB1*1414 appears to be closely related to DRB1*1407 and is proposed to have arisen by intragenic recombination. A novel DR-DQ haplotype, DRB1*1402-DRB3*0101-DQA1*0501-DQB1*0402, was also identified. This haplotype may be ancestral to the DRB1*1409-DQB1*0402 haplotype present in these populations. The presence of alleles and haplotypes apparently confined to Australian aboriginal populations and differences in the distribution of these alleles in different populations suggests that evolution has occurred in the class II region in the period since colonization of Australia, an estimated 50,000 years ago.

Amino Acid Sequence↗

PubMed1995

T cell response to grass pollen allergens: correlation with skin test reactivity and serum IgE levels.

T cell proliferative responses to rye and Bermuda grass pollen allergens have been studied in a series of 51 atopic and 18 non-atopic subjects. Mean T cell responses were higher in the atopic group than in the non-atopic group (P < 0.001), and there was a strong correlation between the magnitude of reaction in the T cell assay and in the skin test (rye P < 0.01, Bermuda P < 0.05). A similar association was shown between T cell reactivity and serum levels of allergen-specific IgE (rye P < 0.05, Bermuda P < 0.05), but no relationship was found between serum allergen-specific IgG levels and any other parameter studied. T cell reactivity was not found in three cord blood samples tested. Discordance between positivity for T cell responses and skin test reactions in some cases might reflect reactivity by T cell subsets that promote IgG antibody or cell-mediated responses without IgE antibody production. A precise knowledge of T cell recognition of grass pollen allergens will provide exciting new prospects for more effective and safer immunotherapy strategies for allergic diseases including asthma.

Adult↗

PubMed1995

MHC proteins and heparan sulphate proteoglycans regulate murine cytomegalovirus infection.

Factors influencing MCMV infection mediated by MHC class 1 molecules were analysed further as previous studies showed that the effects of the MHC genotype on sensitivity to this virus are important in vivo. Here we show that H-2d, H-2b, H-2r and H-2v macrophages are highly sensitive to MCMV. Moreover, transfection of H-2k L-cells with Kb or Dd conferred sensitivity to MCMV. This was not affected by amino acid substitutions in Kb alpha 1 or alpha 2, although previous studies demonstrated that exchange of the alpha 1 domain of Dd with Ld alpha 1 compromised sensitivity. Here replacement of Kb alpha 3 with Ld alpha 3 reduced susceptibility to low doses of MCMV. In addition, extracellular beta 2-microglobulin (beta 2m) promoted infection of beta 2m-negative RIE/TL8X.1 cells transfected with Db with or without a beta 2m gene. Hence MCMV infection can involve beta 2m and the alpha 1 and alpha 3 domains of MHC heavy chains. MCMV infection of L-cells expressing Dd or Kb was also inhibited by heparin, but infection of the parental L-cell line was not reproducibly affected. A role for heparan sulphate proteoglycan in MHC-mediated MCMV infection was confirmed using cells pre-treated with heparinase I or III, or propagated in chlorate to inhibit the sulphation of the glycosaminoglycan chains.

Animals↗

PubMed1995

Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle.

In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111-242) containing the RNA recognition motif (RRM, aa 111-187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.

Autoantibodies↗