Catalytic activity of human ADAM33

Area of Science:

• Biochemistry and Molecular Biology
• Genetics and Genomics
• Pharmacology

Background:

• ADAM33 (a disintegrin and metalloproteinase) is a recently identified asthma susceptibility gene.
• Understanding its catalytic properties is crucial for elucidating its role in disease pathogenesis.

Purpose of the Study:

• To characterize the catalytic properties of the metalloproteinase domain of human ADAM33.
• To identify ADAM33 peptide substrates and determine its cellular activity.
• To investigate the inhibitor profile of ADAM33.

Main Methods:

• Expression and purification of the metalloproteinase domain of human ADAM33 in Drosophila S2 cells.
• Testing of synthetic peptides as potential substrates, including beta-amyloid precursor protein (APP) and Kit-ligand-1 (KL-1).
• Utilizing a transfected cell-based sheddase assay to assess cellular activity.
• Evaluating inhibition by hydroxamate inhibitors and tissue inhibitors of metalloproteinase (TIMPs).

Main Results:

• ADAM33 cleaved specific peptides derived from APP, KL-1, and other proteins, with catalytic activity dependent on the E346A site.
• APP cleavage occurred inefficiently at a non-alpha-secretase site, while KL-1 cleavage occurred at a physiologically relevant site.
• ADAM33 negatively regulated APP shedding and mediated constitutive KL-1 shedding.
• ADAM33 activity was inhibited by hydroxamate inhibitors and differentially by TIMPs (TIMP-3, TIMP-4, TIMP-2), but not TIMP-1.

Conclusions:

• The study identified specific peptide substrates and characterized the cellular activity of ADAM33.
• ADAM33 exhibits a distinct inhibitor profile compared to other ADAMs.
• These findings provide a foundation for further functional studies of ADAM33 in asthma and other conditions.