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. 2007 Dec;125(3):247-56.
doi: 10.1016/j.clim.2007.07.016. Epub 2007 Sep 17.

Clinical and serological features of patients with autoantibodies to GW/P bodies

Rahima A Bhanji  1 , Theophany Eystathioy, Edward K L Chan, Donald B Bloch, Marvin J Fritzler
Affiliations

Clinical and serological features of patients with autoantibodies to GW/P bodies

Rahima A Bhanji et al. Clin Immunol. 2007 Dec.

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, 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 (16%). 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 were 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.

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Figures

Figure 1
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 serum that contains both nuclear and CDS staining is shown to illustrate that some anti-GW/PB sera have separate antibodies directed to nuclear and cytoplasmic antigens. The secondary antibodies included Alexa Fluor 568 anti-human IgG (shown in the first column), Alexa Fluor 488 anti-mouse IgG (shown in the second column), and Alexa Fluor 647 anti-chicken IgG (shown in the third column). The arrows in the right column, where the images to the left have been merged, show that the CDS pattern of the human serum colocalized with the Ago2 and LSm4 markers. Nuclei were stained with DAPI (not shown) dissolved in glycerol mounting medium (VectaShield).
Figure 2
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 that IP recombinant Ge-1. C: Sera that IP recombinant GW182. D: Sera that IP recombinant RAP55. Normal human serum (NHS) did not precipitate any of the proteins (lane 1 in all panels). As described in Methods, the luciferase protein was included in the IP reaction to serve as a control for antibody binding specificity but none of the sera reacted with this protein.
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 the reactive peptides. A positive signal was scored after reactivity by NHS was subtracted from the reactivity produced by the patient’s sera. B) Table representing the peptide sequence, the amino acid positions and the reactivity of patient sera. Only the most reactive domain represented as the N-terminus, is included in the table. The normal human serum also reacted with two peptides that did not overlap with any of those of the anti-GW/PB sera. C) Bar graph with the y-axis indicating the number of patients with positive reactivity to the various domains of the protein (x-axis). The numbers in parenthesis are the amino acid residues represented within each domain.
Figure 4
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 signal was scored after reactivity by NHS was subtracted from the reactivity produced by the patient’s sera. B) Table representing the peptide sequence, the amino acid residues and the reactivity of patient sera to WD40, the Ser rich domain (S), the NLS containing (NLS-C) and the Psi domains. The Psi domain represents the second of four Psi domains in Ge-1 [48]. C) Bar graph with the y-axis indicating the number of patients with positive reactivity to the various domains of the protein (x-axis). The numbers in parenthesis are the amino acid residues contained within each domain. Though patients have reactive epitopes within each domain, the NLS-C is the most reactive.
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