Activity profile of dust mite allergen extract using substrate libraries and functional proteomic microarrays.
Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.
Harris
Jennifer
J
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, San Diego, California 92121, USA. harris@scripps.edu
Mason
Daniel E
DE
Li
Jun
J
Burdick
Keith W
KW
Backes
Bradley J
BJ
Chen
Teresa
T
Shipway
Aaron
A
Van Heeke
Gino
G
Gough
Lucy
L
Ghaemmaghami
Amir
A
Shakib
Farouk
F
Debaene
François
F
Winssinger
Nicolas
N
D000595:Amino Acid Sequence; D039741:Antigens, Dermatophagoides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D019151:Peptide Library; D025941:Protein Interaction Mapping / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D039741:Antigens, Dermatophagoides; D019151:Peptide Library; D017346:Protein Serine-Threonine Kinases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases