Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein

“…Antibodies should be a powerful tool to solve this question. Despite the insufficient biochemical characterization of PrP Sc , many attempts have been made to establish antibodies with fine specificity using mice immunized with partially purified PrP Sc from prion-infected cells or brain extracts (6,10,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). However, most antibodies were specific to PrP C or cross-reactive to both PrP C and PrP Sc but not monospecific to PrP Sc , although more recently a PrP Sc -specific murine IgG1 antibody, W261, has been established (10).…”

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2

“…Helix 1 of mouse PrP is located in 1 of these recognition regions, which has 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) [14]. In our previous study, we reported that the mutations introduced into Asp146 and Arg150 decrease the reactivity of the resultant protein with the T2 antibody [13]. To systematically analyze the influence of these 2 salt bridges on the recognition of the PrP120-230 fragment by the T2 antibody, we introduced mutations into the 2 salt bridge residues (Asp143-Arg147 and Asp146-Arg150) to change their charges to be neutral or significantly reduced ( Fig.…”

“…The T2 antibody recognizes 2 contiguous regions of the PrP120-230 fragment located far apart from each other along the primary structure, indicating that the antibody recognizes the tertiary structure of PrP120-230 [13]. Helix 1 of mouse PrP is located in 1 of these recognition regions, which has 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) [14].…”

“…Our previous experiments have revealed that 4 mutations (C178M, C213M, D146N, and R150H) reduced PrP reactivity with the T2 antibody compared to wild-type PrP; in particular, the salt bridge mutants D146N and R150H were found to have almost completely lost reactivity to the T2 antibody [13]. Most specific antibodies, which inhibit the conversion and accumulation of PrP Sc , interact with the helix 1 region of PrP C [7][8][9][10][11], which is the case for the T2 antibody [13]. Therefore, characterizing the mechanisms by which the T2 antibody recognizes the salt bridges will provide an insight into the PrP C -to-PrP Sc conversion mechanism.…”

“…Additionally, it was reported that the T2 antibody inhibits PrP Sc accumulation in cultured cells [12]. Recently, we reported that this antibody recognized both a disulfide bond and salt bridges in PrP [13]. Nuclear magnetic resonance (NMR) analysis has revealed that the recombinant mouse PrP fragment PrP120-230 consists of 2 short β-strands (strand 1, 126-129 and strand 2, 159-162) and 3 α-helices (helix 1, 142-152; helix 2, 177-191; and helix 3,[198][199][200][201][202][203][204][205][206][207][208][209][210][211][212][213][214][215] [14].…”