NR AWLD
AU Notari,S.; Capellari,S.; Langeveld,J.P.M.; Giese,A.; Gambetti,P.; Kretzschmar,H.A.; Parchi,P.
TI Human PrPsc "typing" pitfalls associated with the use of type 1 selective antibodies combined with relative inefficient hydrolysis of PrPsc by proteinase K
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions PR-27
PT Konferenz-Poster
AB The bulk of current molecular CJD classification is based on the detection of either one of two protease-resistant PrPsc core fragments which differ in molecular mass and primary site of Proteinase K (PK) cleavage (i.e. 21 kDa and residue 82 for type 1, 19 kDa and residue 97 for type 2). Using antibodies recognizing an epitope between residues 82 and 96 (not detecting type 2), Polymenidou et al and Yull et al recently found at least some type 1 in all type 2 samples, and reached the conclusion that multiple PrPsc types regularly coexist in CJD. At variance, we explored the possibility that the "type 1-like"/type 2 co-occurrence detected by these selective antibodies represents, in most cases, an inefficient hydrolysis of type 2 rather than real, strain related, type 1. To verify this hypothesis we analysed the PrPsc core in 50 sCJD and 3 vCJD subjects using a wide range of PK activity, 15 cm long gels with high resolution, and both 3F4 and 12B2 (epitope 89-93) antibodies. When exposed to a relatively moderate PK activity (i.e. 1U/ml corresponding to 50 g/ml when specific PK activity is 20 U/ml, for 1 h at 37 deg C, pH 6.9, 6 mg proteins/ml) both types 1 and 2 showed, in addition to the 21 or 19 kDa band respectively, a tight group of slower migrating bands. In type 2, 12B2 only recognized these bands which appeared as a single one, migrating similarly to type 1, in a standard 5-7 cm gel. The degree of PK resistance of these fragments ("type 1-like") was significantly lower than that of types 1 or 2 resistant cores. We conclude that the "type 1-like" signal detected by the 12B2 antibody in CJD type 2 is not a PrPsc resistant-core but instead matches the physicochemical properties of partially cleaved fragments with ragged N-terminus generated in both types 1 and 2 by relative inefficient hydrolysis.
AD S. Notari, S. Capellari, P. Parchi: Dipartimento di Scienze Neurologiche, Università di Bologna, Italy; J.P. Langeveld: CIDC-Lelystad, The Netherlands; A. Giese, H.A. Kretzschmar: Institut fuer Neuropatologie, LMU Muenchen, Germany; P. Gambetti: Institute of Pathology, CWRU, Cleveland, OH, USA
SP englisch
PO Italien