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Clin Immunol. Author manuscript; available in PMC Dec 1, 2008.
Published in final edited form as:
PMCID: PMC2147044
NIHMSID: NIHMS35637

Clinical and Serological Features of Patients with Autoantibodies to GW/P Bodies

Abstract

GW bodies (GWBs) are unique cytoplasmic structures involved in messenger RNA (mRNA) processing and RNA interference (RNAi). GWBs contain, mRNA, components of the RNA-induced silencing complex (RISC), microRNA (miRNA), Argonaute proteins (i.e. Ago2), the Ge-1/Hedls protein and other enzymes involving mRNA degradation. The objective of this study was to identify the target GWB autoantigens reactive with 55 sera from patients with anti-GWB autoantibodies and to identify clinical features associated with these antibodies. Analysis by addressable laser bead immunoassay (ALBIA) and immunoprecipitation of recombinant proteins indicated that autoantibodies in this cohort of anti-GWB sera were directed against Ge-1/Hedls (58%), GW182 (40%) and Ago2 (9%). GWB autoantibodies targeted epitopes that included the N-terminus of Ago2 and the nuclear localization signal (NLS) containing region of Ge-1/Hedls. Clinical data was available on 42 patients of which 39 were female and the mean age was 61 years. The most common clinical presentations were neurological symptoms (i.e. ataxia, motor and sensory neuropathy) (33%), Sjögren’s syndrome (SjS) (31%) and the remainder had a variety of other diagnoses that included systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC). Moreover, 44% of patients with anti-GWB antibodies had reactivity to Ro52. These studies indicate that Ge-1 is a common target of anti-GWB sera and the majority of patients in a GWB cohort had SjS and neurological disease.

Keywords: autoantibodies, P bodies GW182, Ago2, Ge-1

Introduction

Historically, human autoantibodies have been used to identify novel cellular proteins and were linked to clinical subsets of autoimmune diseases (reviewed in [1]). Small nuclear ribonucleoprotein (snRNPs), components of the spliceosome complex involved in pre-mRNA splicing, were discovered with the aid of anti-Sm autoantibodies from systemic lupus erythematosus (SLE) patients [2-5]. Several other RNA binding autoantigens were also identified using patient sera including U1RNP, SS-A/Ro and SS-B/La [6-10]. More recently, use of human autoantibodies led to the discovery of a novel cellular compartment named GW bodies (GWB) which contained a glycine/tryptophan (G/W)-rich mRNA binding protein GW182 [11].

GWB, also known as mammalian processing (P) bodies and hereafter referred to as GW/PB, are sites for mRNA processing and degradation, and are important in the RNA interference (RNAi) pathway (reviewed in [12;13]). Some of the proteins identified in GWBs include the decapping proteins hDcp1, the human homolog of the yeast decapping enzyme subunit Dcp1; hDcp2 which has the enzymatic decapping activity; the exonuclease hXrn1, a 5′-3′ exonuclease active in deadenylation dependent and independent cytoplasmic mRNA turnover pathways; members of the heptameric decapping activator complex LSm1-7; hCcr4, a yeast homolog of deadenylase shown to have exonuclease activity in vitro; the human enhancer of decapping large subunit (Hedls, also known as Ge-1 or RCD8), and Edc3; eukaryotic translation initiation factor and cap binding protein (eIF4E) and the eIF4E binding protein, eIF4E-T; cytoplasmic polyadenylation element-binding protein CPEB1; proteins that comprise the RNA induced silencing complex (RISC) that include Argonaute 2 (Ago2), and human Rck/p54, a homolog of yeast Dhh1p, that is involved in decapping and translation initiation (reviewed in [12-14]). More recently, additional components of GW/PB were identified using an approach referred to autoantigenomics [15].

GW/PBs contain a unique family of proteins referred to as GW182 (TNRC6-A), GW2 (TNRC6-B), and GW3 (TNRC6-C), all of which have a RNA recognition motif (RRM) and multiple GW repeats throughout most of the protein (reviewed in[12;13]). GW182 associates with the Ago2 protein, a subset of mRNA, and has a role in miRNA-mediated gene regulation (reviewed in[12-14]). Recent data suggests that GW2 (TNRC6-B) may also interact with Ago2[16], whereas GW3 remains to be characterized.

