Comparative two-dimensional mapping of prion protein isoforms in human cerebrospinal fluid and central nervous system.
The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. Although its precise function is unknown, PrP(C) represents the substrate for the generation of a conformational pathogenic isoform (PrP(Sc)) in human and animal transmissible spongiform encephalopathies, or prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrP(C) by immunoblot of two-dimensional (2-D) gel electrophoresis preparations, using specific antibodies. Here, we show that PrP(C) from brain and CSF is composed of several charge isomers of differently glycosylated isoforms of the full-length PrP(C) and two N-terminally truncated fragments of 20 and 18 kDa. In the CSF, substantial amounts of the highly glycosylated PrP(C) isoforms and of the unglycosylated 18 kDa fragment are detected. Our study, for the first time, provides a detailed 2-D map of human PrP(C) both in brain and CSF, and establishes an innovative and sensitive method that might help in detecting the CSF pathological PrP(Sc) isoform in vivo. It also shows the incredible microheterogeneity of such isoforms (ca. 60 spots!), as revealed in 2-D mapping, as opposed to 3-4 main zones by mono-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Castagna
Annalisa
A
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Campostrini
Natascia
N
Farinazzo
Alessia
A
Zanusso
Gianluigi
G
Monaco
Salvatore
S
Righetti
Pier Giorgio
PG
D001923:Brain Chemistry; D007562:Creutzfeldt-Jakob Syndrome / Q000134:cerebrospinal fluid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D010449:Peptide Mapping / Q000379:methods; D018621:PrPC Proteins / Q000032:analysis*; D018620:PrPSc Proteins / Q000032:analysis* / Q000134:cerebrospinal fluid; D020033:Protein Isoforms / Q000032:analysis
D018621:PrPC Proteins; D018620:PrPSc Proteins; D020033:Protein Isoforms