Proteolytic cleavage and shedding of the bovine prion protein in two cell culture systems.
We have compared the processing, turnover and release of bovine PrP (boPrP) in transfected baby hamster kidney (BHK) and mouse neuroblastoma (N2a) cells. In BHK cells, boPrP was subjected to two distinct proteolytic cleavage events, the first was mapped between K(121) and H(122) generating an N-terminal and a C-terminal PrP fragment. Transport block experiments, cell surface biotinylation and PIPLC analyses showed that the bulk of boPrP on the cell surface was the C-terminal fragment and indicated that the first cleavage of boPrP took place prior to or very soon after it appears at the cell surface. The second cleavage was situated at the extreme C-terminus of the boPrP GPI-anchored C-terminal fragment and as a result of this was shed into the medium rapidly. The kinetics, the migration in SDS-PAGE of the released fragment and protease inhibition studies indicate that a proteolytic activity was responsible for the release of the boPrP fragment from its GPI-anchor. Both N- and C-terminal fragments of boPrP could be detected in the medium. Moreover, in normal bovine brain, a C-terminal fragment was identified, suggesting that similar proteolytic processing events occur in vivo. In N2a cells, the majority of boPrP was subjected to a more complete degradation process, and only trace amounts of full length boPrP was shed into cell culture medium in a process which also indicated a release by proteolytic cleavage.
Zhao
Hongxing
H
Department of Molecular Biosciences, MBV, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Biomedical Centre, Box 588, S-751 23 Uppsala, Sweden.
Klingeborn
Mikael
M
Simonsson
Magnus
M
Linné
Tommy
T
D000818:Animals; D002417:Cattle; D006224:Cricetinae; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D012995:Solubility; D013045:Species Specificity
D011328:Prions; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase