Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies

Clinical Immunology 110 (2004) 30 – 44 www.elsevier.com/locate/yclim

Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies Laura M. Stinton, a Theophany Eystathioy, a Sanja Selak, b Edward K.L. Chan, c and Marvin J. Fritzler a,* a

Faculty of Medicine, University of Calgary, Calgary, AB, Canada b Department of Neural Plasticity, Cajal Institute, Madrid, Spain c Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL 32610-04024, USA Received 4 September 2003; accepted with revision 20 October 2003

Abstract Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and this was quickly followed by the introduction of the LE cell test and indirect immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical laboratories. Recently, attention has turned to the identification of the autoantigens that bind to cytoplasmic organelles such as the Golgi complex, endosomes and other ‘‘cytoplasmic somes’’. Three endosome autoantigens include early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170 (CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1 were seen in a variety of conditions but approximately 40% of the patients had a neurological disease. Despite the prominence of lysosomes in cells and tissues, reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130, and golgin-67. More recently, there has been an interest in autoantibodies that bind components of the ‘‘SMN complex’’ or the ‘‘assemblyosome’’. Arginine/glycine (RG)-rich domains in components of the SMN complex interact with Sm, like-Sm (LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are antigen targets in a variety of diseases. More recently, components of a novel cytoplasmic structure named GW bodies (GWBs) have been identified as targets of human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn) enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic processing of mRNAs. Autoantibodies to the ‘‘cytoplasmic somes’’ are relatively uncommon and serological tests to detect most of them are not widely available. D 2003 Elsevier Inc. All rights reserved. Keywords: Autoantibodies; Autoimmunity; Endosome; Proteasome; Lysosome; Review

Introduction Abbreviations: AGA, anti-Golgi antibodies; ANA, antinuclear antibody; cANCA, cytoplasmic antineutrophil cytoplasmic antibodies; CLIP, cytoplasmic linker protein; EEA1, early endosome antigen 1; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; GWBs, GW bodies; IIF, indirect immunofluorescence; HIV, human immunodeficiency virus; LBPA, lysobisphosphatidic acid; LE, lupus erythematosus; NMD, nonsense-mediated decay; PM, polymyositis; RG, arginine/glycine; SjS, Sjo¨gren’s syndrome; SLE, systemic lupus erythematosus; SMA, spinal muscular atrophy; SMN, survival of motor neuron; SRP, signal recognition particle; SSc, systemic sclerosis. * Corresponding author. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, Canada T2N 4N1. Fax: +1-403-283-5666. E-mail address: [email protected] (M.J. Fritzler). 1521-6616/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.10.005

Historically, autoantibodies directed to nuclear antigens (ANA) have been the focus of clinicians and scientists interested in systemic rheumatic diseases [1– 3]. In the past decade, however, considerably more attention has been given to cytoplasmic antigens. Among these are antibodies localized in mitochondria [4– 7], the Golgi complex [8,9], centrosomes [10 – 12], ribosomes [13 – 15], endosomes [16,17], lysosomes [18], proteasomes [19,20], exosomes [21,22], and GW bodies [23,24]. These cytoplasmic antigens have different subcellular localizations and diverse functions. Through the use of expression cloning, mass spectros-

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copy, and other technologies, the molecular identity of these autoantigens is being clarified and is rapidly expanding. Although the ANA test continues to be a screening test for patients with suspected autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjo¨gren’s syndrome (SjS), the ANA test is based on direct observer interpretation of indirect immunofluorescence staining patterns and there is a concern that many sera with antibodies to cytoplasmic antigens may be reported as ‘‘ANA negative’’ [25,26]. Therefore, clinicians who receive such a report may presume that the patient under investigation did not have autoantibodies to intracellular antigens at all. It is important to appreciate that autoantibodies to certain cytoplasmic antigens (i.e. mitochondria, lysosomes) hold as much diagnostic and prognostic significance as the timehonored ‘‘ANAs’’. The prevalence of ‘‘ANA-negative’’ rheumatic diseases ranges from up to 60% in RA, to 25% in SjS, 20% in SSc, and 5% in SLE [25, 26]. These numbers are based on conventional autoantibody assays using IIF performed on commercially prepared HEp-2 cells with parameters that are adjusted and performed to achieve an appropriate level of sensitivity and specificity. In an effort to enhance turnaround times and cost measures, some laboratories have adopted an ELISA that uses native or recombinant antigens coated on microtiter plates [27]. However, these kits are difficult to validate and there are growing concerns that the high rate of false negatives may preclude their use as a screening test [28 –30]. If other techniques such as immunoblotting [31; 32] or line assays [31,33,34] are used, it is apparent that many of the so-called ‘‘ANA negative’’ sera do indeed express autoantibodies that react with intracellular antigens including extracellular constituents, cell surface antigens, and the cytoplasmic antigens such as the Golgi complex, endosomes, lysosomes, proteasomes, assemblyosomes, and GW bodies. Although the precise frequency of autoantibodies to these cytoplasmic components has not been studied in detail, the Advanced Diagnostics Laboratory at the University of Calgary recently conducted a review of autoantibody specificity in sera received in a 6-month audit period (Table 1). Out of a total of 2724 sera referred by clinicians from a broad spectrum of specialties and tested for autoantibodies, 1102 (40.5%) showed a nuclear IIF staining pattern and 408 (15%) a cytoplasmic staining pattern on HEp-2 cells. The IIF titers of the cytoplasmic antibodies ranged from 1/160 to 1/5120. Further analysis showed that 0.1% of these were directed to the Golgi complex, 0.3% to endosomes, 2.7% to mitochondria, and 0.4% to GWBs. This is compared to a frequency of 3.4% for anti-dsDNA, 1.1% anti-Scl-70 (topoisomerase I), and 0.6% anticentromere proteins (CENPs). In a recent published study, 18 of 65 sera (28%) that displayed a vesicular cytoplasmic staining pattern obtained over an approximately 3-year period immunoprecipitated the recombinant EEA1 [17,35]. In this study the other sera had antibodies to ribosomal RNP [36] and cytoplasmic linker protein

