Molecular characterization of the group 4 house dust mite allergen from Dermatophagoides pteronyssinus and its amylase homologue from Euroglyphus maynei.
BACKGROUND
Of the ten recognised groups of Dermatophagoides pteronyssinus allergens, the group 4 is the only group that has not been characterised at the molecular level.


METHODS
Primers were designed to PCR amplify Der p 4 (D. pteronyssinus) and Eur m 4 (Euroglyphus maynei) cDNA. These fragments were used to screen the corresponding cDNA libraries and the cDNA clones obtained were subsequently sequenced. The coding regions of Der p 4 and Eur m 4 were cloned into the pET expression vector and recombinant histidine-tagged proteins expressed in Escherichia coli.


RESULTS
cDNA clones which included the mature protein coding sequence for Der p 4 and Eur m 4 were sequenced. The Der p 4 and Eur m 4 genes were found to code for 496 amino acid mature proteins with residues important for the function of alpha-amylase highly conserved. Der p 4 and Eur m 4 were calculated to be 90% identical and a BLAST search of the GenBank database found these sequences to be approximately 50% identical to insect and mammalian alpha-amylases. The calculated molecular weights of Der p 4 and Eur m 4 were approximately 57,000, although recombinant Der p 4 and Eur m 4 migrate on SDS-PAGE at about 60,000. Der p 4 recombinant protein was found to bind specific IgE in 3 of the 10 house dust mite allergic patients tested.


CONCLUSIONS
This paper describes the first cDNA sequence of Der p 4 and Eur m 4 confirming that this allergen is house dust mite alpha-amylase.
Mills
K L
KL
TVW Telethon Institute for Child Health Research, West Perth, Australia.
Hart
B J
BJ
Lynch
N R
NR
Thomas
W R
WR
Smith
W
W
D000485:Allergens / Q000276:immunology; D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics; D000818:Animals; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D001483:Base Sequence; D004391:Dust / Q000032:analysis; D015723:Gene Library; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D019476:Insect Proteins; D008925:Mites / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D004391:Dust; C079269:Eur m I protein, Euroglyphus maynei; D006023:Glycoproteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D000681:Amylases