Abstract
Eighteen severe human diseases have so far been associated with trinucleotide repeat expansions coding for either polyalanine (encoded by a GCN repeat tract) or polyglutamine (encoded by a CAG repeat tract). Among them, oculopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxia type-3 (SCA3), and Huntington’s disease (HD) are late-onset autosomal-dominant disorders characterized by the presence of intranuclear inclusions (INIs). We have previously identified the OPMD causative mutation as a small expansion (from 2 in normal to 7 in disease) of a GCG repeat tract in the PABPN1 gene. In addition, -1 ribosomal frameshifting has been reported to occur in expanded CAG repeat tracts in the ATXN3 (SCA3) and HTT (HD) genes, resulting in the translation of a hybrid CAG/GCA repeat tract and the production of a polyalanine-containing peptide. Previous studies on OPMD suggest that polyalanine-induced toxicity is very sensitive to the dosage and length of the alanine stretch. Here we report the characterization of a polyclonal antibody that selectively recognizes pathological expansions of polyalanine in PABPN1. Furthermore, our antibody also detects the presence of alanine proteins in INIs of SCA3 and HD patient samples.
Highlights
Expansion of trinucleotide repeated sequences within the coding regions of distinct genes has been established to cause a number of severe human diseases [for reviews, see 1–4
Using oculopharyngeal muscular dystrophy (OPMD) as the disease model and Western blot immunodetection as a first assay, our analyses revealed that the antibody was able to produce a strong signal from whole protein lysates prepared from HeLa cells that transiently expressed a vector encoding a GFP-tagged hPABPN1 cDNA bearing alanine repeat lengths of 13, 17, 30, and 40 (Figure 1a)
antibody 4340 (Ab4340) was assessed for its ability to selectively detect alaninecontaining proteins in disease models of OPMD, spinocerebellar ataxia type-3 (SCA3), and Huntington’s disease (HD), while confirming that unaffected control individuals would not present significant levels of these same polyalanine peptides
Summary
Expansion of trinucleotide repeated sequences within the coding regions of distinct genes has been established to cause a number of severe human diseases [for reviews, see 1–4. The so-called “polyglutamine” diseases share a number of genetic and molecular events/features; among which are their mutation process (dynamic expansion of their respective CAG repeat), intergenerational repeat instability, anticipation, and a disease course that is progressive following a late onset (10 to 20 years)[5]. For these reasons, it has been proposed that expanded CAG repeat tract diseases share, to some extent, a common pathogenic mechanism, whereas the phenotypic variability of each disease would reflect the intrinsic properties the protein in which the repeat resides and the cellular environment where the affected protein is expressed. This could either be due to native properties of each protein, or could be explained by novel interactions of the mutant species with other cellular factors, specific for each cell type
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