Trypsin-like protease of mites: purification and characterization of trypsin-like protease from mite faecal extract Dermatophagoides farinae. Relationship between trypsin-like protease and Der f III.
A serine protease from mite faecal extract, Dermatophagoides farinae, was purified using DEAE-Sephacel anion exchange chromatography and Superdex 75 pg gel chromatography. The molecular weight of this protease was 34 kD on SDS-PAGE under reducing conditions. The optimal pH and temperature of the protease were 8.0 and 47 degrees C, respectively. In addition, this protease cleaved arginyl or lysyl residue containing substrates selectively and was only inhibited by aprotinin, FUT-175, and soy bean trypsin inhibitor and not by chymostatin, E-64 and iodoacetic acid. These results show that our purified serine protease belongs to the trypsin-type. Purified trypsin-like protease was shown to be allergenic by enzyme-linked immunosorbent assay. Antigenicity of trypsin-like protease was completely different from those of Der f I and Der f II. Both, 20 N-terminal amino acid sequence and amino acid compositions of the purified protease were very similar to those of Der f III. Good similarities were found between trypsin-like protease and Der f III concerning physicochemical properties such as molecular weight on SDS-PAGE and ammonium sulphate solubility. Summarizing the above data, it can be concluded that a trypsin-like protease from mite faecal extract is actually the Der f III allergen and that it may be involved in the digestive process of the mite as it was found not in mite body but in mite faeces.
Ando
T
T
Section of Allergology, Research Laboratories, Torii & Co. Ltd, Ichikawa, Japan.
Homma
R
R
Ino
Y
Y
Ito
G
G
Miyahara
A
A
Yanagihara
T
T
Kimura
H
H
Ikeda
S
S
Yamakawa
H
H
Iwaki
M
M
D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D052179:Antigens, Plant; D007611:Aprotinin / Q000494:pharmacology; D060829:Arthropod Proteins; D001550:Benzamidines; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004064:Digestive System / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000201:enzymology; D005260:Female; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008875:Middle Aged; D008925:Mites / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000485:Allergens; D000596:Amino Acids; D052179:Antigens, Plant; D060829:Arthropod Proteins; D001550:Benzamidines; D006146:Guanidines; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D007611:Aprotinin; C409649:Der f III allergen; D012697:Serine Endopeptidases; C032855:nafamostat