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Table 1 Frequency of autoantibodies detected in the Advanced Diagnostics Laboratory during a 6-month time frame (January – June 2003) IIF staining pattern Nuclear Homogeneous Speckled

Specificitya

Frequency (%), n = 2724

chromatin dsDNA U1-RNP SS-A/Ro SS-B/La Sm matrixb Scl-70 PCNAb CENPb otherc

240 (8.8) 93 (3.4) 65 (2.4) 140 (5.1) 51 (1.9) 75 (2.8) 56 (2.1) 31 (1.1) 0 (0) 19 (0.6) 64 (2.4) 268 (9.8) 1102 (40.5)

Nucleolar Total nuclear Cytoplasmic Golgi Speckled/dots Ribosome Mitochondria Diffuse

endosomes/lysosomesb GW bodiesb Rib P PDC Jo-1 otherc Total cytoplasmic

3 (0.11) 7 (0.3) 11 (0.4) 38 (1.40) 74 (2.7) 6 (0.2) 224 (8.3) 402 (14.8)

a Specificity of autoantibodies determined by an addressable laser bead assay using a QuantaPLEXk ENA8 kit (INOVA, San Diego, CA) that includes chromatin, Jo-1 (histidyl tRNA synthetase), Ribosomal P protein, Scl-70 (topoisomerase I), Sm, SS-A/Ro, SS-B/La, and U1RNP. Antibodies to dsDNA were determined by IIF on a Crithidia luciliae assay (ImmunoConcepts Inc., Sacramento, CA). Antibodies to pyruvate dehydrogenase complex (PDC) were detected by Western blot of purified proteins (Sigma). b Antibodies detected by IIF pattern recognition and when required endosomes/lysosomes and GW bodies by colocalization with control antibodies. c Other autoantibody targets represented by a nuclear speckled staining pattern include coiled bodies (p80 coilin) [195], SP100 and PML [196], and others that remain unidentified. Autoantibodies that produce a diffuse pattern of cytoplasmic staining include p95c [197] and others that remain unidentified.

(CLIP170) [37] and other as yet unknown endosome or lysosome antigens. Thus although the observed frequency of antibodies to cytoplasmic antigens is approximately 40% of antinuclear antibodies, the frequency of some specific cytoplasmic autoantibodies (e.g. anti-GWB 0.4%) is approximately the frequency of other well-known nuclear antigens (e.g. CENPs 0.6%). In this audit period antibodies to proliferating cell nuclear antigen (PCNA) were not seen. This review will discuss endosomes, lysosomes, Golgi complex, the protein processing pathway involving the proteasome, and the RNA processing pathways in the cytoplasm such as those in assemblyosomes, exosomes, and GWBs. Autoantibodies to mitochondria, centrosomes, and primary granules (cANCA) will not be discussed in detail because they are the subject of recent comprehensive reviews [5,10,38]. It is important to appreciate that

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the ‘‘cytoplasmic somes’’ that are targets of the human autoimmune response are represented by diverse cellular morphology and structure (Fig. 1). Some of the ‘‘somes’’ are 50 –200 Am in diameter and are distinguishable as distinct cytoplasmic structures using appropriate staining and antibody markers by conventional microscopy while others are much smaller and not distinguishable a discrete entities.

Endosomes Endosomes are membrane-bound cytoplasmic vesicles that are involved in the transport of materials and macromolecules between the exterior of the cell and various compartments within the cell. By IIF, they appear as distinct cytoplasmic dots that are adjacent to the cell membrane, such as the unidentified autoantigen (Fig. 1A) that partially

Fig. 1. Indirect immunofluorescence patterns (green stain) produced by human autoantibodies to different cytoplasmic autoantigens. (A) Antibodies to an endosome/lysosome antigen. (B) Antibodies to GW182 and GW bodies. (C) Antibodies to CLIP-170. (D) Antibodies to early endosome antigen 1 (EEA1). (E) antibodies to golgin 160. (F) Antibodies to pericentrin in the centrosome. Green nuclear staining seen with the human anti-CLIP-170 (C) is an unrelated autoantibody. Nuclei are counterstained blue with DAPI.

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some vesicle fusion during endocytosis [41 – 44]. The Nand C-terminal zinc-binding domains interact with Rab5GTP [43,45] and phosphatidylinositol-3-phosphate (PI3P) [41,46] on adjacent vesicles thereby promoting membrane fusion. The C-terminal zinc finger, also termed the FYVE finger, is required for targeting of EEA1 to endosomes [39,47]. In addition to these motifs, EEA1 also contains a calmodulin-binding motif, protein kinase C and tyrosine kinase phosphorylation sites, a cAMP/cGMP-dependent protein kinase phosphorylation site, and a leucine zipper [16]. A methyl-accepting (MA) domain, also called chemotaxis sensory transducer signature (amino acids 558– 771), was also identified [35]. Autoantibodies to early endosome antigen 1 (EEA1) have been associated with neurological diseases, and a variety of systemic and organ-specific autoimmune diseases [16,17,35,48] (Table 2). Further study of the sera that reacted with EEA1 showed that 94% reacted with the partial length EEA1 constructs that included the C-terminal zinc finger (+FYVE) and the MA domain (LeuMA: amino acids

colocalizes with the endosome marker early endosome antigen 1 (EEA1). Although other autoantibodies, such as anti-GWB, produce a similar staining pattern (Fig. 1B), immunoelectron microscopy and colocalization studies using antibody markers have shown that these structures are not membrane-bound and do not associate with endosomes [23]. By comparison, antibodies to EEA1 are characterized by dots that are more widely dispersed throughout the cytoplasm of HEp-2 cells (Fig. 1D). Other endosome antigens, such as CLIP-170, appear as much smaller speckles throughout the cytoplasm (Fig. 1C). Early endosome antigen 1 (EEA1) Early endosome antigen 1 (EEA1) is a 162-kDa hydrophilic peripheral membrane protein that is located on the cytoplasmic face of early endosomes [16, 39]. EEA1 adopts a highly ordered quaternary structure composed of multiple a-helical coiled-coil motifs [16,40] (Table 2). In EEA1, the coiled-coil structures play a key role in promoting endo-

