Cockroach allergen Bla g 2: an unusual aspartic proteinase.
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 microg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific.


CONCLUSIONS
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.
Wünschmann
Sabina
S
INDOOR Biotechnologies, Inc, Charlottesville, VA 22903, USA. sabina@inbio.com
Gustchina
Alla
A
Chapman
Martin D
MD
Pomés
Anna
A
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003058:Cockroaches / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010436:Pepstatins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D010436:Pepstatins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C094699:allergen Bla g 2