NR AOTM
AU Groschup,M.H.; Geissen,M.; Neuendorf,E.; Garner,K.; Eiden,M.; Mella,H.; Schätzl,H.M.; Saalmüller,A.; Reifenberg,K.; Pfaff,E.
TI Involvement of structural regions and glycan attachments of PrP in the conversion process
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Oral sessions OS-07
PT Konferenz-Vortrag
AB
The conversion of the cellular prion protein (PrPc or PrPsen) into a proteinase K resistant conformer designated PrPsc or PrPres which is rich in b-sheet structure is a central event in prion diseases. PrPc is composed largely of two structural areas, the flexible non-structured amino-terminus (up to amino acid (aa) 120) and the structured carboxy-terminal region (~aa 121-230). The carboxy-terminal region comprises three a-helices (spanning aa 144-154, aa 174-192 and aa 199-218) and two short anti-parallel ß-sheets (aa 127-130, aa 160-163). Helices II and III are connected by a disulfide bond (C178 and C213), and helix II bears a N-glycosylation at N180. The second N-glycosylation is sited at N196, with a heterogenous population of glycans at both sites. However, it is still unclear to which extend particular PrP subdomains and the glycan attachments contribute to the conversion process and infection cycle of prions.
To get a better understanding of the involved protein structures, we have introduced mutations at either one of the two glycosylation consensus sites. These mutations rendered PrPc molecules glycosylation defective, respectively. Using the scrapie infected and non-infected neuroblastoma cell system as well as by generating transgenic mice, we could show that both glycosylation sites are non-essential for the conversion process itself and for de-novo infections with BSE or scrapie.
Moreover, we have generated a series of insertion mutants, where a heptapeptide insert was introduced at evenly spaced sites through out the whole sequence of murine 3F4-tagged PrP. This insertion led to deviant cellular processing in some mutant proteins while others behaved similar to 3F4moPrP as judged by FACS and western-blotting analysis. Furthermore mutants were probed in in vitro conversion assays (ScN2a and SMB cell cultures) and in a cell-free conversion system. Four regions were identified where the insertion of a foreign sequence still allowed conversion: the amino-terminal region; the stretch just before the second ß-sheet; the loop region between helix II and III and the carboxy-terminus past helix III. A number (but not all) of non-convertible insertion mutants displayed a dominant negative inhibition of the wild-type PrP conversion in infected cells. Influences of the site of mutation on cellular processing, conversion and inhibitory effects are discussed in the context of homology structure models of PrP mutants.
AD Martin H. Groschup, Markus Geissen, Erdmute Neuendorf, Kathleen Garner, Martin Eiden, Harriet Mella, Federal Research Centre for Virus Diseases of Animals, Greifswald-Island of Riems, Germany; Hermann Schätzl, Technical University Munich, Germany; Armin Saalmüller, Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany; Kurt Reifenberg, Eberhardt Pfaff, Johannes-Gutenberg-University, Mainz, Germany
SP englisch
PO Deutschland