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

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.

Keywords: ADAM17; Exosites; Glycosylation; Metalloprotease.

Figure 1. Western blotting of the hADAM17_ECD recovered from the conditioned media after Ni-NTA agarose purification

P1 and P2, HEK293 are incubated in the presence of serum (FBS (+)). P3-P6, HEK293 are incubated in the absence of serum (FBS (−)).

Figure 2. Deglycosylation of insect and mammalian ADAM17 using PNGase F glycosidase

(A) His-Tag antibody and ADAM17_CD B). ADAM17-specific antibody and ADAM17_CD. C). ADAM17-specific antibody and ADAM17_ECD. 1 - insect non-treated; 2 - insect treated; 3 - mammalian non-treated; 4- mammalian treated.

Figure 3. Structures of substrates and inhibitors used to evaluate ADAM17 expressed in insect and mammalian cells

(A) Structures of substrates; (B) Structures of small molecular weight inhibitors. Substrate numbering according to [25].

Figure 4. Lineweaver-Burke plots of insect- and mammalian-produced ADAM17 ECD in presence of small molecule inhibitors

(A) ADAM17_ECD expressed in mammalian cells and TAPI-2 (B). ADAM17_ECD expressed in insect cells and TAPI-2. (C) ADAM17_ECD expressed in mammalian cells and CalBiochem MMP9/13 (D). ADAM17_EctoD expressed in insect cells and CalBiochem MMP9/13. Legend units are inhibitor concentration in μM.

Figure 5. Determination of concentration of active ADAM17_ECD using MMP9/13 inhibitor

(A) Titration of insect-produced ADAM7_ECD from R&D Systems; (B) Stoichiometry determination for insect-produced ADAM7_ECD from R&D Systems and MMP9/13 inhibitor; (C) Titration of mammalian-produced ADAM7_ECD; (D) Dose response study with insect- and mammalian-produced ADAM7_ECD and MMP9/13 inhibitor. Once the concentration of inhibitor for which V_i/V₀ is determined, the stoichiometric coefficient was calculated by dividing the label amount of R&D Systems enzyme present in the reaction. To determine the concentration of mammalian-produced ADAM17_ECD the stoichiometry of insect-produced ADAM17_ECD and MMP9/13 inhibitor interaction was used.

Figure 6. Inhibition profile of insect- and mammalian-expressed ADAM17_ECD

(A) ADAM17_ECD inhibition by N-acetohydroxamic acid; (B) ADAM17_ECD inhibition by TAPI-2; (C) ADAM17_ECD inhibition by compound #15 using substrate #4 ((EDANS) E-PLAQAVRSS*S-K (DABCYL). Note: * - glycosylation); (D) ADAM17_ECD inhibition by compound #15 using substrate ES010 (Mca-KPLGL-Dpa-AR-NH2).