In this report we identify target autoantigens in GW/PB and the retrospective clinical diagnoses for 55 patients with autoantibodies to GW/PBs. Previous studies have independently identified GW182[17], Ge-1 [18;19], RAP55[20], hAgo2[21] and diacyl-phosphatidylethanolamine[22] as GW/PB autoantigens. However, this is the first report of the relative frequency of autoantibodies directed to specific GW/PB components and then studied clinical features associated with anti-GW/PB.

Materials and Methods

Serum, antibodies and clinical information

All sera used in the present study were obtained from the serum bank stored in the Mitogen Advanced Diagnostics Laboratory (University of Calgary, Calgary, AB). Sera were selected for study based upon a cytoplasmic discrete speckled (CDS) pattern of staining that was then confirmed to be anti-GW/PB by co-localization with established markers of GW/PB (see below). Patient diagnoses were obtained by retrospective chart review as approved by the Conjoint Medical Ethics Review Board at the University of Calgary.

Indirect immunofluorescence

Patient sera with reactivity to GW/PBs were initially identified as the CDS pattern observed by indirect immunofluorescence (IIF) using HEp-2 cell substrates (ImmunoConcepts, Sacramento, CA) using a protocol as previously described [11]. Reactivity with GW/PB was confirmed by colocalization using a murine monoclonal to Ago2[23], chicken polyclonal antibodies to LSm4 (Abcam, Cambridge, MA) and/or murine monoclonal anti-GW182 (4B6: CytoStore, Calgary, AB). The secondary antibodies were AlexaFluor conjugated anti-mouse/chicken/human IgG, (Molecular Probes, Eugene, OR). Nuclei in the cell substrates were stained with 4′,6-diamidino-2-phenylindole (DAPI), which was included in the glycerol mounting medium (VectaShield, Vector Laboratories, Burlingame, CA).

In vitro transcription/translation and immunoprecipitation

Immunoprecipitation (IP) using various recombinant proteins was performed to test and confirm the reactivity of patient sera to GW182, Ago2, Ge-1 and RAP55. The full-length cDNA of GW182 [11], Ago2 (gift from Dr. Tom Hobman, University of Alberta); Ge-1 and RAP55 cDNAs [18;20] were used as templates to synthesize the respective proteins using an in vitro transcription and translation (TnT) rabbit reticulocyte lysate kit (TnT, Promega Biotec, Madison, WI) in the presence of 35S methionine at 30°C for 4 hours as previously described [17]. A 2 - 5 μl the labeled sample was separated using SDS-PAGE and analyzed by autoradiography to confirm the presence of the TnT product. The TnT product was then used in IP reactions as described previously [17]. To ascertain the specificity of the individual recombinant proteins, in vitro translated luciferase protein was added to the IP mix to serve as a control for nonspecific co-precipitation.

Recombinant Protein and Addressable Laser Bead Immunoassay (ALBIA)

Recombinant GW proteins GW182, GW2, GW3 were prepared and purified as previously described [17;24]. Briefly, the respective cDNAs were subcloned into the expression vector pET28 (Novagen, WI) and transformed to E. coli JM109 (DE3) for recombinant protein production. The N-terminal 6X histidine fusion recombinant protein was purified from a 1-L culture using Ni2+ affinity chromatography as per the manufacturer’s instructions (Qiagen, Valencia, CA). A set of addressable beads bearing laser reactive dyes (Luminex, Austin, TX) were coupled to the purified GW182, GW2, and GW3 as previously described [17;25]. The sera were diluted in HRP diluent (INOVA, San Diego, CA) to a final concentration of 1:100. Thirty microlitres of HRP diluent was added to each well followed by 10 μl of the diluted patient serum and then incubated on an orbital shaker for 30 minutes at room temperature. This was followed by addition of 40μl of phycoerythrin-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:50 to each well and incubated on the orbital shaker for an additional half hour. The reactivity of the antigen-coated beads was determined on a Luminex 100 dual-laser flow cytometer (Luminex). Each assay included negative and positive controls. The tests were semi-quantitative, and the results were expressed as median fluorescent units of the test sample.

