10.1Introduction

Over the past several years, different laboratories have tried to deÞne the Þne speciÞcity of

A.G. Tzioufas ( )

Department of Pathophysiology, School of Medicine, University of Athens, Athens, Greece

e-mail: agtzi@med.uoa.gr

autoantibodies to intracellular antigens by identifying within the antigen moiety the antigenic determinants (or B-cell epitopes), preferentially recognized by autoantibodies. Characterization of B-cell epitopes might give useful information on putative mechanisms of autoantibody production, such as molecular mimicry (sequence or structural molecular similarity that leads to crossreactivity between antigens from a foreign agent

and self-proteins) and epitope spreading (expansion of a B-cell autoreactive clone from a single determinant to other sites on the autoantigen) [1]. Furthermore, deÞnition of B-cell epitopes with high sensitivity and speciÞcity may allow development of immunoassays based on single epitopes (usually produced as synthetic peptides) that can be utilized for detection of autoantibodies. When used as antigenic substrates in diagnostic assays, synthetic peptides have several advantages over recombinant antigens. In contrast to in vivo production of recombinant proteins, peptide synthesis is a controlled chemical process that leads to high purity and homogenous, stable antigen preparations, allowing generation of highly sensitive and speciÞc assays [2]. Such laboratory test systems can be useful for deÞning subgroups of a disease and may offer important information on the prognosis and natural course of the disease [3]. Furthermore, clinically relevant peptides, corresponding to B-cell epitopes, have been proposed as potentially useful in the treatment of autoimmune diseases via the use of immobilized peptides to remove pathogenic autoantibodies or as vaccine components [4, 5].

The identiÞcation of B-cell epitopes of autoantigens has raised an array of questions. Do autoimmune epitopes constitute a few dominant sequences or do they represent multiple disparate regions on a single autoantigen? Are

frequency and signiÞcance of epitope spreading important for maintenance and perpetuation of the autoimmune response? Are the molecular structures of epitopes suitable to provide support for the hypothesis of epitope mimicry as a trigger for autoimmunity? Does knowledge of epitopes provide information on the regulation of the autoimmune response? In this review, we will discuss the clinical and biological relevance of epitopes to Ro/SSA and La/SSB, major autoantigens in SjšgrenÕs syndrome (SS).

10.2Classification of B-cell Epitopes of Intracellular Autoantigens

The B-cell epitopes are classiÞed according to their structure as follows (Fig. 10.1):

1.Primary structure epitopes (identified also as linear epitopes) are composed of sequential amino acids. Such epitopes have been identiÞed by synthetic peptide mapping in the majority of autoantigens discussed here, including Ro60, Ro52, La, Sm B, Sm D, RNP70, and Scl-70.

2.Secondary structure epitopes are formed by amino acids distributed in simple threedimensional structures, such as α(alpha)-

stretch with all amino acids relevant for antibody binding located on one side of the helix. Other secondary structure epitopes have been described in Ro52 and Ro60 leucine zipper and zinc Þnger motifs, in patients with neonatal lupus and primary SS, respectively.

3.Tertiary structure epitopes are formed by distant regions of the protein sequence coming together in the tertiary structure. Such conformational epitopes may be the main targets of some autoantibodies (e.g., anti-Ro60).

4.Quaternary structure epitopes consist of amino acids distributed over different subunits within a macromolecular complex, interacting transiently or permanently to form a structure recognized by the autoantibody. Such epitopes have been identiÞed in the Ro/La RNP complexes as well as in nucleosome subunits composed of histones and DNA elements.

5.Cryptic epitopes (cryptotopes) are usually linear epitopes hidden in the native structure of the autoantigen. They become accessible to antibody binding after disruption of the three-dimensional structure by denaturation, proteolytic degradation, or chemical modiÞcation of the autoantigen. These epitopes are observed in a number of nuclear autoantigens.

6.Modified epitopes (neoepitopes) contain posttranslationally modiÞed amino acid residues

[6].Examples of these modiÞcations include

(1)serine, threonine, and tyrosine phosphorylation; (2) lysine acetylation or ubiquitination; (3) cysteine lipidation or oxidation (disulÞde-bond formation); (4) glutamic acid methylation or gamma-carboxylation; (5) glutamine deamidation; (6) asparagine (N-linked) and serine/threonine (O-linked) glycosylation; (7) arginine citrullination or symmetric/asymmetric dimethylation; and (8) proteolytic cleavage or degradation. In some instances, side chain modiÞcations of speciÞc amino acids, such as citrullination of arginine residues, are responsible for epitope highafÞnity binding [7]. Such modiÞed amino acids have been reported in a variety of human nuclear proteins, including the Sm antigens D1 and D3 [8], Þbrillarin [9], and nucleolin

10.2.1 Ro/La RNP Particles

In patients with systemic lupus erythematosus (SLE) and SS, the Ro/La RNP is one of the main targets of autoantibodies (Fig. 10.1). Human Ro/La RNP is composed of one of the four small, uridine-rich hY RNAs (human cYtoplasmic RNAs) non-covalently associated with at least three proteins, the Ro52, La, and Ro60 autoantigens [11, 12]. Additional components of the complex have been identiÞed recently as the proteins calreticulin [13] and nucleolin [14]. The localization of these complexes is exclusively cytoplasmic but their protein components can be found in the nucleus as well.

