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Cloning and sequencing of the Dermatophagoides pteronyssinus group III allergen, Der p III
Abstract:
House dust mites are widely recognized as major factors involved in the triggering of allergic diseases such as asthma. It is now apparent that the group III allergens of the Dermatophagoides mite species may play a significant role in a number of house dust mite allergic cases. Natural Der p III was isolated by gel filtration of salt precipitated Dermatophagoides pteronyssinus extract and as reported previously ran as a doublet of Mr 28 and 30 K on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Natural Der fIII was isolated by affinity purification with the 5A12 monoclonal antibody. Amino acid sequence data was generated for both these proteins which was used to construct DNA probes to screen a Dermatophagoides pteronyssinus cDNA library by hybridization and resulted in the isolation of a recombinant Der p III cDNA clone, P3WS1. The 1059 bp cDNA fragment included a 786 bp open reading frame which encodes a pre-pro region of 29 amino acids and a mature protein of 232 amino acids with a calculated Mr 24,985. A search of the BLAST protein database has confirmed that the Der pIII P3WS1 clone is approximately 50% homologous with other trypsin proteins. We have confirmed with both our natural protein sequence and the P3WS1 amino acid sequence data that the group III allergens are trypsin-like proteins.
Insights
House dust mite allergens, specifically Group III Dermatophagoides allergens, are linked to asthma and allergic diseases. Research confirms these allergens are trypsin-like proteins, aiding in understanding allergic responses.
Area of Science:
- Immunology
- Molecular Biology
- Allergology
Background:
- House dust mites are significant triggers for allergic diseases like asthma.
- Group III allergens from Dermatophagoides mite species are implicated in house dust mite allergies.
Purpose of the Study:
- To isolate and characterize Group III allergens from Dermatophagoides pteronyssinus (Der p III) and Dermatophagoides farinae (Der f III).
- To determine the protein sequence and genetic basis of Der p III.
- To investigate the enzymatic nature of Group III allergens.
Main Methods:
- Isolation of natural Der p III and Der f III using gel filtration and monoclonal antibody affinity purification.
- Amino acid sequencing of natural allergens.
- Construction of DNA probes and screening of a cDNA library for Der p III.
- Sequencing of the recombinant Der p III cDNA clone (P3WS1).
- BLAST protein database search for homology analysis.
Main Results:
- Natural Der p III appeared as a doublet (28 and 30 kDa) on SDS-PAGE.
- A recombinant Der p III cDNA clone (P3WS1) of 1059 bp was isolated, containing a 786 bp open reading frame.
- The P3WS1 clone encodes a mature protein of 232 amino acids with a calculated molecular weight of 24,985 Da.
- Sequence analysis confirmed Der p III is approximately 50% homologous to other trypsin proteins.
- Both natural and recombinant protein sequences indicate Group III allergens are trypsin-like.
Conclusions:
- Group III allergens from Dermatophagoides mites are trypsin-like proteins.
- The characterization of Der p III provides insights into the molecular mechanisms of house dust mite allergy.
- Understanding the enzymatic nature of these allergens may inform future therapeutic strategies for allergic diseases.