Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors

Abstract

BackgroundThe infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease.ResultsTo explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion.ConclusionsOur results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation

“…The cloning of the bank vole PrP109M or PrP109I (BVPrP109M or BVPrP109I) genes were carried out based on the previously described [20]. The DNA coding for full-length BVPrP-109M and BVPrP-109I was amplified by PCR using a template plasmid of BVPrP-109M/pOPINE or BVPrP-109I/pOPINE.…”

“…RT-QuIC assay was conducted as previously described [20, 22, 24]. Briefly, the reaction mix was composed of 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/mL recombinant bank vole PrP23-231, 10 μM thioflavin T (ThT), 1 mM ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA), and 0.001% SDS.…”

Applications of catalyzed cytoplasmic disulfide bond formation

“…Subsequent publications sometimes replaced the oxidase (Erv1p) with either another sulfhydryl oxidase (e.g. QSox [11,12,13] or inverted the E.coli periplasmic transmembrane disulfide bond forming enzyme DsbB [14] such that its active site pointed to the cytoplasm or used a transmembrane disulfide bond forming enzyme VKOR from a hyperthermophilic organism that naturally makes disulfide bonds in the cytoplasm [14]. However, Erv1p remains the most widely used oxidase in engineered systems for cytoplasmic disulfide bond formation.…”