RESUMO
Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure.
Assuntos
Proteína ADAMTS13/genética , Dicroísmo Circular , Células HEK293 , Humanos , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Nuclear and cytoplasmic endometrial expression of Indian hedgehog increased from the late proliferative to mid and late secretory phases in 26 healthy volunteers compared with 30 women with endometriosis. The abnormal expression of Indian hedgehog protein in women with endometriosis suggests a resistance to P action.