Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesis.
The cellular prion protein (PrP(C)) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrP(C) functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrP(C), the newly synthesized GFP-PrP(C)K81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrP(C), the mature GFP-PrP(C)K81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrP(C) at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrP(C)DeltaGPI and GFP-PrP(C)octa mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrP(C) for its correct biosynthesis.
van Rosmalen
Jos W G
JW
Department of Molecular Animal Physiology, Nijmegen Center for Molecular Life Sciences, Institute for Neuroscience, Faculty of Science, Radboud University Nijmegen, 6525 GA Nijmegen, The Netherlands.
Martens
Gerard J M
GJ
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D049452:Green Fluorescent Proteins; D047468:Immunoprecipitation; D052717:Melanotrophs / Q000378:metabolism* / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission; D016296:Mutagenesis; D009154:Mutation; D011328:Prions / Q000096:biosynthesis* / Q000235:genetics; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014982:Xenopus laevis
D017261:Glycosylphosphatidylinositols; D011328:Prions; D049452:Green Fluorescent Proteins; D011333:Pro-Opiomelanocortin