Heterologous expression and epitope mapping of a human small nuclear ribonucleoprotein-associated Sm-B'/B autoantigen.

The U snRNP associated B'/B polypeptides are primary targets of Sm autoantibodies in patients with systemic lupus erythematosus. We have bacterially expressed a Sm-B'/B autoantigen from Raji cells as a fusion with the anthranilate synthase protein from Escherichia coli. The recombinant Sm-B'/B fusion displays comparable immunologic reactivity to the native protein when tested with both monoclonal and polyclonal antibodies. To map Sm-B'/B epitopes, we constructed a series of 12 anthranilate synthase fusions spanning different regions of Sm-B'/B and tested such fusions on immunoblots against a panel of characterized sera. In this manner, we have identified six epitopes, five of which overlap the proline-rich carboxyl-terminus of the protein. Some of these epitopes appear to be conformational. The human sera tested can be divided, according to the epitopes they recognize, into six groups. Finally, we have shown that anti-Sm recognition of the (U1)RNP-specific A protein is attributable to cross-reactivity between the Sm-B'/B and A autoantigens.     

Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis.

Immunization with recombinant human pyruvate dehydrogenase (PDH)-E2, the major autoantigen of primary biliary cirrhosis, readily induces a vigorous murine antibody response but does not generate hepatic disease. To determine the fine specificity of this response, 18 mAb were generated from three strains of mice and the reactive epitopes mapped. An initial examination of mAb suggested that they behaved similarly to the antimitochondrial autoantibodies in primary biliary cirrhosis (PBC) because i) all polyclonal antisera and 2 of 18 mAb reacted with all species of mammalian PDH-E2 examined including mouse PDH-E2, ii) 15 of 18 mAb inhibited PDH enzyme function, and iii) the reactivity of mAb toward rPDH-E2 were blocked by PBC sera. However, fine examination of the reactive sequences of the PDH-E2 complex revealed that antibodies identical to those in PBC patients were not produced by experimental immunization. In contrast to PBC, none of the mAb or murine polyclonal sera were able to react with protein X, a lipoic acid-containing component of the PDH complex previously shown to cross-react with PDH-E2 when probed with PBC sera. Although the epitopes for 12 mAb were localized within the inner lipoyl domain, none reacted with mouse PDH-E2 or cross-reacted with the outer lipoyl domain as observed in PBC. In addition, the epitopes of the two mAb which did react with all mammalian species of mitochondria were not localized within the PBC epitope. These findings indicate the highly immunogenic nature of the inner lipoyl domain of PDH-E2. The inability to elicit antibodies of the same specificity in mice, considered together with the highly localized autoantibody response in humans, suggests that antimitochondrial autoantibodies are most likely the result of specific breakdown of tolerance to a unique autoepitope.     

The nuclear autoantigen La/SS-B: mapping and sequencing of the gene and the three retropseudogenes

One target of autoantibodies in sera of patients with systemic lupus erythematosus or primary Sjögren's syndrome is the nuclear autoantigen La/SS-B. Lambda clones and cosmids were isolated, which contained the sequences of the La gene and the three La pseudogenes. They were used for preparation of a physical map. Finally, the La gene and pseudogenes were sequenced. The pseudogenes were characterized as retropseudogenes. Their evolutionary ages were estimated to be approx. 4, 4.5 and 5 million years. Inserts of 4, 16 and 24 nucleotides, which were mostly A-residues, were found in exon 7 of the respective pseudogene. The oldest pseudogene contained the longest insert, the youngest pseudogene contained the smallest insert. The oligonucleotides seem to be the result of repeated inserts of A-residues in a hot spot region of the La genes. Two La cDNAs were isolated which contained either a deletion or an insert of an A-residue at the same position.     

The autoantigen La/SSB is a calmodulinmbinding protein

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca²⁺-dependent way. [¹²⁵I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca²⁺ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.     

Mutational mapping of a cloned adenovirus origin

We have developed a standardized, quantitative assay to study the function of a cloned adenovirus origin. We have shown that the adenovirus origin is located within the first 20 bp of the adenovirus inverted terminal repetition (ITR), a region containing a sequence conserved among human, simian, murine, and avian adenoviruses. Deletions removing or penetrating from either direction into the conserved sequence inactivated the cloned adenovirus origin. A point mutation within the conserved sequence impaired the adenovirus origin, but point mutations outside the conserved sequence had no effect. These results strongly suggest that the conserved sequence within the first 20 bp of the ITR alone constitutes the adenovirus origin (ori) signal.     