Enzyme-linked immunosorbent assay (ELISA)

Patient sera were tested for reactivity to each of SSA/Ro60 and Ro52 using commercially available QUANTA Lite kits (INOVA, San Diego, CA) and the protocol provided by the manufacturer. Results were calculated and interpreted using manufacturer’s recommended formula.

Epitope mapping

Membranes containing in situ synthesized sequential peptides of 15 amino acids offset by five amino acids, representing full-length GW182, GW2, Ago2 and Ge-1 proteins were prepared (Eve Technologies, Calgary, AB) as previously described [17;25] and then used to map the epitopes on the respective proteins. The membranes were prepared for immunoblotting by soaking in 100% ethanol for 10 minutes followed by rehydration in Tris-buffered saline (TBS; 10mM Tris-HCl pH 7.6, 150 mM NaCl) for 10 minutes at room temperature. The membranes were then blocked in a solution of 2% milk/TBS at room temperature for one hour. Human sera were diluted 1:100 in 2% milk/TBS and overlayed on the membrane for 2 hrs at room temperature after which the membranes were washed three times with 2% milk/TBS. A horseradish-peroxidase (HRP) conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:12000 according to the manufacturer’s protocol was used as the secondary antibody, and reactivity was visualized using enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ). The intensity of each reactive peptide on the membrane was scored from 0 to 4 (0 being negative, 1 being weakly reactive and 4 the highest intensity). The assignment of a peptide as being reactive or non-reactive was determined after subtraction of the reactivity by a pooled normal human serum (NHS) control.

Results

Sera were identified with a CDS pattern of staining and the presence of anti-GW/PB antibodies was confirmed by IIF studies on HEp-2 cells using each patient serum in a colocalization reaction with Ago2, chicken polyclonal antibodies to LSm4 (Figure 1) and/or murine monoclonal anti-GW182 [24]. In a typical six month audit period at Mitogen Advanced Diagnostics Laboratory, 2500 samples are received for autoantibody analysis and of these 240 (9.6%) display a CDS pattern. Further verification that these sera had anti-GW/PB antibodies using the approach described above showed that 14/240 (5.8%) co-localized with these GW/PB markers. The frequency of anti-GW/PB is equivalent to antibodies to endosomes (i.e. early endosome antigen 1 - EEA-1), Sm and centromere proteins in this same cohort and more common than antibodies to proliferating cell nuclear antigen or the Golgi complex[25;26]. Using this approach, over four years 55 patient sera with anti-GW/PB antibodies were obtained for further study. The other sera displaying a CDS pattern had antibodies to endosome and lysosome autoantigens as previously reported [25;26], while other sera had antibodies to autoantigens yet to be identified.

Figure 1
Human anti-GW/PB sera that showed a CDS pattern of staining (left column) were identified as having anti-GW/PB on the basis of IIF colocalization studies using murine anti-Ago2, and chicken anti-LSm4 antibodies were performed using HEp-2000 cells. A human ...

Retrospective inquiry and chart review indicated that clinical and demographic information was available on 42/55 patients (Table 1). The age range of the patients was 36 to 90 yrs and the mean age was 60 and 69 yrs for the 39 female and 3 male patients, respectively. The demographic and clinical characteristics for these patients are summarized in Table 2. The most common diagnosis for this cohort of patients was neuropathies (33%) (motor and sensory neuropathies, ataxia) and Sjögren’s syndrome (SjS) (31%). The other disease groups for patients with anti-GW/PB antibodies included SSc (14%), arthritis (14%), SLE (12%), primary biliary cirrhosis (PBC) (10%), rheumatoid arthritis (RA) (7%), cancer (5%), multiple sclerosis (2%) and other diagnoses (10%).

