Characterization of the prion protein 3F4 epitope and its use as a molecular tag.
The monoclonal antibody (MAb) 3F4 has for nearly two decades been one of the most commonly used tools in prion research. This MAb has contributed significantly to our understanding of the normal cell biology of the prion protein (PrP(C)), as well as the disease related abnormalities occurring in prion diseases. The 3F4 antibody binds strongly to human and hamster PrP, with a specific requirement of two Met residues at positions 109 and 112 in the human PrP. Other species in which PrP lack one of the Met residues, like cattle and sheep, or both, like rat and mouse, do not react with the 3F4 antibody. These and other observations have led to the commonly accepted notion that the 3F4 epitope consists of the tetra-peptide Met-Lys-His-Met. In this study, we have identified the minimal epitope for 3F4 by studying its binding to synthetic peptides and by analysis of mutated ovine PrP::GFP constructs expressed in cell culture. We have found that the 3F4 epitope consists of a hepta-peptide (Lys-Thr-Asn-Met-Lys-His-Met), which in sheep encompass residues 109-115. We found that Lys 109 is critically important for 3F4 binding, as omission, or substitution of this residue to Ala resulted in no binding. We also demonstrate that the hepta-peptide constituting the minimal 3F4 epitope, can be used as a discrete, moveable high-affinity molecular tag. Thus, the 3F4 antibody can find its use beyond prion research.
Lund
Christoffer
C
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway.
Olsen
Christel Moraeus
CM
Tveit
Heidi
H
Tranulis
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000915:Antibody Affinity; D001665:Binding Sites / Q000276:immunology; D045744:Cell Line, Tumor; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007118:Immunoassay / Q000379:methods; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D015336:Molecular Probe Techniques; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D017096:Prion Diseases / Q000175:diagnosis / Q000276:immunology; D011328:Prions / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D011328:Prions; D008239:Lysine