Glycosylation of a disintegrin and metalloprotease 17 affects its activity and inhibition Article

Chavaroche, A, Cudic, M, Giulianotti, M et al. (2014). Glycosylation of a disintegrin and metalloprotease 17 affects its activity and inhibition . ANALYTICAL BIOCHEMISTRY, 449(1), 68-75. 10.1016/j.ab.2013.12.018

cited authors

  • Chavaroche, A; Cudic, M; Giulianotti, M; Houghten, RA; Fields, GB; Minond, D

authors

abstract

  • ADAM17 (a disintegrin and metalloprotease 17) is believed to be a tractable target in various diseases, including cancer and rheumatoid arthritis; however, it is not known whether glycosylation of ADAM17 expressed in healthy cells differs from that found in diseased tissue and, if so, whether glycosylation affects inhibitor binding. We expressed human ADAM17 in mammalian and insect cells and compared their glycosylation, substrate kinetics, and inhibition profiles. We found that ADAM17 expressed in mammalian cells was more heavily glycosylated than its insect-expressed analog. To determine whether differential glycosylation modulates enzymatic activity, we performed kinetic studies with both ADAM17 analogs and various TNFα-based substrates. The mammalian form of ADAM17 exhibited 10- to 30-fold lower kcat values than the insect analog, while the KM was unaffected, suggesting that glycosylation of ADAM17 can potentially play a role in regulating enzyme activity in vivo. Finally, we tested ADAM17 forms for inhibition by several well-characterized inhibitors. Active-site zinc-binding small molecules did not exhibit differences between the two ADAM17 analogs, while a non-zinc-binding exosite inhibitor of ADAM17 showed significantly lower potency toward the mammalian-expressed analog. These results suggest that glycosylation of ADAM17 can affect cell signaling in disease and might provide opportunities for therapeutic intervention using exosite inhibitors. © 2013 Elsevier B.V. All rights reserved.

publication date

  • March 15, 2014

published in

Digital Object Identifier (DOI)

start page

  • 68

end page

  • 75

volume

  • 449

issue

  • 1