Table 1
Demographic and Detailed Clinical Characteristics of 42 patients with GW/PB autoantibodies
Table 2
Summary of demographic and clinical characteristics of patients

To determine which antigens were the primary targets of the GW/PB autoantibodies we chose GW/PB proteins that included the GW family proteins GW182, GW2, GW3, the decapping activating protein Ge-1, the putative decapping coactivator RAP55, and the RNAi associated protein Ago2 for further analysis. Two assays were employed to determine reactivity to the GW/PB proteins: addressable laser bead immunoassay [27] for only the GW family of proteins, and IP of recombinant GW182, Ago2, Ge-1, RAP55 generated by TnT using the respective cDNAs (Table 3). No reactivity was obtained with the pooled normal human serum in TnT/IP, ALBIA or ELISA. Analysis of antibody reactivity to GW182, GW2, GW3, Ago2, Ge-1, and RAP55 indicated that the two most common autoantigens were Ge-1 (58%) and GW182 (40%) (Table 3 and Figure 2). Recognition of the Ago2, RAP55, GW2, GW3 proteins by the anti-GW/PB sera was 16%, 18%, 16%, and 9%, respectively (Table 3 and Figure 2). Only one patient (#3) reacted with all the GW/PB proteins and eight (#s 8, 26, 27, 28, 33, 36, 52, 53) did not react with any of the proteins employed in the assays. Sera that did not react with any of the tested antigens had a broad range of anti-GW/PB titers ranging from 1/320 - 1/5120 (Table 3).

Figure 2
Immunoprecipitation analysis using human sera positive for anti-GW/PB antibodies. These studies identified sera that recognized all, one or none of the in vitro translated proteins (Ago2, Ge-1, GW182 and RAP55). A: Sera that IP recombinant Ago2. B: Sera ...
Table 3
Autoantibody Profile of 55 Patients with GW/PB autoantibodies*

To determine the reactive epitopes in Ago2 and Ge-1, epitope mapping was conducted with an established strategy and sera with autoantibodies to these proteins [17]. Since the epitopes on GW182 were previously reported using this technology [17], we report here the epitopes on other GW/PB autoantigens, Ge-1 and Ago2 by probing membranes containing spots of overlapping peptides spanning the full-length sequence of the Ago2 and Ge-1 proteins with the patient sera. Figure 2A illustrates that the reactive epitopes obtained for 3 patient sera containing autoantibodies to Ago2 were predominantly localized to the N- terminal region (Figure 3). When the peptide membrane containing Ge-1 peptides was probed, (8/9, 89%) of patients recognized the region represented as amino acids 890-939 and referred to as the NLS containing region (NLS-C), but also reacted with the WD40, Ser and Psi domains (Figure 3).

Figure 3
Epitope mapping obtained using membranes that contained spots of 15mer peptides overlapping by 5 amino acids representing the full-length Ago2 protein. A) Membranes probed using normal human serum, and sera from patients 1, 3 and 15. The asterisks highlight ...

It was noted that some sera containing anti-GW/PB had antibodies to other nuclear and cytoplasmic antigens. By ELISA, 44% of the GW/PB positive sera had reactivity to Ro52, and 9% to SSA/Ro60. Moreover, 100% and 64% of the SjS patients had reactivity to SSA/Ro60 and Ro52, respectively. Since 10/20 of the patients with anti-Ro52 did not have SjS, the presence of anti-Ro52 was not always related to SjS. In addition, the presence of anti-Ro52 was not correlated with any of the other diagnostic categories. All 4 PBC patients had anti-mitochondrial antibodies and one had anti-CENP (Table 3). All 3 patients with limited cutaneous SSc had anti-CENP and 1/3 patients with diffuse cutaneous SSc had anti-topoisomerase I antibodies.