Table 2 Autoantibody targets in endosomes, lysosomes, proteasomes, exososomes, assemblyosomes, and GW bodies Cytoplasmic organelle autoantigen target

MW (kDa)

Function/molecular feature

Disease association

Reference

Assemblyosome Sm LSm4 Fibrillarin RNA helicase II (Gu) p80 coilin

12 – 34 14 34 100 80

mRNA processing mRNA processing rRNA processing mRNA processing rRNA processing

SLE SLE SSc SSc, SLE, UCTD SLE

[121] [124] [126] [128,129] [130,132]

Endosome Early endosome antigen 1 (EEA1) Cytoplasmic linker protein (CLIP-170) Lysobisphosphatidic acid

160 170 –

coiled coils coiled coils phospholipid

Neurological disease SLE APS

[16,40] [55] [58,59]

Exosome PM/Scl-75 PM/Scl-100

75 100

NLS coiled coil

PM/SSc overlap PM/SSc overlap

[138,143] [21,139,142]

Golgi complex Golgin-67 Golgin-95/gm130 Golgin-97 Golgin-160 Golgin-245 Macrogolgin/giantin

67 95/130 97 160 245 345

coiled coiled coiled coiled coiled coiled

SjS SjS, SLE, ataxia SjS SjS, SLE, ataxia SjS SjS

[8,9] [72]

GW bodies GW182

182

GW repeats, RRM, NLS

SjS, Neurological disease, SLE

[23,24]

Lysosome hLAMP-2 cANCA

170/80-110 PR3, other

membrane GP proteolytic enzymes

GN WG, vasculitis

[18] [38,198]

Proteasome a3-HC9

29.5

NLS

SLE, SjS, PM

[19,99,100]

coils, coils coils, coils coils, coils,

TMD GRIP GRIP TMD

Abbreviations: APS, antiphospholipid syndrome; GP, glycoprotein; GRIP, conserved domain present in golgins; GW, glycine (G) tryptophan (W); NLS, nuclear localization signal; PCD, paraneoplastic cerebellar degeneration; PM, polymyositis; RRM, RNA recognition motif; PVA, postvaricella ataxia; SjS, Sjo¨gren’s syndrome; SLE, systemic lupus erythematosus; snRNP, small nuclear ribonucleoproteins; TMD, trans-membrane domain; UCTD, undifferentiated connective tissue disease.

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82 –781) in an addressable laser bead assay [35]. A study of the epitopes bound by sera from patients with neurological diseases and patients with other conditions suggested that the latter sera from patients recognized epitopes in the central and C-terminal EEA1 domains, whereas the patients with neurological disease recognized a more restricted set of epitopes in the C-terminal domain [35].

suggest that there is no association between EEA1 and cardiolipin antibodies. Of possible relevance to the pathogenicity of these autoantibodies, it was shown that the antibody binding to LBPA perturbed both the structure and function of late endosomes [60]. The frequency of anti-LBPA in unselected or disease cohorts has not been thoroughly studied.

Cytoplasmic linker protein (CLIP-170) Lysosomes Cytoplasmic linker proteins (CLIPs) are a class of proteins that facilitate the interaction of endosomes and other cellular organelles with cytoplasmic microtubules [49 – 51]. CLIP-170 was localized to the plus ends of microtubules, a feature that is facilitated by its binding to newly polymerized tubulin, prometaphase kinetochores, and an activator of the microtubule-based motor, dynactin [51 – 54]. CLIP-170 has three functional domains: an N-terminal microtubule binding domain characterized by two ‘‘CAPGly’’ motifs, a long a-central helical coiled coil, and two ‘‘zinc-knuckles’’ in the C-terminal [49,51,55,56] (Table 2). The C-terminal domain is implicated in binding to kinetochores in prometaphase cells [54] and the second zincknuckle motif binds to the molecular motor dynactin [57]. The cytoplasmic linker protein-170 [37] was identified as an autoantigen when the serum from a patient with SLE and myositis was used to screen a HeLa cell expression library (Table 2). The human autoantibodies reacted with the purified recombinant protein in a Western immunoblot and immunoprecipitated the in vitro translated recombinant protein. Furthermore, antibodies affinity-purified with the recombinant CLIP-170 protein, the prototype human serum and a monoclonal antibody raised against CLIP-170 exhibited identical speckled staining of the cytoplasm in HEp-2 cells [37] (Fig. 1C). The clinical diagnoses in three patients were limited SSc, glioblastoma, and idiopathic pleural effusion. Although our studies suggest that antibodies to CLIP-170 are not common (Table 1), it is interesting that two patients in one study were considered to be ‘‘ANA negative’’ and the attending clinicians presumed that they did not have autoantibodies at all [37]. The frequency of antibodies to CLIP170 is not known in any disease group, but studies are underway to test sera from large cohorts of patients to derive more accurate frequency data for the major autoimmune diseases seen in clinical practice. Lysobisphosphatidic acid (LBPA) It has also been found that sera from some patients with antiphospholipid syndrome contained anti-LBPA antibodies [58]. LBPA is associated with late endosomes [59], whereas Rab 5 and EEA1 [16] localize to early endosomes, [59]. The sera of the patients with anti-EEA1 antibodies were tested by ELISA for the presence of anticardiolipin antibodies and were found to be negative (unpublished data). These results

