Comparative enzymology of native and recombinant house dust mite allergen Der p 1.
BACKGROUND
The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool.


METHODS
Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined.


RESULTS
Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism.


CONCLUSIONS
The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens.
Zhang
J
J
Ion Channels and Cell Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, London, UK.
Saint-Remy
J-M
JM
Garrod
D R
DR
Robinson
C
C
D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry*; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D055808:Drug Discovery; D004591:Electrophoresis, Polyacrylamide Gel; D039981:Pyroglyphidae / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1