Discussion

In this report we studied a cohort of 55 anti-GW/PB sera and extend previous findings in smaller or other disease cohorts [17-22] to include several target antigens recognized by human anti-GW/PB autoantibodies that included GW182, GW2, GW3, Ge-1, Ago2, RAP55 and Ro52. The most common clinical diagnoses in patients with anti-GW/PB antibodies were neuropathies that included ataxia, sensory and/or motor neuropathies (33%), and Sjögren’s syndrome (31%). At this time we can find no evidence that the spectrum of neurological signs and symptoms constitute a unique neurological syndrome or that SjS patients have occult neurological features or autoimmune liver disease. This study relied on retrospective clinical data but, in the future, prospective studies in which a uniform clinical evaluation for SjS, neuological disease, autoimmune liver disease (e.g. PBC) and other features of autoimmunity would be desirable.

Among the proteins surveyed, the most common target autoantigens for anti-GW/PB antibodies were Ge-1 and GW182 with a frequency of 58% and 40%, respectively. Only one serum reacted with all GW/PB proteins and eight did not react with any. Because even sera with high titer anti-GW/PB by IIF did not react with any of the antigens employed in our assay, it is unlikely that this finding is due to low titer autoantibodies. More likely, this finding suggests that there are other target autoantigens or that the assay conditions employed may not have contained the reactive epitopes on some proteins. In this study we used three different assays to identify GWB and other autoantibody reactivity: IIF on HEp-2 cells, TnT/IP, ALBIA and ELISA. Studies conducted on a large number of autoantigen systems have shown that there is high correlation between ALBIA and ELISA (Dr. R. Burlingame, INOVA Diagnostics and our unpublished observations). In addition, we have found good correlation of ALBIA with the TnT/IP assay and for most systems studied (including GW182 here) ALBIA has a slightly higher sensitivity than TnT/IP. Hence, if anything, in this study the frequency of anti-Ge-1 in this cohort could be even higher if we had the full length recombinant protein to use in an ALBIA.

The finding that Ge-1 is the most common autoantigen target in GW/PB was an unexpected finding since most of our attention had focused on GW182 and Ago2. It is notable that none of the sera in this study targeted one ‘universal’ GW/PB autoantigen and, therefore, it does not appear that the human anti-GW/PB response is initiated toward a single component in these macromolecular structures. Perhaps this finding is not surprising considering the variety of diagnoses and clinical findings. In other studies some serological cohorts (e.g. anti-centromere, anti-fibrillarin) are associated with specific MHC markers but are also seen in a narrower diagnostic framework (anti-centromere with limited cutaneous SSc; anti-fibrillarin with diffuse cutaneous SSc) [28;29]. Genetic studies of patients with GW/PB antibodies may shed light their association with a variety of conditions, most notably SjS and neurological diseases.

Ge-1 is important for GW/PB integrity and is required for functional interaction between Dcp1 and decapping catalyst Dcp2 [18;30-34]. Of note, autoantibody reactivity to Ge-1 did not coincide with reactivity to GW182 for the majority of samples tested (only 8/32 as determined by TnT/IP analysis). This might be explained by evidence that the decapping complex may only transiently interact with GW182 bound mRNA (see review[12]) thereby, limiting intermolecular epitope spreading. In other autoantigen systems, intermolecular epitope spreading is most usually observed with the components of “stable” macromolecular complexes such as snRNPs (i.e. Sm) and hY RNP (i.e. Ro) [35]. In support of this conclusion it is noted that 89% patients with antibodies to Ago2 also had antibodies to GW182 (see also[21]) a finding that might be expected considering that GW182 has been shown to associate/interact with Ago2 [36;37].

The frequency of antibodies to Ro52 in the present study was 44% suggesting that Ro52 is a common target in patients with anti-GW/PB autoantibodies, but the presence of antibodies to Ro52 did not correlate with the diagnosis of SjS or SLE. It has been reported that Ro52 localized to “dot-like” structures in the cytoplasm [38], however it has not yet been confirmed whether these dot-like structures are GW/PBs. Ro52 is known to be a RING-dependent E3 ligase [39;40], and the presence of an ubiquitin-associated (UBA) domain in GW3 and a putative UBA domain in GW182 [12] may be related to the generation of antibodies to Ro52 as a result of interaction between Ro52 and GW182 during GW protein degradation in the proteasome pathway [26;41].