Lysosomes are membranous organelles containing approximately 40 different hydrolytic enzymes that participate in the controlled breakdown of macromolecules. These enzymes include proteases, nucleases, lipases, glycosidases, phospholipases, phosphatases, and sulfatases, which they receive as vesicular packages that bud from the trans-Golgi network. The pH in the lumen of lysosomes is maintained at approximately 5.0 by an H+ pump in the lysosomal membrane. The lysosomal membrane transports the products of enzymatic digestion (amino acids, sugars, nucleotides) into the cytosol where they can be excreted or reutilized by the cell. Lysosomes are heterogeneous in size and shape, and their content is reflected by the components being degraded at any one time. Targeting of the hydrolytic enzymes to the lysosome occurs via the mannose 6-phosphate receptor, which binds to the mannose 6-phosphate groups present on the N-linked oligosaccharides of these enzymes. Lysosomes are found at the intersection of several organelle traffic pathways in the cell. The endocytic pathway is defined by molecules that are taken up by the cell and initially transported in early endosomes. Some of the molecules in early endosomes are retrieved and recycled, whereas others pass on to late endosomes or multivesicular bodies where acid hydrolysis begins in a mildly acidic (pH f6) lumen. Mature lysosomes are formed as the lumenal pH decreases even further, a feature that enhances complete hydrolysis of the contents. A second pathway that depends on lysosomes is autophagocytosis where obsolete components of the cell (i.e. worn out mitochondria, Golgi complex, cell membrane receptors) are ubiquitinated and disposed of through the formation of autophagosomes that eventually fuse with late endosomes to form lysosomes [61]. The third pathway is responsible for engulfing and processing extraneous materials such as bacteria, viruses, and particulate matter, including cell remnants of apoptosis and necrosis. This material is removed from the extracellular milieu through a process of phagocytosis into a membrane-bound vesicle known as the phagosome. Like the autophagosome, the phagosome fuses with a late endosome to form mature lysosomes where this ‘‘foreign’’ material is processed. This pathway is particularly important in macrophages, neutrophils, and dendritic cells. Of relevance to autoimmunity, antigens presented by classical MHC class II and the nonclassical MHC class I-like molecule CD1D requires their entry into the endosomal/

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lysosomal compartment and processing by lysosomal cysteine proteases [62]. Except for the antigens that are targets of the anticytoplasmic neutrophil antibodies (cANCA), surprisingly few other lysosomal autoantigens have been identified. One novel lysosome antigen, the membrane glycoprotein hlamp-2, was described in the sera of 15/16 patient(s) with necrotizing and crescentic glomerulonephritis and cANCA antibodies [18]. In contrast to h-lamp-2, which is ubiquitous in most cell types, autoantibodies to better-known autoantigens that are relatively restricted to the lysosomes of monocytes [63,64], and to primary granules or lysosomes in polymorphonuclear leukocytes (neutrophils) [38,65,66]. The primary target antigen is proteinase 3 (PR3) [67] and autoantibodies to this enzyme are primarily associated with Wegener’s granulomatosis but are also seen in other conditions [38,68]. Anti-EEA1 antibodies also produce a cANCA staining pattern that partially colocalizes with, but do not bind to, PR3 [69]. Since several excellent reviews have been written on the biochemical, clinical, and laboratory features of antibodies to cANCA [68,70], this review will not discuss this topic further.

Golgi complex The Golgi complex is localized in the perinuclear region of most mammalian cells (Fig. 1E) and is characterized by membranous stacks spatially and functionally organized as distinct cis-, medial-, and trans-Golgi networks [8,9,71]. The Golgi complex has a prominent function in the processing, transporting, and sorting of newly synthesized proteins from the rough endoplasmic reticulum. In the last decade, the identity of Golgi complex autoantigens has been elucidated by human autoantibodies that were used as probes in expression cloning to identify a unique family of proteins called golgins [9,72]. The golgin autoantigens include giantin/macrogolgin/GCP372, golgin245/p230, golgin-160/GCP170, golgin-95/gm130, golgin97, p115, and golgin-67 [8,9,14,15,71,73 –75]. All of the golgins, except giantin/macrogolgin and perhaps golgin-67, are peripheral Golgi components that are bound on the cytoplasmic face of Golgi membranes. Giantin/macrogolgin has a single trans-membrane domain in the C-terminus but the majority of the molecule projects into the cytosol [76]. Golgin-245 and golgin-97 were localized to the trans-Golgi compartment [77] and gm130/golgin-95 was reported in the cis-Golgi compartment [78]. It was shown that golgins such as golgin-95/GM130 and p115 bind to Rab GTPases via their coiled-coil domains, and that golgin95/GM130 is dynamically involved in exchange between the membrane surface and the cytoplasm. Golgin-245 and golgin-97/ GM130 attach to Golgi membranes through a GRIP domain in their C-termini [79] and rather than binding Rabs, interact with, and are recruited to membranes by another class of GTPase known as the Arls [72]. Current evidence suggests

