Molecular and functional characterization of a putative PA28γ proteasome activator orthologue in Schistosoma mansoni

PA28 gamma is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28 gamma of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28 gamma, this transcript has a 753 bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28 gamma with multiple PA28 gamma orthologues revealed an average similarity of similar to 40% among the investigated organisms, and 90% similarity with PA28 gamma from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28 gamma to its sister group that contains well-characterized PA28 gamma sequences from Drosophila spp., as well as sharing the same branch with PA28 gamma from S. japonicum. Gene expression profiling of SmPA28 gamma using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28 gamma antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28 gamma from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28 gamma transcript and protein in these stages may be correlated with an important role of the PA28 gamma in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28 gamma double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28 gamma dsRNA treatment resulted in gene and protein knockdown of similar to 60% and similar to 80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28 gamma in larval growth/development of the S. mansoni. (c) 2013 Elsevier B.V. All rights reserved.