Use of thermolysin in the diagnosis of prion diseases.
The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala, and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPC into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27-30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.
Owen
Jonathan P
JP
ADAS UK, Department of Biology, Adrian Building, University of Leicester, University Road, Leicester, LE1 7RH, UK.
Maddison
Ben C
BC
Whitelam
Garry C
GC
Gough
Kevin C
KC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001709:Biotechnology; D001923:Brain Chemistry; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis; D019286:Endopeptidase K / Q000378:metabolism; D018604:Epitope Mapping; D066298:In Vitro Techniques; D019151:Peptide Library; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017096:Prion Diseases / Q000175:diagnosis / Q000662:veterinary*; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012608:Scrapie / Q000175:diagnosis; D012756:Sheep; D013820:Thermolysin / Q000378:metabolism*
D000596:Amino Acids; D019151:Peptide Library; D018620:PrPSc Proteins; D011328:Prions; D019286:Endopeptidase K; D013820:Thermolysin