La autoantigen enhances translation of BiP mRNA

Translational initiation of the human BiP mRNA is directed by an internal ribosomal entry site (IRES) located in the 5′-untranslated region (5′-UTR). In order to understand the mechanism of the IRES-dependent translation of BiP mRNA, cellular proteins interacting with the BiP IRES were investigated. La autoantigen, which augments the translation of polioviral mRNA and hepatitis C viral mRNA, bound specifically to the second half of the 5′-UTR of the BiP IRES and enhanced translation of BiP mRNA in both in vitro and in vivo assays. This finding suggests that cellular and viral IRESs containing very different RNA sequences may share a common mechanism of translation.     

Methods of epitope mapping

Concluding remarks It is important to remember that the merits of the different approaches to epitope mapping should be judged against the purpose of the study. A peptide specifically recognized by nearly all sera containing a certain autoimmune specificity [20, 21, 26], would most likely be selected for the detection of the anti-linear/continuous epitope fraction among those autoantibodies and could be highly useful for diagnostic purposes. This is true even if a majority of the antibodies were directed against conformational/discontinuous protein epitopes. If the purpose is to study the induction or maintenance of the autoimmune response, one would also have to look at and account for the conformation-dependent autoantibodies. There is also the possibility that some pathogenic autoantibodies could constitute a small fraction requiring the complete nucleic acid-protein complex as an antigen. In that case, the pathogenic epitope would not be identified nor mapped using the techniques discussed in this review.     

Mapping of epitopes on the La(SS-B) autoantigen of primary Sj?gren's syndrome: identification of a cross-reactive epitope.

Autoepitopes on the ribonucleoprotein La(SS-B) were identified by using recombinant La(SS-B) polypeptides and sera from 166 patients with the antinuclear autoantibody anti-La(SS-B). The La(SS-B) polypeptides were encoded by polymerase chain reaction-derived overlapping or nonoverlapping fragments of the La(SS-B) gene, which encodes a protein of 408 amino acids (aa). Of the 166 sera tested, 99% reacted with a fusion protein comprising the first 107 N-terminal aa (LaA); 91% reacted with a fusion protein comprising aa 111 to 242 (LaC), and 91% reacted with a fusion protein comprising aa 346 to 408 (LaL2/3) at the C terminus of La(SS-B). The order of immunodominance as assessed by the number of sera reacting with each epitope and the strength of the reactivity was LaA (aa 1 to 107) greater than LaC (aa) 111 to 242) much greater than LaL2/3 (aa 346 to 408). Cross-reactivity was observed between antibodies eluted from LaC (aa 111 to 242) and LaL2/3 (aa 346 to 408), but there was no significant primary sequence homology between the two regions. The LaC region contained at least two epitopes, one encompassing a putative RNA-binding motif (aa 112 to 187) which was recognized by 83% of patient sera. Serial serum samples from three patients showed that the antibody response to La(SS-B) was initially directed to the N terminus (LaA, aa 1 to 107), but over a period of time all three major epitopes, including that encompassing the putative RNA-binding motif, were recognized. This result suggests that the primary immune response to La(SS-B) is restricted to an immunodominant epitope. As the specificity of the autoantibody response broadens, it includes the RNA-binding motif, which may have important implications for the expression of disease.     

The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif.     

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.     

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.     

Regional mapping of six cloned DNA sequences on human chromosome 7.

The regional localization of six cloned DNA sequences on human chromosome 7 was assessed by molecular hybridization to human/rodent cell hybrid DNAs. The allelic distribution and familial segregation of two frequent polymorphisms revealed by two probes are presented.     

Preparation of human recombinant kinesin heavy chain and epitope mapping of its structural domains

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.     

Isolation and subregional mapping of an arbitrary cloned probe detecting a common RFLP on human chromosome 2.

In the course of studies on restriction fragment length polymorphisms (RFLPs) being conducted in our laboratory, several single-copy cloned probes have been generated from specific human chromosomes using murine-human hybrid cell libraries. The following describes the isolation and subregional localization of an arbitrary single-copy cloned probe for human chromosome 2. This probe, designated pXG-18, has detected a common TaqI polymorphism in addition to two other RFLPs using the restriction enzymes MspI and HindIII. This sequence maps to the interval q32-q36 of chromosome 2, a region of the human genome to which very few markers have been assigned.