Cryptic epitopes in N-terminally truncated prion protein are exposed in the full-length molecule: dependence of conformation on pH.
Prion diseases are diseases of protein conformation. Structure-dependent antibodies have been sought to probe conformations of the prion protein (PrP) resulting from environmental changes, such as differences in pH. Despite the absence of such antibodies for full-length PrP, a recombinant Fab (D13) and a Fab derived from mAb 3F4 showed pH-dependent reactivity toward epitopes within the N-terminus of N-terminally truncated PrP(90-231). Refolding and maintaining this protein at pH > or =5.2 before immobilization on an ELISA plate inhibited reactivity relative to protein exposed to pH < or =4.7. The reactivity was not affected by pH changes after immobilization, showing retention of conformation after binding to the plate surface, although guanidine hydrochloride at 1.5-2 M was able to expose the cryptic epitopes after immobilization at pH > or =5.2. The alpha-helical CD spectrum of PrP(90-231) refolded at pH 5.5 was reduced somewhat by these pH changes, with a minor shift toward beta-sheet at pH 4 and then toward coil at pH 2. No covalent changes were caused by the pH differences. This pH dependence suggests titration of an acidic region that might inhibit the N-terminal epitopes. A similar pH dependence for a monoclonal antibody reactive to the central region identified an acidic region incorporating Glu152 as a significant participant.
Matsunaga
Y
Y
Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94143-0446, USA.
Peretz
D
D
Williamson
A
A
Burton
D
D
Mehlhorn
I
I
Groth
D
D
Cohen
F E
FE
Prusiner
S B
SB
Baldwin
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D002942:Circular Dichroism; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D008647:Mesocricetus; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D011328:Prions / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; D019791:Guanidine