Protease activity of Der p 1: Cysteine, serine or both?

Abstract

Rationale Shared sequence homology with the enzyme papain has identified the major house dust mite allergen, Der p 1, as a cysteine protease. Immunoaffinity-chromatography purified Der p 1, however, has both cysteine and serine activity. The serine activity is considered a contaminant and removed; yet its identity has not been elucidated. We have evaluated the protease activity (PA) of immunopurified Der p 1 and assessed its purity. Methods PA of Der p 1 was calculated by measuring the rate of hydrolysis of N-benzoyl-Phe-Val-Arg-p-nitroanilide. Purity was assessed by SDS-PAGE, Western blot analysis, N-terminal amino-acid sequencing, mass spectrometry and 2D gel electrophoresis. Results PA of Der p 1 was inhibited by both E64 (21.4%) and APMSF (84.13%), specific cysteine and serine protease inhibitors. SDS-PAGE and silver stain analysis demonstrated a single band and the protein was recognised by the monoclonal antibodies 4C1, 5H8 and 10B9 on Western blot. Its N-terminal matched the published primary sequence: TNACSINGNA. MALDI-TOF mass spectrometry of both tryptic peptides and 2D gel electrophoresis separated material, resulted in 8 matched sequences with Der p 1, encompassing 49% overall sequence coverage. No other protein sequences could be detected. Conclusions Immunopurified Der p 1 has both cysteine and serine protease activity. A contaminating source to account for the serine activity, however, is not evident. This would suggest that Der p 1 may represent a novel protease with a mixed mechanistic class of action.