A novel epitope for the specific detection of exogenous prion proteins in transgenic mice and transfected murine cell lines.
Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow future in vitro conversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPC includes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPC was strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPC was correctly posttranslationally processed and translocated to the cell surface.
Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, 72076 Tübingen, Germany.
I
I
Vorberg
A
A
Buschmann
S
S
Harmeyer
A
A
Saalmüller
E
E
Pfaff
M H
MH
Groschup
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002415:Cats; D002417:Cattle; D006224:Cricetinae; D004285:Dogs; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006041:Goats; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012756:Sheep; D014162:Transfection; D014364:Tryptophan / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D011328:Prions; D014443:Tyrosine; D014364:Tryptophan