Many ribonucleoprotein (RNP) particles, such as Sjögren's syndrome antigen A (SS-A/Ro), Sjögren's syndrome antigen B (SS-B/Ia), and components of U2-U6 RNP complexes (first named the Smith (Sm) antigen) have been identified and characterized through the use of human autoantibodies. (1-4) Recently human autoantibodies were used to identify and characterize a novel 182 kDa protein autoantigen named GW182. (5) The GW182 protein contains multiple glycine(G)/tryptophan(W) repeats and a RNA recognition motif (RRM) near the COOH-terminus. This protein was found to be a marker for novel cytoplasmic structures designated GW bodies (GWBs). (5) This protein, which harbors an RNA recognition motif (RRM), was demonstrated to bind a discrete subset of mRNAs from HeLa cells. The GW182 protein binds to a subset of mRNAs and is one of a growing number of messenger ribonucleoproteins (mRNP) that include ELAV/Hu, elF4E, and polyA binding protein (PABP). (6,7)
A novel autoantigen named GW182 was recently identified when the serum from a patient with a sensory ataxic polyneuropathy was used to immunoscreen a HeLa cDNA library. Unique features of the GW182 protein include 39 repeats of glycine (G) and tryptophan (W) residues, binding to a subset of messenger RNA and localization to unique structures within the cytoplasm that were designated GW bodies (GWBs).
Cytoplasmic proteins associated with mRNA are involved in storing, degrading, transport or stabilization of mRNAs once they are transferred from the nucleus. (6,8-11) These processes have been referred to as components of the ribonome or post-transcriptional operon. (7,9,12) Because the processing and regulation of mRNA is an intense area of research, molecular tools to study GW182 and GW bodies would be valuable reagents. Although human autoantibodies to the GW182 protein and GW bodies are available, they have limited use because they often bind to other intracellular components. Hence, we sought to generate murine monoclonal antibodies (MAbs) to GW182 that could subsequently be used to conduct more thorough studies of GW bodies and their biology in a variety of tissues and species.
It is postulated that the GW182 protein, which contains both a putative RRM domain and nuclear localization signal (NLS)(5), may bind subsets of mRNAs at the nuclear pore complex and then participate in their storage or degradation. The composition of GW bodies as observed by IIF and immunoelectron microscopy, (5) could include other proteins involved in regulation or processing of specific mRNAs. Among the intriguing possibilities is that GW182 and related proteins may be involved in the storage(9,12) or controlled degradation of mRNA.(23,24) Recent reports of cytoplasmic sites of mRNA decapping and degradation bear similarities to structures that contain GW182.(25,26) Pilot studies based on exchange of reagents have suggested that the cytoplasmic structures referred to as stress granules(27) are not present in the HEp2 cells used in these studies nor do the MAbs described here co-localize with markers of stress granules (unpublished observations). Taken together, these observations suggest that the GW182 protein and GWBs are involved in mRNA metabolism and, more specifically, GWBs may be functional sites within the cytoplasm involved in mRNA degradation. Interesting features of the GW182 protein include 39 repeats of glycine (G) and tryptophan (W) residues and its localization in unique cytoplasmic structures that have been designated as GW bodies (GWBs). The GW182 protein, which has an RNA recognition motif and binds specific mRNAs, is thought to be part of a mRNA-protein macromolecular complex. It has been postulated that GWBs provide an additional level of posttranscriptional gene regulation and function in mRNA processing in a cell compartment referred to as the ribosome or posttranscriptional operon [12, 13]. More recent evidence implicates the GW182 protein and GWBs in mRNA degradation pathways [14]. The goal of the present study was to characterize the B-cell immune response in patients with antibodies to GWBs and the GW182 protein which resides within the GWBs and to assess the clinical features of these patients. This is the first report of the clinical features of patients with anti-GWB antibodies and a description of the GW182 epitopes bound by these sera.
Notwithstanding the usefulness of the above-described compositions and methods, a need still exists for monoclonal antibodies that specifically bind to GW182 and to diagnostic assays using those monoclonal antibodies.