Novel Prion Protein in BSE-affected Cattle, Switzerland
Figure. Molecular typing of the pathologic prion protein from 2 cows with bovine spongiform encepalopathy (BSE), Switzerland. Brain tissue homogenates of medulla oblongata from cows 1 and 2 and from controls with C-BSE (Switzerland), H-BSE, and L-BSE (Poland ), and a BSE-negative sample were treated with proteinase K, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto membranes according to the protocol of the Prionics AG (Schlieren, Switzerland) Check Western test. On the basis of the antibody binding results, the N terminus of the PrPres fragment in the samples from cows 1 and 2 lies between aa 111 and aa 154. Predicted PrPres fragments of C-BSE, H-BSE (PrPres 1 and 2), and L-BSE were adopted from the literature (2,6). A) Epitope mapping using antibodies 9A2, Sha31, 94B4, and JB10. B) Confirmatory Western blotting using antibody 6H4. C) Comparison PrPres in cow 2 and H-BSE with (+) and without (–) deglycosylation with PNGaseF (antibody 94B4, SDS-PAGE with NuPAGE MES instead of NuPAGE MOPS running buffer [Invitrogen, Carlsbad, CA, USA]). PrPres 1 and 2 in H-BSE samples are indicated. Molecular mass standards are shown in kiloDaltons. D) The illustration at the top represents the full-length, mature bovine prion protein and the binding sites of the antibodies used for epitope mapping (black boxes). N-terminal and C-terminal residues are indicated by numbers. PrPres fragments are partially mono- and di-glycosylated, which results in the characteristic 3-band patterns in the Western immunoblot. Sites of N-linked glycosylation are shown at positions 192 and 208 (-GlcNAc). C-BSE, classic BSE; cow 1, an 8-year-old BSE-positive cow; cow 2, a 15-year-old BSE-positive cow; PrPres, proteinase K–resistant fragment of the prion protein; H-BSE, atypical BSE with higher molecular mass of PrPres; L-BSE, atypical BSE with lower molecular mass of PrPres. Lane 1, C-BSE; lane 2, cow 1; lane 3, cow 2; lane 4, H-BSE; lane 5, L-BSE; lane 6, negative.