Related Experiment Videos
A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae
G A Stewart1, M R Kollinger, C M King
1Western Australian Research Institute for Child Health, Princess Margaret Hospital, Subiaco, Perth.
Abstract:
Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.
Insights
Dust mites Dermatophagoides pteronyssinus and D. farinae possess multiple serine proteases, including a newly identified elastase-like enzyme. This enzyme, distinct from known allergens like trypsin and chymotrypsin, shows higher activity in mite fecal extracts.
Area of Science:
- Allergen research
- Biochemistry
- Immunology
Background:
- Dust mites Dermatophagoides pteronyssinus and D. farinae are significant sources of allergens.
- Known allergenic serine proteases include mite trypsin (Group III) and chymotrypsin (Group VI).
- Other serine proteases exist in mites, but their characterization is less complete.
Purpose of the Study:
- To identify and characterize other serine proteases in dust mites.
- To differentiate a novel elastase-like enzyme from known mite proteases.
- To investigate the distribution and activity of mite serine proteases in different extracts.
Main Methods:
- Enzyme characterization using substrate specificity and inhibition assays.
- Molecular mass determination via gel filtration.
- Isoelectric focusing for isoform analysis.
- Detection and activity measurement in whole mite and fecal extracts.
- Immunochemical analysis using a monoclonal antibody.
Main Results:
- An elastase-like serine protease was identified in both D. pteronyssinus and D. farinae.
- This enzyme differs from chymotrypsin in charge, substrate specificity, and cation inhibition.
- Its molecular mass is approximately 30 kDa, with distinct isoelectric points (10.5 for D. pteronyssinus, 9.8 for D. farinae).
- Trypsin and the elastase-like enzyme showed higher activity in fecal extracts compared to whole mite extracts.
- A monoclonal antibody for Group III allergen cross-reacted with mite trypsin and slightly with chymotrypsin.
Conclusions:
- Dust mites contain an elastase-like serine protease distinct from known allergens.
- Protease activity, particularly trypsin and elastase-like enzyme, is concentrated in mite fecal material.
- Culture conditions may influence protease production in dust mites.
- Limited cross-reactivity exists between mite trypsin, chymotrypsin, and Group III allergen.