Background: Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs.
Objective: We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1.
Methods: We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD.
Results: The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD.
Conclusions: This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.
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Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the () gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology.
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The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle.
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Background: Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs.
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