Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites.
ChPrP is the chicken homologue of PrPC, the cellular isoform of the mammalian prion protein. We have used sequence-specific antibodies to immunoprecipitate and immunoblot chPrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. We have also used mass spectrometry to characterize fragments of the protein purified from conditioned medium. The majority of chPrP protein present in neuroblastoma cells and on isolated brain membranes can be released by incubation with phosphatidylinositol-specific phospholipase C, indicating that these molecules are attached to the cell surface by a glycosylphosphatidylinositol anchor. Surprisingly, most of the surface-anchored molecules are truncated at their N-terminus distal to the proline/glycine-rich repeats. The corresponding N-terminal fragments are found in medium conditioned by neuroblastoma cells, as well as in cerebrospinal fluid and a postmicrosomal supernatant of brain. One of these fragments extends from Lys25 to Phe116. 35-45-kDa forms of chPrP that can be metabolically labeled with [3H]ethanolamine can also be found in extracellular media. We propose that the chPrP molecule undergoes at least two cleavages as part of its normal metabolism: one within the glycosylphosphatidylinositol anchor and one within or just N-terminal to the central hydrophobic domain. The second cleavage lies within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that may have physiological as well as pathological significance.
Harris
D A
DA
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Huber
M T
MT
van Dijken
P
P
Shyng
S L
SL
Chait
B T
BT
Wang
R
R
D000818:Animals; D001921:Brain / Q000378:metabolism; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D007106:Immune Sera; D051379:Mice; D018620:PrPSc Proteins; D011233:Precipitin Tests; D011328:Prions / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014407:Tumor Cells, Cultured
D017261:Glycosylphosphatidylinositols; D007106:Immune Sera; D018620:PrPSc Proteins; D011328:Prions