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that the golgins are necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. However, other potential functions include negative regulation of membrane fusion events by keeping membranes apart, providing transient binding sites for vesicles to attach to limit their diffusion, compartmentalization and organization of membrane proteins, and cytoskeletal linking [72]. The evidence that the golgins have rather unique biochemical features is of interest to cell biologists but how this relates to them as preferred Golgi complex targets of autoantibody targets is not known. Anti-Golgi complex autoantibodies (AGA) were first identified in the serum of a SjS patient with lymphoma [80]. This was followed by other reports that described AGAs in SjS [81] and diseases such as SLE [82], rheumatoid arthritis [83], mixed connective tissue disease [84], Wegener’s granulomatosis [85], and in HIV infection [73,86] (Table 2). Immunoblotting and immunoprecipitation studies have shown that the proteins recognized by human AGAs are remarkably heterogeneous [87]. Although AGAs are generally considered to be rare, at the Advanced Diagnostics Laboratory at the University of Calgary they are at least as common as antibodies to Sm (Table 1). The presence of high-titer AGAs may constitute an early sign of systemic autoimmune diseases even in the absence of clear clinical manifestations [88]. In a study of 80 sera, the frequency of AGA was correlated with the molecular masses of the golgins [89]. Thus autoantibodies to giantin/macrogolgin, the highest molecular weight golgin, were the most frequent, being found in 50% of the AGA sera. By contrast, antibodies to golgin 97 were the least common, being found in only approximately 4% of the AGA sera. The most reactive of the giantin/macrogolgin epitopes were those that encompasses the C-terminal trans-membrane domain [89].

Protein processing pathways: signal recognition particles and proteosomes During protein synthesis the cell uses pathways and signals that are key components in the transport of proteins from the rough endoplasmic reticulum to the Golgi complex and then to compartments that store, secrete, or target molecules to the correct location. One such pathway involves components called the signal recognition particle (SRP) [90]. Remarkably, during protein synthesis and processing, the cell has a ‘‘protein quality control’’ that functions to rapidly degrade aberrant proteins through a pathway that involves the proteasome [91]. Signal recognition particle (SRP) The SRP, first discovered in the early 1980s, is a cytoplasmic ribonucleoprotein complex that includes a membrane-associated signal receptor (SR) that is involved in mediating the movement of newly synthesized peptides to

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the endoplasmic reticulum (ER) [90,92]. Both secretory and membrane proteins are transported via the SRP to the SR that is embedded in the ER [90,92]. The SRP is composed of six proteins that have molecular masses of 72, 68, 54, 19, 14, and 9 kDa [93]. As mRNAs are translated and newly synthesized polypeptides emerge from the ribosome, SRP54 binds to a region of the protein called the signal sequence and brings proteins to the ER where they are modified or exported [92]. Antibodies to SRP antibodies as detected by IIF give a homogeneous cytoplasmic pattern on HEp-2 cells [92]. Since other autoantibodies, such as anti-Jo-1 (histidyl tRNA synthetase), can give a similar IIF staining pattern, anti-SRP autoantibodies should be confirmed by immunoprecipitation of the recombinant or radiolabeled native proteins or by immunoblotting [94]. ELISA assays have been used [95], but they are only available at a few research centers. Autoantibodies to the SRP were first described in 1986 in the serum of a patient with polymyositis [96]. The patient’s serum in this report immunoprecipitated a protein that was identified as the 54-kDa SRP [96] and this appears to be the primary target of human autoantibodies [92,95]. Anti-SRP autoantibodies are present in about 4% of myositis patients and were most common in black females [92]. Anti-SRP autoantibodies are considered ‘‘disease-specific’’ because they were found exclusively in the sera of polymyositis and dermatomyositis patients. Proteasome The proteasome is a 26S complex responsible for most nonlysosomal protein degradation [97]. The core of the proteasome is in the nucleus and the cytoplasm and is an ATP-dependent protease that constitutes nearly 1% of the cellular protein. The structure is described as a hollow cylinder formed by four heptameric rings of seven different subunits with a configuration of a7h7h7a7. The outer rings belong to the a-type subunit, while the inner rings, which carry the active sites of these subunits, face the inner corridor of the macromolecule and are composed of the htype family [19]. At the ends of the cylinder is a complex of approximately 20 proteins that serve as the gatekeeper for entry into the proteasome and bind targeted aberrant proteins. For the most part, proteins marked for destruction in the proteasome are covalently bound to multiple copies of a small protein called ubiquitin. Of relevance to autoimmunity, the 26S ubiquitin/proteasome pathway that is under the control of interferon gamma generates antigenic peptides for the MHC class I pathway [19, 98]. In 1991, autoantibodies to different components of the proteasome were reported in the sera of patients with SLE [99]. This was followed by a report that the 29.5-kDa, outerring subunit HC9 (a3) is the primary target of autoantibodies in sera of patients with myositis, SjS, SLE, and other connective tissue diseases [19,100,101]. Less-frequent reactivities were observed against other a-proteasomal subunits,

such as HC8 and HC2, and h-type subunits, such as the active subunits MECL-1, LMP-7, and Z [19]. In addition to the presence of autoantibodies, some evidence suggests that circulating proteasomal components may be markers of cell damage and immunologic activity in autoimmune diseases [102]. It has been shown that affinity-purified antiproteasomal antibodies stain the cytoplasm and nucleus of HEp-2 cells with a pattern that is distinct from antibodies to Jo-1 (histidyl-tRNA synthetase [20].

Messenger RNA processing pathways: assemblyosomes, exosomes, and GW bodies The cellular mechanisms that process, transport, and perform quality control of proteins is remarkably mirrored in pathways and processes involving messenger RNA (mRNA) [103,104]. Compartments representing these pathways include the assemblyosome [105], the exosome [106], and GW bodies [107]. The process of mRNA production and processing begins in the nucleus where the removal of introns from premRNAs is an essential step in eukaryotic mRNA processing that is performed by the spliceosome. The small nuclear RNPS (snRNPs: U1, U2, U4/U6, U5) are essential and major components of the spliceosome. Each snRNP consists of one uridine-rich snRNA (U snRNA) molecule, a core composed of a ring of seven Sm proteins, and snRNPspecific proteins [108]. SnRNP biogenesis, which occurs in the cytoplasm, requires the assembly of the Sm proteins on an UsnRNA to form the Sm core [109]. For the snRNPs to recruit the necessary import receptors and translocate into the nucleus, a properly assembled Sm core and a methylated 5V cap are both required [108,109]. Once in the nucleus, snRNPs associate with specific proteins that are unique to each snRNA, and function in pre-mRNA splicing. Assemblyosome The assemblyosome is a recently described cellular compartment that includes components of the survival of motor neuron (SMN) complex [105]. It is an ATP-dependent 50S macromolecular complex in the cytoplasm that plays a critical role in the assembly and restructuring of small nuclear RNP (snRNP) and possibly small nucleolar (snoRNP) complexes [110 –112]. Key components of the assemblyosome complex include the SMN protein, a family of proteins called gemins, and the argonaute protein eIF2C2 [105,113 –116]. The SMN protein is the product of the spinal muscular atrophy (SMA)-determining disease gene [117], and reduced levels of SMN protein result in SMA, a neurodegenerative disease of the motor neurons (reviewed in Ref. [115]). A role of the assemblyosome complex is to ensure that the Sm cores assemble on correct RNA targets and prevent promiscuous association with other RNAs. Thus the assemblyosome functions as a specificity factor

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essential for the efficient assembly of Sm proteins on UsnRNAs and likely protects cells from nonspecific binding and potentially deleterious binding of Sm proteins to other RNAs [115]. The MicroRNAs are part of a novel class of RNPs termed miRNPs or mi-like RNPs that are characteristically short (20 – 24 nt) single-stranded noncoding regulatory RNAs [118,119] that are thought to modulate gene expression by partially base pairing with target mRNA sequences [116]. The precursors of miRNAs are predicted to form imperfect stem-loop structures from which the mature miRNAs are excised by a process that involves the RNAse III enzyme Dicer [120]. Also, several miRNAs identified as part of miRNPs appear to constitute two distinct subfamilies that are located on different chromosomes, suggesting an important function in the regulation of gene expression. Of interest, some of the human autoantigens are bound to the assemblyosme through RG-rich domains. These include Sm (U2-U6 RNP) [121 – 123], like-Sm (LSm) [124], fibrillarin [125 – 127], RNA helicase A (Gu) [128,129], and p80 coilin [130 – 132]. Since human autoantibodies tend to bind various components of macromolecular complexes [1,133] it will be interesting to see if other components of the assemblyosome are identified as human autoantigens in the future. Exosomes If certain steps of mRNA production (transcription, scanning, editing) go awry, the RNA may be degraded by 3V– 5Vexonucleases in a macromolecular complex known as the exosome [21,104,106]. The exosome is a large protein complex that includes 10 or more exonucleases and several other proteins that are involved in nonsense-mediated decay (NMD) [106]. One of the critical features of mRNA monitored by this proofreading system is the spatial relationship between the first in-frame stop codon and the exon – exon boundaries formed by pre-mRNA splicing, particularly in mRNAs bearing AU-rich elements [104,106]. Current evidence suggests that components of the exosome are found in the nucleolus, nucleus, and cytoplasm [21,134,135]. The nuclear exosome is involved in processing many different types of RNA including ribosomal RNA (rRNA), spliceosomal small nuclear RNAs, and small nucleolar RNAs. It is also implicated in the degradation of pre-rRNA spacers and unspliced premRNAs. The cytoplasmic exosome is involved in mRNA degradation in the 3V! 5Vpathway [134]. The structure of the exosome is not fully understood, although several models have been proposed [21,103,136]. The polymyositis/scleroderma (PM/Scl) autoantigen, first described in 1971, was initially named PM-1 [137] and was subsequently shown to be a complex of at least two proteins, the 75-kDa PM/Scl-75 [138] and a 100-kDa PM/Scl-100 [139] antigens. Both of these components are homologous to bacterial exoribonuclease proteins RNase D and RNase PH

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[140] and were recently shown to be subunits of the human exosome [21,22]. The originally described PM/Scl-75 cDNA [138] and epitopes [141] encoded a polypeptide that failed to interact with the exosome complex [135], but recently, a PM/ Scl-75 cDNA encoding an additional 84 amino acids at the N terminus was described that is required for interaction with the exosome complex [141]. Additional evidence suggested that this interaction was mediated by protein – protein interactions with two other exosome subunits, hRrp46p and hRrp41p. In addition, it was demonstrated that the putative nuclear localization signal (NLS) present in the C-terminal region of PM/Scl-75 is sufficient, although not essential, for nuclear localization of the protein. Moreover, deletion of the NLS did not disrupt interaction with the exosome but abrogated the nucleolar accumulation of PM/Scl-75. Taken together, these studies suggest that PM/Scl-75 plays a role in targeting the exosome to the nucleolus. Autoantibodies toward the anti-PM/Scl autoantigens are found in 5 – 8% of patients with polymyositis (PM), 3% of SSc, and in 24% of patients with a PM/SSc overlap syndrome [21] (Table 2). Initial studies suggested that most patients (90 – 98%) with anti-PM/Scl positive sera react by Western blot with the 100– 110-kDa (PM/Scl-100) protein [142]. Some patients (50 –63%) react with a 39-kDa acidic protein (PM-Scl-75) that migrates aberrantly on Western blots at 75 – 80 kDa [22, 142]. More recent clinical studies found that antibodies from 28% of polymyositis/SSc patients bound to the longer PM/Scl-75 protein [143]. This is higher than the frequency of antibodies to PM/Scl-100 and a significantly higher frequency than reported for the shorter or truncated PM/Scl-75 protein. In contrast to earlier reports, a significant number of patients had anti-PM/Scl-75, but not anti-PM/Scl-100 antibodies. There are no commercially available kits to detect autoantibodies to PM/Scl/exosome antigens and the most commonly used screening technique for anti-PM/Scl autoantibodies is double immunodiffusion [142]. Immunoprecipitation using [35S]methionine-labeled human tissue culture cells and immunoblotting with the patient sera can also be used [142]. Preliminary studies using an ELISA with recombinant PM-Scl-100 antigen indicated a promising low false-positive result, but not all epitopes may be identified [142]. More recently, both ELISA and line immunoassays have been shown to hold promise for commercial kits based on these autoantigens [143]. IIF staining produced by antiPM/Scl sera is typically a homogeneous nucleolar pattern with weaker staining of the nucleoplasm. This pattern can suggest that anti-PM/Scl antibodies are present, but is not conclusive [142]. Of note, anti-PM/Scl antibodies have not been reported to produce significant cytoplasmic staining [142]. GW bodies (GWBs) The processing and degradation of mRNA has recently achieved scientific prominence [144 – 148]. Recent reports

