Cockroach allergen Bla g 2: An unusual aspartic proteinase
Received 10 February 2005; received in revised form 24 March 2005; accepted 18 April 2005. published online 31 May 2005.
Background
Enzymatic activity of mite, fungal, and bee venom allergens is thought to potentiate their allergenicity. Bla g 2 is a potent cockroach allergen, but despite sharing sequence homology with aspartic proteinases, it contains critical amino acid substitutions that impair proteolytic activity. The biologic function of Bla g 2 remains unclear.
Objective
We sought to investigate the effects of specific amino acid substitutions on enzymatic activity, and the peptide-binding capability of Bla g 2.
Methods
Site-directed mutagenesis was used to produce a recombinant Bla g 2 mutant (Mut) with corrected canonical triads and a flap region. Another mutant (MutF−) was expressed after additional mutations in the flap region of Mut. Bla g 2 wild-type (Wt), Mut, and MutF− were assayed for aspartic proteinase activity, and Bla g 2 Wt was tested for pepstatin binding.
Results
Recombinant Bla g 2 Wt and Mut did not show enzymatic activity in a milk-clotting and hemoglobin assay. By using a modified hemoglobin assay, residual activity inhibited by pepstatin was detected for MutF− and Wt at 20 μg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific.
Conclusion
Residual proteolytic activity was found for Bla g 2 at concentrations of approximately 4 mM. This weak activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Bla g 2. Bla g 2 has a cleft that might specifically bind ligands other than pepstatin.
Presented in part at the 60th annual meeting of the American Academy of Allergy, Asthma and Immunology, San Francisco, CA, March 19-23, 2004.
Supported by INDOOR Biotechnologies, Inc, and Philip Morris USA, Inc.
Disclosure of potential conflict of interest: Drs Wünschmann and Pomés have received research support from Philip Morris; Dr Chapman has received research support from Philip Morris and is owner of INDOOR Biotechnologies, Inc; and Dr Gustchina has no conflict of interest to disclose.
ABSTRACT