10.2.2 La/SSB

The La/SSB antigen is a 47-kDa phosphoprotein that associates with a variety of small RNAs, including 5S cellular RNA and tRNA, 7S RNA, and hY RNAs, all transcribed by RNA polymerase III. In molecular level, it binds a short poly-uridylate sequence (poly-U) that exists at the 3 end of almost all nascent pol III transcripts (and in mature hY RNAs) [15Ð17]. In this regard La mediates transcript release from RNA polymerase III and facilitates multiple rounds of transcription/reinitiation by RNA polymerase III. In addition to immature RNA polymerase III products and hY RNAs, La can bind to viral RNAs (e.g., adenovirus VA, EpsteinÐBarr encoded RNAs (EBER)) and the RNA component of telomerase complex [18, 19]. La/SSB autoantigen has additional cellular functions: (1) La is an essential factor for cap-independent translation (La binds to internal ribosomal entry site (IRES) elements of the 5 -untranslated region of viral or human mRNAs promoting their capindependent translation at the correct AUG) [20]; (2) La acts as an adenosine triphosphate (ATP)-dependent helicase able to melt RNAÐ DNA hybrids [21]; (3) La unwinds doublestranded RNA inhibiting the double-stranded RNA-dependent activation of protein kinase PKP [22]; (4) La can associate with telomerase and inßuence telomere homeostasis in vivo [18]; and

(5) La is an RNA chaperone capable of transient

bipartite (5 -end and 3 -end) binding of nascent RNA transcripts (e.g., tRNA precursors) [23].

Structurally, human La is a multidomain protein that contains the La motif in its N-terminal region, a typical RNA recognition motif (RRM) in its central part, and an unusual RRM, encompassing residues 229Ð326 (RRM_3). The latter is followed by a long, ßexible polypeptide that contains a short basic motif (SBM), a regulatory phosphorylation site on Ser366, and a nuclear localization signal (NLS). Recently, the three-dimensional structure of the La motif, the central RRM, and the carboxyl-terminal RNA recognition domain of the autoantigen was solved [24, 25] (Fig. 10.2). The La motif folds into a winged-helix motif elaborated by the insertion of three helices. The central RRM consists of a four-strand β(beta)-sheet attached to

Fig. 10.2 Modeled structure of the human La region 5Ð 326aa in complex with part of the hY1-RNA (shown in yellow ribbons), containing the La motif, the central RRM, and carboxyl-terminal RRM (RRM_3). The epitopes 145Ð164 and 289Ð308 are represented with green and blue wire surface

two α(alpha)-helices, while the C-terminal RRM folds to generate a Þve-stranded, anti-parallel β(beta)-sheet surface that is terminated by a long α(alpha)-helix. It seems that both the La motif and the adjacent central RRM are required for high-afÞnity poly-U RNA binding and that the C-terminal RRM, in conjunction with the SBM downstream, contributes to La interactions with non-poly-U RNA targets such as viral RNAs and TOP (terminal oligopyrimidine) mRNAs [24, 26].

During the last decade, the target epitopes of anti-La/SSB autoantibodies have been mapped (Fig. 10.3). Early efforts to identify epitopes on the La antigen began by using enzymatic digestion of the native protein and large recombinant fragments. In this regard, antigenic sites covering the larger part of La autoantigen were identiÞed. These sites were called LaA (amino acids 1Ð107, 1Ð107aa), LaC (111Ð242aa), and LaL2/3 (346Ð408aa) [27]. Later, more detailed and analytical epitope mapping revealed the exact localization of its antigenic determinants. Some of the La epitopes were found to reside in functional regions of the autoantigen, like the RNA recognition motif (RRM) and the ATP-binding site [28Ð30]. The interaction of hY RNA with the RRM motif, however, did not affect autoantibody binding in the same region [28]. In contrast, the interaction of the ATP-binding site with ATP abolishes autoantibody binding at the same part of the protein [29]. B-cell epitope mapping of La/SSB was also performed in our laboratory using 20-mer synthetic peptides overlapping

Fig. 10.3. Schematic representation of autoepitopes of the La48 kDa protein. a. [27, 30]. b. [28]. c. [93]. d. [29]. e. [94]. f. [31]. g. [34, 35] (apotopes). Domain

organization: LA = La motif (N-terminal RRM), RRM: RNA = recognition motif (central RRM), RRM_3: RNA recognition motif (N-terminal RRM)