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have indicated that certain mRNAs and mRNA-processing molecules are localized in distinct cytoplasmic foci [23,107,147,149]. We have identified autoantibodies that target these components involved in this mRNA processing and degradation in cytoplasmic structures called GW bodies (GWBs) [23,107]. One novel protein of GWBs is GW182, which was named for the presence of multiple glycine (G) – tryptophan (W) repeats and for its molecular mass of 182 kDa, was localized by indirect immunofluorescence (Fig. 1B) and immunoelectron microscopy to distinct foci within the cytoplasm [23]. This protein, which harbors an RNA recognition motif (RRM), was shown to bind a discrete subset of mRNAs from HeLa cells and was localized to GWBs. Recently, discrete cytoplasmic foci that contained hLSm complex proteins 1 – 7, as well as the hDcp1 and Xrn proteins, were described; interestingly, cytoplasmic foci containing these proteins resembled GWBs [147,149]. GWBs were later shown to contain LSm4 and hDcp1 [107]. Since the hLSm1 –7 complex [148,150,151], hDcp, and Xrn [147,152,153] are involved in mRNA decapping, it was suggested that GWBs are functional sites within the cytoplasm involved in mRNA degradation. Recent evidence in yeast described a major pathway of eukaryotic messenger RNA (mRNA) turnover that begins with deadenylation, followed by decapping and 5V to 3V exonucleolytic decay that occurred in discrete cytoplasmic foci called processing bodies (P bodies) [154]. It may be that the P bodies in yeast are analogous to GWBs in mammalian cells. In mammalian cells, mRNAs might localize to the GWBs as part of surveillance and proofreading processes that include the decapping activity of hDcp1 and the hLSm1 – 7 complex [155,156]. These observations, and those that GW182 protein is localized to the same cytoplasmic compartment as hLSm4 and hDcp1 proteins, elucidate the emerging theme of coordinated mRNA regulation, as several mRNA binding proteins have been found to associate with different and discrete subsets of mRNAs from a total mRNA population [157 – 159]. The most common clinical diagnosis of patients with antibodies to GWBs was SjS followed by mixed motor/ sensory neuropathy and SLE (Table 2). Of interest, 9/18 (50%) and 3/18 (17%) of the sera that react with GWBs had autoantibodies to the 52 and 60-kDa SS-A/Ro autoantigen, respectively [24]. Epitopes bound by the human autoantibodies were mapped to the GW-rich mid-part of the protein, the non-GW-rich region, and the C-terminus of GW182 protein. None of the GW182 epitopes had significant sequence similarities to other known proteins. GW182 represents a new category of ribonucleoprotein autoantigens. The association of anti-GWB antibodies with antibodies to the 52-kDa SS-A/Ro antigen, particularly in the patients with no evidence of SjS and SLE, was an unexpected finding. Although the 52-kDa SS-A/Ro antigen has been localized to both the nucleus and cytoplasm, antibodies from a variety of sources directed to the 52-kDa SS-A/Ro

autoantigen do not produce a GWB staining pattern [160,161]. The function of the 52 kDa SS-A/Ro antigen is not clear [162], and the observation that it is associated with GWB antibodies may help clarify its function. Although GW182 does not have coiled-coil domains, it is interesting that approximately 50% of sera with anti-GW182 have antibodies to the 52-kDa SS-A/Ro antigen that does have coiled-coil domains [163]. The tie that binds: coiled coils As indicated in this review, several human autoantigens are characterized by the presence of coiled-coil domains (Table 2) (Fig. 2). These include all Golgi complex autoantigens identified to date [8,9,72], early endosome antigen 1 (EEA1) [16], cytoplasmic linker protein (CLIP-170) [55], the 52-kDa SS-A/Ro [163], nuclear mitotic antigen (NuMA) [164 – 166], lamin B [167,168], heavy chain of myosin [169], the 52-kDa SS-A/Ro [163], the 70/80-kDa Ku autoantigens [170], the Purkinje cell antigen APCA-1/Yo) [171], and the centrosomal autoantigens pericentrin and ninein [10,172,173]. The coiled-coil rod domains in Golgi and many other autoantigens identified to date are located throughout the molecule except for small non-coiled-coil or globular domains at both termini (Fig. 2). The number and length of coiled-coil domains tend to be correlated with the molecular mass of the protein and giantin, the most dominant human autoantigen contains a large number of coiled-coil domains compared to lower molecular mass Golgi autoantigens such as golgin-97. An exception to this generalization is the Ku, 75-kDa PM/Scl, and 52-kDa SS-A/Ro antigens where the coiled-coil regions are relatively short (Fig. 2). Studies to date have shown that the autoimmune response is not simply to coiled-coil motifs because there is no demonstrated crossreactivity among autoantigens that bear this motif and even within the Golgi complex autoantigens, the human autoim-

Fig. 2. Coiled-coil domains of 52-kDa SS-A/Ro, 70-kDa Ku, 100-kDa PM/ Scl, golgin 67, golgin-95/GM130, golgin-97, golgin-160, EEA1, golgin245/p230, and giantin. Coiled-coil domains are depicted as black boxes and calculated by the COILS program [194] available from the EMBnet server (http://www.ch.embnet.org/software/COILS_form.html).

