Processing body autoantibodies reconsidered

Abstract

Processing bodies (P-bodies) are cellular structures that have critical roles in mRNA degradation and post-transcriptional gene silencing. Some patients with autoimmune disease have high titer antibodies directed against P-bodies, and certain sera have been used as markers for the GW182 component of these structures. This study shows that available reference sera are unreliable markers for GW182 because the sera contain antibodies directed against Ge-1, a second P-body autoantigen.

FIGURE 1.

Detection of anti-Ge-1 and anti-GW182 antibodies in human reference sera by indirect immunofluorescence. (Panel I) GFP-Ge-1 was expressed in Hep-2 cells and cells were stained with anti-GFP antiserum (a,d,g) and human serum Ge (b), 18033 (e), or IC-6 (h). The human sera stained larger and more numerous P-bodies in cells expressing GFP-Ge-1 compared with nontransfected cells, indicating the presence of anti-Ge-1 antibodies in all three sera. (Panel II) GFP-GW182 was expressed in Hep-2 cells and cells were stained with anti-GFP antiserum (a,d,g) and human reference sera. The number of P-bodies recognized by GW182 reference serum 18033 (b) was increased in transfected, compared with nontransfected cells, indicating the presence of anti-GW182 antibodies. Reference sera Ge (e) and IC-6 (h) stained P-bodies (indicated by arrows) in transfected cells weakly (in the case of Ge) or not at all (IC-6), indicating that these sera contained little or no anti-GW182 antibodies. DAPI staining (c,f,i, in Panels I and II) indicates the location of cell nuclei.

FIGURE 2.

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FIGURE 2.

(Panel I) Detection of anti-Ge-1 and anti-GW182 antibodies in human reference sera by immunoblot. Extracts were prepared from Hep-2 cells transfected with GFP-Ge-1, GFP-GW182, or GFP (control) as indicated. Reference sera Ge, 18,033, and IC-6 reacted with GFP-Ge-1, as well as endogenous Ge-1 in Hep-2 cells. Note that rabbit anti-GW182 did not react with a ∼180-kD band, consistent with the absence of detectable, endogenous GW182 in these cells. Serum 18033, but not Ge or IC-6, reacted with GFP-GW182. (Panel II) GW182, but not Ge-1, recruits Ago2 to P-bodies. Hep-2 cells were transfected with either myc-Ago2 and GFP-GW182 (a–c) or myc-Ago2 and GFP-Ge-1 (d–f) and cells were stained with anti-myc and anti-GFP antiserum. DAPI staining in c and f indicates the location of cell nuclei.