Reactivities Of The Recombinant Prion Protein Of Myodes Glareolus And Effects Of Two Amino-acid Alternations In RT-QuIC

Prion diseases,also known as transmissible spongiform encephalopathy?TSEs?,are a group of rare,fatal neurodegenerative diseases,including Creutzfeldt-Jakob disease?CJD?,Kuru,fatal familial insomnia?FFI?and--Scheinker syndrome?GSS?in human,bovine spongiform encephalopathy?BSE?in cow,scrapie in sheep and so on.The clinical manifestations of TSEs include progressive dementia,ataxia,tremor,cognitive impairment,and eventually lead to death.Pathological features show characteristic spongy change,loss of neurons and glial cell proliferation in central nervous system,etc.A possible pathogenesis of TSEs is a transformation of normal prion protein?PrP^C?into an abnormal form(PrP^Sc)via misfolding of the protein and the accumulation of the abnormal prion protein in central nervous system,eventually leading to neuron death.Bank vole is susceptible for the infections of the prions from many different species.The mature PrP protein?aa23-230?of Myodes glareolus?bank vole?shows two amino-acid differences at the C-terminus of aa227 and aa230 compared with that of the mouse and hamster.To assess the potential influence of these two amino acids on the reactivity in RT-QuIC,two PrP mutants in Myodes glareolus,Vole-E227D and Vole-S230R,were generated,in which the amino acids of PrP at aa227 and aa230 were replaced with those of the hamster and mouse.Together with the PrP of Myodes glareolus?Vole-WT?and PrP from a hamster?Ha-WT?,the reactivities of Vole-E227D and Vole-S230R in RT-QuIC using a strain?263K?adapted from a hamster suffering from scrapie as the seed were tested.The expressions of PrP constructs were performed in TB medium.The denatured PrP proteins were purified by chromatograph of Ni-NTA Superflow resin,in a column.RT-QuIC was conducted with a black 96-well,optical-bottomed plate on a BMG FLUO star plate reader?BMG LABTECH?.To calculate the main biochemical parameters of the PrP proteins,such as theoretical p I and hydropathicity,an open-accessible website ExPASy was used.All three PrPs from Myodes glareolus showed much stronger reactivities in RT-Qu IC than the PrP from a hamster,even under attenuated reaction conditions.Moreover,compared with Vole-WT,the RT-Qu IC reactivity of Vole-E227D was stronger,whereas that of Vole-S230R was slightly weaker.Our results indicate that exchange of amino acids at positions aa227 and aa230 in the PrP of Myodes glareolus with hamster-and mouse-derived ones have little influence on their strong RT-QuIC reactivities.Nevertheless,special amino-acid changes at these two positions offer the constructs limited but stable RT-QuIC reactivity.This result also shows that Vole PrP protein has a strong tendency to fibrillate in RT-QuIC,and even spontaneous fibrosis,this characteristic may be the reason that bank voles are susceptible to many strains,but it also affects the application of protein in the future diagnosis of CJD disease.