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mune response appears to be highly specific as many sera react with only one of these autoantigens [89]. The coiled-coil motifs also seem to participate in the formation of unique epitopes that are exposed to immune system only on the native protein, since protein denaturation frequently results in the loss of antigenicity. For example, only 2 out of 19 antiEEA1 sera bind denatured EEA1 protein, whereas all 19 sera bind nondenatured recombinant and native EEA1 in a variety of immunological assays [35]. The reactivity with the native epitopes may also imply that, at least in the case of EEA1, the autoimmune response is primarily driven by B cells, which contrary to T cells, bind exclusively native protein epitopes. One of the striking features of many coiled-coil cytoplasmic autoantigens is that they reside on the cytoplasmic face of their respective organelles. How and why coiled-coil proteins become autoimmune targets remains to be determined. An explanation is that they may be recognized as surface structures on the organelle that is exposed to immune system in aberrant diseases states associated with unregulated cell death (apoptosis or necrosis) and subsequent defective clearance of dying cells. We have recently noted that during apoptosis the golgins were localized to a unique indentation of the nucleus [174] and antigenic fragments with distinctive characteristics were produced [175]. These observations suggested that the golgins may play a role in sustaining autoantibody production in systemic autoimmune diseases. The clinical conundrum For approximately three decades, diseases such as SjS and SLE were commonly associated with a group of autoantibodies that have been called ‘‘linked sets’’ [176,177]. These included antibodies to protein and RNA moieties on a family of ribonucleoprotein complexes known as small cytoplasmic ribonucleoprotein complexes (scRNPs) in SjS and to small nuclear cytoplasmic ribonucleoprotein complexes (snRNPs) in SLE [2,178,179]. Although there is some overlap of antibody reactivity in these diseases, the frequency of autoantibodies to components of the scRNPs such as SS-A/Ro and SS-B/La in SjS is approximately 70% and 30%, respectively [180,181]. The frequency of antibodies to components of snRNPs, such as Sm and U1-RNP, in SLE is approximately 15% and 40%, respectively [2]. Because these autoantibodies are seen relatively frequently in a clinical rheumatology setting, assays to detect them are widely available, for the most part in the form of commercial kits [27]. However, autoantibodies to exosomes, the Golgi complex, and GWBs discussed in this review present a clinical conundrum because studies of cohorts of SjS and SLE patients suggest that autoantibodies to these autoantigens are relatively rare. For example, studies of autoantibodies in cohorts of 100 SjS and 300 SLE patients have shown that antibodies to the Golgi complex and GWBs are seen in less than 5% of these sera (unpublished observations). However, studies of serological

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cohorts that contain anti-Golgi or anti-GWB antibodies have shown that over 40% of these sera are associated with SjS [24,89]. This clinical and serological enigma is not unique to the autoantibodies discussed in this review. For example, in studies of serological cohorts of anti-CENP-F, approximately 50% were found to have a malignancy [182,183]. However, in surveys of sera of patients with malignancies, the frequency of antibodies to CENP-F was less than 1% [183]. Similarly, studies have shown that virtually 100% of patients with antibodies to the paraneoplastic antigen have a malignancy, but less than 1% of patients with breast or ovarian cancer have anti-Yo antibodies [184,185]. How should this translate into clinical practice? Autoantibodies to the ‘‘somes’’ discussed in this review are admittedly uncommon (Table 1). By routine IIF, some of them appear quite similar (Fig. 1) and this is further complicated from time to time when they are seen in association with other autoantibodies (Fig. 1C). For example, antibodies to the 52-kDa SS-A/Ro antigens are seen in the sera of 20– 50% of sera that contain anti-Golgi, antiGWB, and anti-Jo-1 antibodies [24,89,186]. It is evident that some SjS, SLE, and other autoimmune conditions, particularly those with neurological diseases, are being missed or misdiagnosed because of failure of clinical laboratories to detect and/or identify autoantibodies to ‘‘cytoplasmic somes’’. In defense of clinical laboratories, budgets do not permit the widespread availability of autoantibodies to every known autoantigen [27]. However, it might be feasible that autoantibody testing for rarer, or what some call esoteric, autoantibodies might be offered in centralized laboratories that have the necessary technology to conduct rapid and economical testing for a wide array of autoantibodies. The rapid development of new technologies that permit detection of multiple autoantibodies in a single platform, such as addressable laser bead assays, microchip arrays, microfluidics (i.e. lab on a chip technology), and nanobarcodes will allow detection of at least 100 different autoantibodies in a single small (< 5 Aml) serum sample [187 –192]. Along with these technological advances will come challenges for the clinician to keep abreast of the clinical relevance of different autoantibodies and the understanding of autoantibody profiles in patients and cohorts of patients with various diseases will take on a new meaning [189,190,193]. For the future of clinical diagnostic immunology to be effectively realized, there will have to be a higher level of cooperation between manufacturers, clinical laboratories, physicians, patients, regulatory agencies, and medical paymasters [27,193].

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