It is of interest that the Ago2 eptiopes were localized to the N-terminal region because analysis of the crystal structure of P. furiosus Ago protein [42] indicates that the N-terminus is exposed and, hence, could be readily accessible during antigen processing making it a likely target for autoantibody reactivity. Further, the connection between RNAi components, specifically Ago2 and Dicer [21;43], and autoimmune reactivity to GW/PBs is intriguing. Recent data has suggested that mammalian viruses interact with and may indeed suppress the RNAi pathway and therefore, RNAi may function as an innate anti-viral response mechanism [44-46]. The association of viral infection and the development of autoimmunity have been proposed [47] and an association between viral infection and the development of autoimmunity has been proposed to be due to the presence of autoantibodies to components of the RNAi pathway and GW/PBs [21;43].

Since previous studies mapped the reactive epitopes of GW182 to the GW rich, the mid-portion, the non-GW rich and the C-terminal domains of the GW182 protein [17] our focus in this study was the other major target autoantigens Ge-1 and Ago-2. We showed that the Ge-1 amino acid residues (890-939) containing the NLS were the most reactive epitopes. This is in agreement with a previous report of Bloch et al. [48] who identified the immunoreactive region localized to amino acids 877-934. In addition, we identified other reactive regions of Ge-1 that included the N-terminal WD40 repeats, the Ser rich domain and the second Psi repeat.

Although Ge-1 antibodies were found in 5% of PBC sera [18] our study indicated that in addition to PBC these antibodies are seen in patients with other clinical diagnoses including those with neuropathies, SjS, RA, SLE and various malignancies. Ge-1 was first discovered using serum from a patient that was initially thought to have SjS [48] but subsequent evaluation and follow-up showed that she had anti-mitochondrial antibodies and sicca complex associated with PBC [18]. This highlights the importance of longitudinal studies and clinical evaluation of all patients with various autoantibodies. For example, patient 26 in our study with disabling headaches and high titers of anti-mitochondrial and nuclear envelope antibodies, both of which are associated with PBC [49], could have sub-clinical autoimmune liver disease that may progress to PBS.

In summary, the present study reports the frequency of autoantigen targets among components of GW/PBs including GW182, Ago2, Ge-1 and RAP55. Human anti-GW/PB antibodies are mostly seen in patients diagnosed with neuropathies and Sjögren’s syndrome. Though Ge-1 and GW182 are common targets of patient antibodies, there are yet unknown antigens to be discovered as 18% (10/55) patients do not react to any of the antigens tested.

Figure 4
Epitope mapping obtained using membranes that contained spots of 15mer peptides overlapping by 5 amino acids representing the full length Ge-1 protein. A) Membranes probed using normal human serum (NHS), and sera from patients 3, 13, and 14. A positive ...

Acknowledgements

We thank Mark Fritzler (Eve Technologies) for establishing the ALBIA and epitope mapping membranes, Riley Sullivan for assistance with the epitope mapping analysis, and Haiyan Hou and Meifeng Zhang for identifying and processing the anti-GW/PB sera. This work was supported by the Canadian Institutes for Health Research Grant MOP-57674, and NIH Grants AI47859. MJF holds the Arthritis Society Chair.

Abbreviations

Ago2
Argonaute protein 2
ALBIA
addressable laser bead immunoassay
CDS
cytoplasmic discrete speckled
GW/PB
glycine(G)-tryptophan(W) repeat/processing bodies
Hedls
human enhancer of decapping large subunit
HRP
horseradish-peroxidase
IIF
indirect immunofluorescence
IP
immunoprecipitation
LSm
like Sm
MFU
median fluorescence units
miRNA
microRNA
NHS
normal human serum
NLS
nuclear localization signal
PBC
primary biliary cirrhosis
RA
rheumatoid arthritis
RAP55
RNA associated protein of 55 kDa
RNAi
RNA interference
RNP
ribonucleoprotein
RRM
RNA recognition motif
SjS
Sjögren syndrome
SLE
systemic lupus erythematosus
snRNP
small nuclear ribonucleoprotein
TnT
transcription and translation

Footnotes

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