Use of thermolysin in the diagnosis of prion diseases

Abstract

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.

Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

“…Here, we quantified the densitometric ratio of thermolysin-resistant PrP Sc generated by mb-PMCA to the total amount of PrP. In a mixture such as a PMCA product containing both PrP Sc and PrP C , thermolysin is known to preserve protease-sensitive and protease-resistant PrP Sc species while destroying PrP C (55, 56) and thus may allow reliable assessment of the conversion yield.…”

Molecular Profiling of Ovine Prion Diseases by Using Thermolysin-Resistant PrP Sc and Endogenous C2 PrP Fragments

“…Western blots of the cerebellar digests from animals 0456 and 0284 were also probed with monoclonal antibody L42, confirming the absence of thermolysin-resistant PrP within these samples under the assay conditions used (data not shown). It should be noted that a 7-kDa P4-reactive band was present in all samples, including those from TSE-affected and healthy animals (data not shown), and represents an N-terminal fragment of PrP that does not contain favored thermolysin cleavage sites (31). To further investigate the presence of disease-associated PrP species within the cerebellum of animals 0284 and 0456, homogenates were analyzed on sucrose gradients (Fig.…”

Concentration of Disease-Associated Prion Protein with Silicon Dioxide

“…Resulting proteaseresistant PrP Sc , or PrP res , is then detected immunologically, usually after denaturation. A further protease, thermolysin, also differentiates PrP Sc from PrP C but, unlike PK, is capable of leaving PrP Sc intact without truncation of the N-terminus [2,3].…”