Characterization of ADAMTS9 proteoglycanase activity: Comparison with ADAMTS1, ADAMTS4, and ADAMTS5.

Area of Science:

• Biochemistry
• Molecular Biology
• Developmental Biology

Background:

• ADAMTS9 (A Disintegrin-like And Metalloprotease domain with Thrombospondin type I motifs 9) is vital for embryogenesis, with null mutations causing embryonic lethality.
• ADAMTS9 deficiency leads to cardiovascular and ocular anomalies, highlighting its critical role beyond proteostasis.
• ADAMTS9 regulates versican, a key proteoglycan in the embryonic extracellular matrix, but its enzymatic activity is poorly understood.

Purpose of the Study:

• To characterize the proteoglycanase activity of ADAMTS9, focusing on its cleavage specificities.
• To compare ADAMTS9 activity with other ADAMTS proteases (ADAMTS1, ADAMTS4, ADAMTS5) using a truncated, functional construct.
• To identify novel cleavage sites in versican and biglycan mediated by ADAMTS9.

Main Methods:

• Expression and purification of a truncated ADAMTS9 construct (ADAMTS9 MDTCS) containing core domains.
• Identification of cleavage sites using label-free quantitation of peptides.
• Quantitative assays to determine enzymatic activity at specific versican cleavage sites.

Main Results:

• ADAMTS9 MDTCS cleaves versican (V1 and V2 isoforms) and biglycan at previously uncharacterized sites.
• ADAMTS9 MDTCS exhibits significantly lower versicanase activity at E441-A442 compared to ADAMTS5 and ADAMTS4, but higher than ADAMTS1.
• ADAMTS9 MDTCS was confirmed to cleave aggrecan at E392-A393.

Conclusions:

• ADAMTS9 possesses distinct proteoglycanase activity compared to other ADAMTS family members.
• Understanding these specificities is crucial for developing targeted inhibitors for diseases involving ADAMTS proteases.
• This study provides a foundation for further research into ADAMTS9 function and its therapeutic potential.